Consensus for EGFR mutation testing in non-small cell lung cancer: results from a European workshop.

PubMed

Pirker, Robert; Herth, Felix J F; Kerr, Keith M; Filipits, Martin; Taron, Miquel; Gandara, David; Hirsch, Fred R; Grunenwald, Dominique; Popper, Helmut; Smit, Egbert; Dietel, Manfred; Marchetti, Antonio; Manegold, Christian; Schirmacher, Peter; Thomas, Michael; Rosell, Rafael; Cappuzzo, Federico; Stahel, Rolf

2010-10-01

Activating somatic mutations of the tyrosine kinase domain of epidermal growth factor receptor (EGFR) have recently been characterized in a subset of patients with advanced non-small cell lung cancer (NSCLC). Patients harboring these mutations in their tumors show excellent response to EGFR tyrosine kinase inhibitors (EGFR-TKIs). The EGFR-TKI gefitinib has been approved in Europe for the treatment of adult patients with locally advanced or metastatic NSCLC with activating mutations of the EGFR TK. Because EGFR mutation testing is not yet well established across Europe, biomarker-directed therapy only slowly emerges for the subset of NSCLC patients most likely to benefit: those with EGFR mutations. The "EGFR testing in NSCLC: from biology to clinical practice" International Association for the Study of Lung Cancer-European Thoracic Oncology Platform multidisciplinary workshop aimed at facilitating the implementation of EGFR mutation testing. Recommendations for high-quality EGFR mutation testing were formulated based on the opinion of the workshop expert group. Co-operation and communication flow between the various disciplines was considered to be of most importance. Participants agreed that the decision to request EGFR mutation testing should be made by the treating physician, and results should be available within 7 working days. There was agreement on the importance of appropriate sampling techniques and the necessity for the standardization of tumor specimen handling including fixation. Although there was no consensus on which laboratory test should be preferred for clinical decision making, all stressed the importance of standardization and validation of these tests. The recommendations of the workshop will help implement EGFR mutation testing in Europe and, thereby, optimize the use of EGFR-TKIs in clinical practice.

Evaluation of the need for routine clinical testing of PALB2 c.1592delT mutation in BRCA negative Northern Finnish breast cancer families.

PubMed

Haanpää, Maria; Pylkäs, Katri; Moilanen, Jukka S; Winqvist, Robert

2013-08-13

Testing for mutations in the BRCA1 and BRCA2 genes among high-risk breast cancer patients has become a routine practice among clinical geneticists. Unfortunately, however, the genetic background of a majority of the cases coming to the clinics remains currently unexplained, making genetic counseling rather challenging. In recent years it has become evident world-wide that also women carrying a heterozygous germline mutation in PALB2 are at significantly increased risk of getting breast cancer. We have previously studied the clinical as well as biological impact of the PALB2 c.1592delT founder mutation occurring in about 1% of Finnish breast cancer patients unselected for their family history of disease, and our results demonstrated a 40% increased breast cancer risk by age 70 for female mutation carriers. Thus, this relatively common mutation in PALB2 is associated with a high risk of developing breast cancer. The aim of the current study was to analyze whether female index individuals of breast cancer families who had tested negative for germline mutations in BRCA1/BRCA2 as part of genetic counseling services should be offered mutation testing for PALB2 c.1592delT. The study cohort consisted of altogether 223 individuals who had contacted the Department of Clinical Genetics at the Oulu University Hospital in Finland between the years 1997 and 2011 for counseling on hereditary breast and/or ovarian cancer risk. 101 of them met our inclusion criteria. Of these, 10 persons were now deceased, but 6 of them had participated in one of our previous studies on PALB2. Seventy (77%) of the remaining 91 persons responded positively to our study invitation. Chart review of updated pedigree data led to the exclusion of 14 further individuals not meeting the selection criteria. Of the 56 alive affected female individuals screened for PALB2 c.1592delT, altogether two (3.6%) tested positive for this mutation. In addition, of the previously tested but now deceased 6 persons eligible for the current study, one more mutation carrier was observed. Therefore, overall 4.8% (3/62) of the tested individuals belonging to the Northern Finnish 1997-2011 study cohort turned out to be carriers of the PALB2 c.1592delT allele. Given the potential benefits versus harms of this testing, the result of our study suggest that PALB2 c.1592delT should be a routine part of the genetic counseling protocol for Finnish high-risk breast cancer cases tested negative for mutations in BRCA1/BRCA2.

Cancer risk and clinicopathological characteristics of thyroid nodules harboring thyroid-stimulating hormone receptor gene mutations.

PubMed

Mon, Sann Y; Riedlinger, Gregory; Abbott, Collette E; Seethala, Raja; Ohori, N Paul; Nikiforova, Marina N; Nikiforov, Yuri E; Hodak, Steven P

2018-05-01

Thyroid-stimulating hormone receptor (TSHR) gene mutations play a critical role in thyroid cell proliferation and function. They are found in 20%-82% of hyperfunctioning nodules, hyperfunctioning follicular thyroid cancers (FTC), and papillary thyroid cancers (PTC). The diagnostic importance of TSHR mutation testing in fine needle aspiration (FNA) specimens remains unstudied. To examine the association of TSHR mutations with the functional status and surgical outcomes of thyroid nodules, we evaluated 703 consecutive thyroid FNA samples with indeterminate cytology for TSHR mutations using next-generation sequencing. Testing for EZH1 mutations was performed in selected cases. The molecular diagnostic testing was done as part of standard of care treatment, and did not require informed consent. TSHR mutations were detected in 31 (4.4%) nodules and were located in exons 281-640, with codon 486 being the most common. Allelic frequency ranged from 3% to 45%. Of 16 cases (12 benign, 3 FTC, 1 PTC) with surgical correlation, 15 had solitary TSHR mutations and 1 PTC had comutation with BRAF V600E. Hyperthyroidism was confirmed in all 3 FTC (2 overt, 1 subclinical). Of 5 nodules with solitary TSHR mutations detected at high allelic frequency, 3 (60%) were FTC. Those at low allelic frequency (3%-22%) were benign. EZH1 mutations were detected in 2 of 4 TSHR-mutant malignant nodules and neither of 2 benign nodules. We report that TSHR mutations occur in ∼5% thyroid nodules in a large consecutive series with indeterminate cytology. TSHR mutations may be associated with an increased cancer risk when present at high allelic frequency, even when the nodule is hyperfunctioning. Benign nodules were however most strongly correlated with TSHR mutations at low allelic frequency. © 2018 Wiley Periodicals, Inc.

False-negative BRAF V600E mutation results on fine-needle aspiration cytology of papillary thyroid carcinoma.

PubMed

Paek, Se Hyun; Kim, Byung Seup; Kang, Kyung Ho; Kim, Hee Sung

2017-11-13

The BRAF V600E mutation is highly specific for papillary thyroid carcinoma (PTC). A test for this mutation can increase the diagnostic accuracy of fine-needle aspiration cytology (FNAC), but a considerably high false-negative rate for the BRAF V600E mutation on FNAC has been reported. In this study, we investigated the risk factors associated with false-negative BRAF V600E mutation results on FNAC. BRAF V600E mutation results of 221 PTC nodules between December 2011 and June 2013 were retrospectively reviewed. BRAF V600E mutation results on both preoperative FNAC and postoperative formalin-fixed, paraffin-embedded (FFPE) samples were compared. We investigated the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of BRAF V600E mutation results on FNAC. And, we identified the risk factors associated with false-negative results. Of 221 PTC nodules, 150 (67.9%) on FNAC and 185 (83.7%) on FFPE samples were BRAF V600E mutation positive. The sensitivity, specificity, PPV, and NPV for BRAF V600E mutation testing with FNAC were 80.5, 97.2, 99.3, and 49.3%, respectively. Thirty-six (16.3%) BRAF V600E mutation-negative nodules on FNAC were mutation positive on FFPE sample analysis. Risk factors for these false-negative results were age, indeterminate FNAC results (nondiagnostic, atypia of undetermined significance (AUS), and findings suspicious for PTC), and PTC subtype. False-negative rate of BRAF mutation testing with FNAC for thyroid nodules is increased in cases of old age, indeterminate FNAC pathology results, and certain PTC subtypes. Therapeutic surgery can be considered for these cases. A well-designed prospective study with informed consent of patients will be essential for more informative results.

Prevalence and Spectrum of Germline Cancer Susceptibility Gene Mutations Among Patients With Early-Onset Colorectal Cancer

PubMed Central

Pearlman, Rachel; Frankel, Wendy L.; Swanson, Benjamin; Zhao, Weiqiang; Yilmaz, Ahmet; Miller, Kristin; Bacher, Jason; Bigley, Christopher; Nelsen, Lori; Goodfellow, Paul J.; Goldberg, Richard M.; Paskett, Electra; Shields, Peter G.; Freudenheim, Jo L.; Stanich, Peter P; Lattimer, Ilene; Arnold, Mark; Liyanarachchi, Sandya; Kalady, Matthew; Heald, Brandie; Greenwood, Carla; Paquette, Ian; Prues, Marla; Draper, David J.; Lindeman, Carolyn; Kuebler, J. Philip; Reynolds, Kelly; Brell, Joanna M.; Shaper, Amy A.; Mahesh, Sameer; Buie, Nicole; Weeman, Kisa; Shine, Kristin; Haut, Mitchell; Edwards, Joan; Bastola, Shyamal; Wickham, Karen; Khanduja, Karamjit S.; Zacks, Rosemary; Pritchard, Colin C.; Shirts, Brian H.; Jacobson, Angela; Allen, Brian; de la Chapelle, Albert; Hampel, Heather

2017-01-01

IMPORTANCE Hereditary cancer syndromes infer high cancer risks and require intensive cancer surveillance, yet the prevalence and spectrum of these conditions among unselected patients with early-onset colorectal cancer (CRC) is largely undetermined. OBJECTIVE To determine the frequency and spectrum of cancer susceptibility gene mutations among patients with early-onset CRC. DESIGN, SETTING, AND PARTICIPANTS Overall, 450 patients diagnosed with colorectal cancer younger than 50 years were prospectively accrued from 51 hospitals into the Ohio Colorectal Cancer Prevention Initiative from January 1, 2013, to June 20, 2016. Mismatch repair (MMR) deficiency was determined by microsatellite instability and/or immunohistochemistry. Germline DNA was tested for mutations in 25 cancer susceptibility genes using next-generation sequencing. MAIN OUTCOMES AND MEASURES Mutation prevalence and spectrum in patients with early-onset CRC was determined. Clinical characteristics were assessed by mutation status. RESULTS In total 450 patients younger than 50 years were included in the study, and 75 gene mutations were found in 72 patients (16%). Forty-eight patients (10.7%) had MMR-deficient tumors, and 40 patients (83.3%) had at least 1 gene mutation: 37 had Lynch syndrome (13, MLH1 [including one with constitutional MLH1 methylation]; 16, MSH2; 1, MSH2/monoallelic MUTYH; 2, MSH6; 5, PMS2); 1 patient had the APC c.3920T>A, p.I1307K mutation and a PMS2 variant; 9 patients (18.8%) had double somatic MMR mutations (including 2 with germline biallelic MUTYH mutations); and 1 patient had somatic MLH1 methylation. Four hundred two patients (89.3%) had MMR-proficient tumors, and 32 patients (8%) had at least 1 gene mutation: 9 had mutations in high-penetrance CRC genes (5, APC; 1, APC/PMS2; 2, biallelic MUTYH; 1, SMAD4); 13 patients had mutations in high- or moderate-penetrance genes not traditionally associated with CRC (3, ATM; 1, ATM/CHEK2; 2, BRCA1; 4, BRCA2; 1, CDKN2A; 2, PALB2); 10 patients had mutations in low-penetrance CRC genes (3, APC c.3920T>A, p.I1307K; 7, monoallelic MUTYH). Importantly, 24 of 72 patients (33.3%) who were mutation positive did not meet established genetic testing criteria for the gene(s) in which they had a mutation. CONCLUSIONS AND RELEVANCE Of 450 patients with early-onset CRC, 72 (16%) had gene mutations. Given the high frequency and wide spectrum of mutations, genetic counseling and testing with a multigene panel could be considered for all patients with early-onset CRC. PMID:27978560

Genetic Testing in Pancreatic Ductal Adenocarcinoma: Implications for Prevention and Treatment.

PubMed

Peters, Mary Linton B; Tseng, Jennifer F; Miksad, Rebecca A

2016-07-01

This article reviews the progress to date and future directions for investigation of germline and somatic genetic testing to inform pancreatic adenocarcinoma (PDAC) treatment, screening, and prevention strategies. We searched PubMed to identify recent articles regarding genetic testing in pancreatic cancer, including both germline and somatic testing, and recent genome-wide association studies. References were specifically hand searched as relevant. Guidelines for testing and screening high-risk individuals were included. We searched clinicaltrials.gov to review the current landscape of active clinical trials. Approximately 10% of PDACs are associated with an identified germline mutation. Although germline mutations may inform treatment options and identify high-risk individuals for screening in other cancers, the data on PDAC are only now emerging. For example, poly adenosine diphosphate ribose polymerase (PARP) inhibitors are under investigation for BRCA-associated PDAC. Somatic mutations have also been identified in PDAC. However, current data are limited regarding treatment for potential PDAC somatic driver mutations. Although erlotinib is used in PDAC, its use is not targeted based on a tumor marker. Many tyrosine kinase inhibitors targeted toward potential driver mutations and critical pathways are in development, including BRAF/MEK, ALK, and CDK4/6. A consensus on screening strategies for individuals at high risk for PDAC is still evolving because of the relatively low prevalence of the disease, the relative invasiveness of endoscopic procedures often used as part of screening, and the lack of a clear survival benefit. Pancreatic cancer has been slower to move toward genomic testing, partially because of a lower prevalence of mutations and partially because of a limited effect of results on treatment choices outside a clinical trial. This is an area of active investigation, and we anticipate that there will be both preventive and therapeutic implications of driver mutations in the coming decade. Copyright © 2016 Elsevier HS Journals, Inc. All rights reserved.

BRCA mutation genetic testing implications in the United States.

PubMed

Bayraktar, Soley; Arun, Banu

2017-02-01

BRCA mutation carriers have a very high risk of breast and ovarian cancer by age 70, in the ranges 47%-66% and 40%-57%, respectively. Additionally, women with BRCA mutation-associated breast cancer also have an elevated risk of other or secondary malignancies. Fortunately, the breast and ovarian cancer outcome for BRCA1/2 mutation carriers is at least as good as for non-carriers with chemoprevention, prophylactic surgeries and appropriate use of therapies. Therefore, identification of those who might have a mutation is important so that genetic counseling, testing, screening and prevention strategies can be applied in a timely manner. This article reviews the impact of genetic testing in general, timing of genetic testing after diagnosis and prior knowledge of mutation status in BRCA carriers with newly diagnosed breast cancer. Additionally, risk-reducing surgeries including the prophylactic contralateral mastectomy, and bilateral salpingo-oophorectomy and the sensitivity of BRCA-defective breast cancer cell lines to differential chemotherapeutic agents will be discussed. Copyright © 2016 Elsevier Ltd. All rights reserved.

Multi-Center Evaluation of the Fully Automated PCR-Based Idylla™ KRAS Mutation Assay for Rapid KRAS Mutation Status Determination on Formalin-Fixed Paraffin-Embedded Tissue of Human Colorectal Cancer

PubMed Central

Solassol, Jérôme; Vendrell, Julie; Märkl, Bruno; Haas, Christian; Bellosillo, Beatriz; Montagut, Clara; Smith, Matthew; O’Sullivan, Brendan; D’Haene, Nicky; Le Mercier, Marie; Grauslund, Morten; Melchior, Linea Cecilie; Burt, Emma; Cotter, Finbarr; Stieber, Daniel; Schmitt, Fernando de Lander; Motta, Valentina; Lauricella, Calogero; Colling, Richard; Soilleux, Elizabeth; Fassan, Matteo; Mescoli, Claudia; Collin, Christine; Pagès, Jean-Christophe; Sillekens, Peter

2016-01-01

Since the advent of monoclonal antibodies against epidermal growth factor receptor (EGFR) in colorectal cancer therapy, the determination of RAS mutational status is needed for therapeutic decision-making. Most prevalent in colorectal cancer are KRAS exon 2 mutations (40% prevalence); lower prevalence is observed for KRAS exon 3 and 4 mutations (6%) and NRAS exon 2, 3, and 4 mutations (5%). The Idylla™ KRAS Mutation Test on the molecular diagnostics Idylla™ platform is a simple (<2 minutes hands-on time), highly reliable, and rapid (approximately 2 hours turnaround time) in vitro diagnostic sample-to-result solution. This test enables qualitative detection of 21 mutations in codons 12, 13, 59, 61, 117, and 146 of the KRAS oncogene being clinically relevant according to the latest clinical guidelines. Here, the performance of the Idylla™ KRAS Mutation Assay, for Research Use Only, was assessed on archived formalin-fixed paraffin-embedded (FFPE) tissue sections by comparing its results with the results previously obtained by routine reference approaches for KRAS genotyping. In case of discordance, samples were assessed further by additional methods. Among the 374 colorectal cancer FFPE samples tested, the overall concordance between the Idylla™ KRAS Mutation Assay and the confirmed reference routine test results was found to be 98.9%. The Idylla™ KRAS Mutation Assay enabled detection of 5 additional KRAS-mutated samples not detected previously with reference methods. As conclusion the Idylla™ KRAS Mutation Test can be applied as routine tool in any clinical setting, without needing molecular infrastructure or expertise, to guide the personalized treatment of colorectal cancer patients. PMID:27685259

Mutation Detection in Patients With Advanced Cancer by Universal Sequencing of Cancer-Related Genes in Tumor and Normal DNA vs Guideline-Based Germline Testing.

PubMed

Mandelker, Diana; Zhang, Liying; Kemel, Yelena; Stadler, Zsofia K; Joseph, Vijai; Zehir, Ahmet; Pradhan, Nisha; Arnold, Angela; Walsh, Michael F; Li, Yirong; Balakrishnan, Anoop R; Syed, Aijazuddin; Prasad, Meera; Nafa, Khedoudja; Carlo, Maria I; Cadoo, Karen A; Sheehan, Meg; Fleischut, Megan H; Salo-Mullen, Erin; Trottier, Magan; Lipkin, Steven M; Lincoln, Anne; Mukherjee, Semanti; Ravichandran, Vignesh; Cambria, Roy; Galle, Jesse; Abida, Wassim; Arcila, Marcia E; Benayed, Ryma; Shah, Ronak; Yu, Kenneth; Bajorin, Dean F; Coleman, Jonathan A; Leach, Steven D; Lowery, Maeve A; Garcia-Aguilar, Julio; Kantoff, Philip W; Sawyers, Charles L; Dickler, Maura N; Saltz, Leonard; Motzer, Robert J; O'Reilly, Eileen M; Scher, Howard I; Baselga, Jose; Klimstra, David S; Solit, David B; Hyman, David M; Berger, Michael F; Ladanyi, Marc; Robson, Mark E; Offit, Kenneth

2017-09-05

Guidelines for cancer genetic testing based on family history may miss clinically actionable genetic changes with established implications for cancer screening or prevention. To determine the proportion and potential clinical implications of inherited variants detected using simultaneous sequencing of the tumor and normal tissue ("tumor-normal sequencing") compared with genetic test results based on current guidelines. From January 2014 until May 2016 at Memorial Sloan Kettering Cancer Center, 10 336 patients consented to tumor DNA sequencing. Since May 2015, 1040 of these patients with advanced cancer were referred by their oncologists for germline analysis of 76 cancer predisposition genes. Patients with clinically actionable inherited mutations whose genetic test results would not have been predicted by published decision rules were identified. Follow-up for potential clinical implications of mutation detection was through May 2017. Tumor and germline sequencing compared with the predicted yield of targeted germline sequencing based on clinical guidelines. Proportion of clinically actionable germline mutations detected by universal tumor-normal sequencing that would not have been detected by guideline-directed testing. Of 1040 patients, the median age was 58 years (interquartile range, 50.5-66 years), 65.3% were male, and 81.3% had stage IV disease at the time of genomic analysis, with prostate, renal, pancreatic, breast, and colon cancer as the most common diagnoses. Of the 1040 patients, 182 (17.5%; 95% CI, 15.3%-19.9%) had clinically actionable mutations conferring cancer susceptibility, including 149 with moderate- to high-penetrance mutations; 101 patients tested (9.7%; 95% CI, 8.1%-11.7%) would not have had these mutations detected using clinical guidelines, including 65 with moderate- to high-penetrance mutations. Frequency of inherited mutations was related to case mix, stage, and founder mutations. Germline findings led to discussion or initiation of change to targeted therapy in 38 patients tested (3.7%) and predictive testing in the families of 13 individuals (1.3%), including 6 for whom genetic evaluation would not have been initiated by guideline-based testing. In this referral population with selected advanced cancers, universal sequencing of a broad panel of cancer-related genes in paired germline and tumor DNA samples was associated with increased detection of individuals with potentially clinically significant heritable mutations over the predicted yield of targeted germline testing based on current clinical guidelines. Knowledge of these additional mutations can help guide therapeutic and preventive interventions, but whether all of these interventions would improve outcomes for patients with cancer or their family members requires further study. clinicaltrials.gov Identifier: NCT01775072.

Population genetic testing for cancer susceptibility: founder mutations to genomes.

PubMed

Foulkes, William D; Knoppers, Bartha Maria; Turnbull, Clare

2016-01-01

The current standard model for identifying carriers of high-risk mutations in cancer-susceptibility genes (CSGs) generally involves a process that is not amenable to population-based testing: access to genetic tests is typically regulated by health-care providers on the basis of a labour-intensive assessment of an individual's personal and family history of cancer, with face-to-face genetic counselling performed before mutation testing. Several studies have shown that application of these selection criteria results in a substantial proportion of mutation carriers being missed. Population-based genetic testing has been proposed as an alternative approach to determining cancer susceptibility, and aims for a more-comprehensive detection of mutation carriers. Herein, we review the existing data on population-based genetic testing, and consider some of the barriers, pitfalls, and challenges related to the possible expansion of this approach. We consider mechanisms by which population-based genetic testing for cancer susceptibility could be delivered, and suggest how such genetic testing might be integrated into existing and emerging health-care structures. The existing models of genetic testing (including issues relating to informed consent) will very likely require considerable alteration if the potential benefits of population-based genetic testing are to be fully realized.

Lynch Syndrome in high risk Ashkenazi Jews in Israel.

PubMed

Goldberg, Yael; Kedar, Inbal; Kariiv, Revital; Halpern, Naama; Plesser, Morasha; Hubert, Ayala; Kaduri, Luna; Sagi, Michal; Lerer, Israela; Abeliovich, Dvorah; Hamburger, Tamar; Nissan, Aviram; Goldshmidt, Hanoch; Solar, Irit; Geva, Ravit; Strul, Hana; Rosner, Guy; Baris, Hagit; Levi, Zohar; Peretz, Tamar

2014-03-01

Lynch Syndrome is caused by mutations in DNA mismatch repair genes. Diagnosis is not always trivial and may be costly. Information regarding incidence, genotype-phenotype correlation, spectrum of mutations and genes involved in specific populations facilitate the diagnostic process and contribute to clinical work-up. To report gene distribution, mutations detected and co-occurrence of related syndromes in a cohort of Ashkenazi Jews in Israel. Patients were identified in dedicated high risk clinics in 3 medical centers in Israel. Diagnostic process followed a multi-step scheme. It included testing for founder mutations, tumor testing, gene sequencing and MLPA. Lynch Syndrome was defined either by positive mutation testing, or by clinical criteria and positive tumor analysis. We report a cohort of 75 Ashkenazi families suspected of Lynch Syndrome. Mutations were identified in 51/75 (68%) families: 38 in MSH2, 9 in MSH6, and 4 in MLH1. 37/51 (73%) of these families carried one of the 3 'Ashkenazi' founder mutations in MSH2 or MSH6. Each of the other 14 families carried a private mutation. 3 (6%) were large deletions. Only 20/51 (39%) families were Amsterdam Criteria positive; 42 (82%) were positive for the Bethesda guidelines and 9 (18%) did not fulfill any Lynch Syndrome criteria. We report C-MMRD and co-occurrence of BRCA and Lynch Syndrome in our cohort. Mutation spectra and gene distribution among Ashkenazi Jews are unique. Three founder Lynch Syndrome mutations are found in 73% families with known mutations. Among the three, MSH2 and MSH6 are the most common. These features affect the phenotype, the diagnostic process, risk estimation, and genetic counseling.

Impact of Clinical Genetics Attendance at a Gynecologic Oncology Tumor Board on Referrals for Genetic Counseling and BRCA Mutation Testing.

PubMed

Cohen, Paul A; Nichols, Cassandra B; Schofield, Lyn; Van Der Werf, Steven; Pachter, Nicholas

2016-06-01

The objectives of this work were to determine the proportion of eligible patients with ovarian cancer discussed at a gynecologic oncology tumor board who were referred for counseling and BRCA mutation testing; to compare referral rates before genetics attendance at the tumor board to referral rates after genetics attendance; and to ascertain the proportions of women with germline BRCA mutations. Eligible cases were identified from the minutes of the weekly Western Australian gynecologic oncology tumor board from July 1, 2013 to June 30, 2015.Patients with ovarian cancer who met eligibility criteria for genetics referral were identified and checked against the records of the genetic services database to ascertain whether a referral was received. Outcomes including attendance for counseling and results of mutation testing were analyzed. Two hundred sixty-one patients were eligible for referral during the 24-month study period. One hundred six patients (40.6%) were referred for counseling and germline mutation testing. Of the eligible patients, 26.7% were referred in the 12 months before genetics attendance at the tumor board compared to 51.7% of the eligible patients in the 12 months after genetics attendance (P ≤ 0.0001). Ninety-seven patients were offered BRCA mutation testing, and 73 underwent testing with 65 results reported to date. Twenty-two patients (33.8 %) tested positive for a germline BRCA mutation. Patients with ovarian cancer had a high rate of BRCA mutations. Attendance of a genetics service at a tumor board was associated with an improved rate of referral of patients for genetic counseling and BRCA mutation testing.

Direct-to-patient BRCA1 testing: the Twoj Styl experience.

PubMed

Gronwald, Jacek; Huzarski, Tomasz; Byrski, Tomasz; Debniak, Tadeusz; Metcalfe, Kelly; Narod, Steven A; Lubiński, Jan

2006-12-01

Ideally, a genetic screening program for cancer should offer testing to all women who qualify, and who wish to participate, and who might benefit from the test. As the number of preventive options for women at high risk for hereditary breast cancer expands, the demand for testing increases. However, many women do not have ready access to testing because of cost, and many others have not been recognized by their physicians to be candidates for testing. It is possible to increase women's awareness about hereditary cancer through the popular press. Genetic testing was offered to 5000 Polish women through an announcement placed in a popular women's magazine (Twoj Styl) in October 2001. A total of 5024 women who qualified received a free genetic test for three mutations in BRCA1 which are common in Poland. Out of these, 198 women (3.9%) were found to carry a BRCA1 mutation. The overall cost per mutation detected was 630 US dollars--approximately 50-100 times less than the equivalent cost in North America. Genetic counseling was offered to women with a positive test or with a significant family history of breast or ovarian cancer. The great majority of women who took part in the program expressed a high degree of satisfaction and after one year approximately two-thirds of identified mutation carriers had complied with our recommendations for breast cancer screening. We found this model of genetic testing and delivery of genetic information to be very efficient in a population in which founder mutations predominate. There is a need for similar studies in other populations.

Clinical applications and implications of common and founder mutations in Indian subpopulations.

PubMed

Ankala, Arunkanth; Tamhankar, Parag M; Valencia, C Alexander; Rayam, Krishna K; Kumar, Manisha M; Hegde, Madhuri R

2015-01-01

South Asian Indians represent a sixth of the world's population and are a racially, geographically, and genetically diverse people. Their unique anthropological structure, prevailing caste system, and ancient religious practices have all impacted the genetic composition of most of the current-day Indian population. With the evolving socio-religious and economic activities of the subsects and castes, endogamous and consanguineous marriages became a commonplace. Consequently, the frequency of founder mutations and the burden of heritable genetic disorders rose significantly. Specifically, the incidence of certain autosomal-recessive disorders is relatively high in select Indian subpopulations and communities that share common recent ancestry. Although today clinical genetics and molecular diagnostic services are making inroads in India, the high costs associated with the technology and the tests often keep patients from an exact molecular diagnosis, making more customized and tailored tests, such as those interrogating the most common and founder mutations or those that cater to select sects within the population, highly attractive. These tests offer a quick first-hand affordable diagnostic and carrier screening tool. Here, we provide a comprehensive catalog of known common mutations and founder mutations in the Indian population and discuss them from a molecular, clinical, and historical perspective. © 2014 WILEY PERIODICALS, INC.

Mutations in epilepsy and intellectual disability genes in patients with features of Rett syndrome.

PubMed

Olson, Heather E; Tambunan, Dimira; LaCoursiere, Christopher; Goldenberg, Marti; Pinsky, Rebecca; Martin, Emilie; Ho, Eugenia; Khwaja, Omar; Kaufmann, Walter E; Poduri, Annapurna

2015-09-01

Rett syndrome and neurodevelopmental disorders with features overlapping this syndrome frequently remain unexplained in patients without clinically identified MECP2 mutations. We recruited a cohort of 11 patients with features of Rett syndrome and negative initial clinical testing for mutations in MECP2. We analyzed their phenotypes to determine whether patients met formal criteria for Rett syndrome, reviewed repeat clinical genetic testing, and performed exome sequencing of the probands. Using 2010 diagnostic criteria, three patients had classical Rett syndrome, including two for whom repeat MECP2 gene testing had identified mutations. In a patient with neonatal onset epilepsy with atypical Rett syndrome, we identified a frameshift deletion in STXBP1. Among seven patients with features of Rett syndrome not fulfilling formal diagnostic criteria, four had suspected pathogenic mutations, one each in MECP2, FOXG1, SCN8A, and IQSEC2. MECP2 mutations are highly correlated with classical Rett syndrome. Genes associated with atypical Rett syndrome, epilepsy, or intellectual disability should be considered in patients with features overlapping with Rett syndrome and negative MECP2 testing. While most of the identified mutations were apparently de novo, the SCN8A variant was inherited from an unaffected parent mosaic for the mutation, which is important to note for counseling regarding recurrence risks. © 2015 Wiley Periodicals, Inc.

Mutations in Epilepsy and Intellectual Disability Genes in Patients with Features of Rett Syndrome

PubMed Central

Olson, Heather E.; Tambunan, Dimira; LaCoursiere, Christopher; Goldenberg, Marti; Pinsky, Rebecca; Martin, Emilie; Ho, Eugenia; Khwaja, Omar; Kaufmann, Walter E.; Poduri, Annapurna

2017-01-01

Rett syndrome and neurodevelopmental disorders with features overlapping this syndrome frequently remain unexplained in patients without clinically identified MECP2 mutations. We recruited a cohort of 11 patients with features of Rett syndrome and negative initial clinical testing for mutations in MECP2. We analyzed their phenotypes to determine whether patients met formal criteria for Rett syndrome, reviewed repeat clinical genetic testing, and performed exome sequencing of the probands. Using 2010 diagnostic criteria, three patients had classical Rett syndrome, including two for whom repeat MECP2 gene testing had identified mutations. In a patient with neonatal onset epilepsy with atypical Rett syndrome, we identified a frameshift deletion in STXBP1. Among seven patients with features of Rett syndrome not fulfilling formal diagnostic criteria, four had suspected pathogenic mutations, one each in MECP2, FOXG1, SCN8A, and IQSEC2. MECP2 mutations are highly correlated with classical Rett syndrome. Genes associated with atypical Rett syndrome, epilepsy, or intellectual disability should be considered in patients with features overlapping with Rett syndrome and negative MECP2 testing. While most of the identified mutations were apparently de novo, the SCN8A variant was inherited from an unaffected parent mosaic for the mutation, which is important to note for counseling regarding recurrence risks. PMID:25914188

KRAS mutation testing in colorectal cancer: comparison of the results obtained using 3 different methods for the analysis of codons G12 and G13.

PubMed

Bihl, Michel P; Hoeller, Sylvia; Andreozzi, Maria Carla; Foerster, Anja; Rufle, Alexander; Tornillo, Luigi; Terracciano, Luigi

2012-03-01

Targeting the epidermal growth factor receptor (EGFR) is a new therapeutic option for patients with metastatic colorectal or lung carcinoma. However, the therapy efficiency highly depends on the KRAS mutation status in the given tumour. Therefore a reliable and secure KRAS mutation testing is crucial. Here we investigated 100 colorectal carcinoma samples with known KRAS mutation status (62 mutated cases and 38 wild type cases) in a comparative manner with three different KRAS mutation testing techniques (Pyrosequencing, Dideoxysequencing and INFINITI) in order to test their reliability and sensitivity. For the large majority of samples (96/100, 96%), the KRAS mutation status obtained by all three methods was the same. Only two cases with clear discrepancies were observed. One case was reported as wild type by the INFINITI method while the two other methods detected a G13C mutation. In the second case the mutation could be detected by the Pyrosequencing and INFINITI method (15% and 15%), while no signal for mutation could be observed with the Dideoxysequencing method. Additional two unclear results were due to a detection of a G12V with the INFINITI method, which was below cut-off when repeated and which was not detectable by the other two methods and very weak signals in a G12V mutated case with the Dideoxy- and Pyroseqencing method compared to the INFINITI method, respectively. In summary all three methods are reliable and robust methods in detecting KRAS mutations. INFINITI, however seems to be slightly more sensitive compared to Dideoxy- and Pyrosequencing.

Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing

PubMed Central

Walsh, Tom; Lee, Ming K.; Casadei, Silvia; Thornton, Anne M.; Stray, Sunday M.; Pennil, Christopher; Nord, Alex S.; Mandell, Jessica B.; Swisher, Elizabeth M.; King, Mary-Claire

2010-01-01

Inherited loss-of-function mutations in the tumor suppressor genes BRCA1, BRCA2, and multiple other genes predispose to high risks of breast and/or ovarian cancer. Cancer-associated inherited mutations in these genes are collectively quite common, but individually rare or even private. Genetic testing for BRCA1 and BRCA2 mutations has become an integral part of clinical practice, but testing is generally limited to these two genes and to women with severe family histories of breast or ovarian cancer. To determine whether massively parallel, “next-generation” sequencing would enable accurate, thorough, and cost-effective identification of inherited mutations for breast and ovarian cancer, we developed a genomic assay to capture, sequence, and detect all mutations in 21 genes, including BRCA1 and BRCA2, with inherited mutations that predispose to breast or ovarian cancer. Constitutional genomic DNA from subjects with known inherited mutations, ranging in size from 1 to >100,000 bp, was hybridized to custom oligonucleotides and then sequenced using a genome analyzer. Analysis was carried out blind to the mutation in each sample. Average coverage was >1200 reads per base pair. After filtering sequences for quality and number of reads, all single-nucleotide substitutions, small insertion and deletion mutations, and large genomic duplications and deletions were detected. There were zero false-positive calls of nonsense mutations, frameshift mutations, or genomic rearrangements for any gene in any of the test samples. This approach enables widespread genetic testing and personalized risk assessment for breast and ovarian cancer. PMID:20616022

Evaluation of BRCA1 and BRCA2 mutation prevalence, risk prediction models and a multistep testing approach in French‐Canadian families with high risk of breast and ovarian cancer

PubMed Central

Simard, Jacques; Dumont, Martine; Moisan, Anne‐Marie; Gaborieau, Valérie; Vézina, Hélène; Durocher, Francine; Chiquette, Jocelyne; Plante, Marie; Avard, Denise; Bessette, Paul; Brousseau, Claire; Dorval, Michel; Godard, Béatrice; Houde, Louis; Joly, Yann; Lajoie, Marie‐Andrée; Leblanc, Gilles; Lépine, Jean; Lespérance, Bernard; Malouin, Hélène; Parboosingh, Jillian; Pichette, Roxane; Provencher, Louise; Rhéaume, Josée; Sinnett, Daniel; Samson, Carolle; Simard, Jean‐Claude; Tranchant, Martine; Voyer, Patricia; BRCAs, INHERIT; Easton, Douglas; Tavtigian, Sean V; Knoppers, Bartha‐Maria; Laframboise, Rachel; Bridge, Peter; Goldgar, David

2007-01-01

Background and objective In clinical settings with fixed resources allocated to predictive genetic testing for high‐risk cancer predisposition genes, optimal strategies for mutation screening programmes are critically important. These depend on the mutation spectrum found in the population under consideration and the frequency of mutations detected as a function of the personal and family history of cancer, which are both affected by the presence of founder mutations and demographic characteristics of the underlying population. The results of multistep genetic testing for mutations in BRCA1 or BRCA2 in a large series of families with breast cancer in the French‐Canadian population of Quebec, Canada are reported. Methods A total of 256 high‐risk families were ascertained from regional familial cancer clinics throughout the province of Quebec. Initially, families were tested for a panel of specific mutations known to occur in this population. Families in which no mutation was identified were then comprehensively tested. Three algorithms to predict the presence of mutations were evaluated, including the prevalence tables provided by Myriad Genetics Laboratories, the Manchester Scoring System and a logistic regression approach based on the data from this study. Results 8 of the 15 distinct mutations found in 62 BRCA1/BRCA2‐positive families had never been previously reported in this population, whereas 82% carried 1 of the 4 mutations currently observed in ⩾2 families. In the subset of 191 families in which at least 1 affected individual was tested, 29% carried a mutation. Of these 27 BRCA1‐positive and 29 BRCA2‐positive families, 48 (86%) were found to harbour a mutation detected by the initial test. Among the remaining 143 inconclusive families, all 8 families found to have a mutation after complete sequencing had Manchester Scores ⩾18. The logistic regression and Manchester Scores provided equal predictive power, and both were significantly better than the Myriad Genetics Laboratories prevalence tables (p<0.001). A threshold of Manchester Score ⩾18 provided an overall sensitivity of 86% and a specificity of 82%, with a positive predictive value of 66% in this population. Conclusion In this population, a testing strategy with an initial test using a panel of reported recurrent mutations, followed by full sequencing in families with Manchester Scores ⩾18, represents an efficient test in terms of overall cost and sensitivity. PMID:16905680

EGFR Mutation Testing in Patients with Advanced Non-Small Cell Lung Cancer: A Comprehensive Evaluation of Real-World Practice in an East Asian Tertiary Hospital

PubMed Central

Cho, Juhee; Rampal, Sanjay; Han, Joungho; Parasuraman, Bhash; Guallar, Eliseo; Lee, Genehee; Lee, Jeeyun; Shim, Young Mog

2013-01-01

Introduction Guidelines for management of non-small cell lung cancer (NSCLC) strongly recommend EGFR mutation testing. These recommendations are particularly relevant in Asians that have higher EGFR mutation prevalence. This study aims to explore current testing practices, logistics of testing, types of EGFR mutation, and prevalence of EGFR mutations in patients with advanced NSCLC in a large comprehensive cancer center in Korea. Methods Our retrospective cohort included 1,503 NSCLC patients aged ≥18 years, with stage IIIB/IV disease, who attended the Samsung Medical Center in Seoul, Korea, from January 2007 through July 2010. Trained oncology nurses reviewed and abstracted data from electronic medical records. Results This cohort had a mean age (SD) of 59.6 (11.1) years, 62.7% were males, and 52.9% never-smokers. The most common NSCLC histological types were adenocarcinoma (70.5%) and squamous cell carcinoma (18.0%). Overall, 39.5% of patients were tested for EGFR mutations. The proportion of patients undergoing EGFR testing during January 2007 through July 2008, August 2008 through September 2009, and October 2009 through July 2010 were 23.3%, 38.3%, and 63.5%, respectively (P<0.001). The median time elapsed between cancer diagnoses and receiving EGFR testing results was 21 days. EGFR testing was most frequently ordered by oncologists (57.7%), pulmonologists (31.9%), and thoracic surgeons (6.6%). EGFR testing was more commonly requested for women, younger patients, stage IV disease, non-smokers, and adenocarcinoma histology. Of 586 cases successfully tested for EGFR mutations, 209 (35.7%) were positive, including 118 cases with exon 19 deletions and 62 with L858R mutations. EGFR mutation positive patients were more likely to be female, never-smokers, never-drinkers and to have adenocarcinoma. Conclusions In a large cancer center in Korea, the proportion of EGFR testing increased from 2007 through 2010. The high frequency of EGFR mutation positive cases warrants the need for generalized testing in Asian NSCLC patients. PMID:23468851

EGFR mutation testing in patients with advanced non-small cell lung cancer: a comprehensive evaluation of real-world practice in an East Asian tertiary hospital.

PubMed

Choi, Yoon-La; Sun, Jong-Mu; Cho, Juhee; Rampal, Sanjay; Han, Joungho; Parasuraman, Bhash; Guallar, Eliseo; Lee, Genehee; Lee, Jeeyun; Shim, Young Mog

2013-01-01

Guidelines for management of non-small cell lung cancer (NSCLC) strongly recommend EGFR mutation testing. These recommendations are particularly relevant in Asians that have higher EGFR mutation prevalence. This study aims to explore current testing practices, logistics of testing, types of EGFR mutation, and prevalence of EGFR mutations in patients with advanced NSCLC in a large comprehensive cancer center in Korea. Our retrospective cohort included 1,503 NSCLC patients aged ≥18 years, with stage IIIB/IV disease, who attended the Samsung Medical Center in Seoul, Korea, from January 2007 through July 2010. Trained oncology nurses reviewed and abstracted data from electronic medical records. This cohort had a mean age (SD) of 59.6 (11.1) years, 62.7% were males, and 52.9% never-smokers. The most common NSCLC histological types were adenocarcinoma (70.5%) and squamous cell carcinoma (18.0%). Overall, 39.5% of patients were tested for EGFR mutations. The proportion of patients undergoing EGFR testing during January 2007 through July 2008, August 2008 through September 2009, and October 2009 through July 2010 were 23.3%, 38.3%, and 63.5%, respectively (P<0.001). The median time elapsed between cancer diagnoses and receiving EGFR testing results was 21 days. EGFR testing was most frequently ordered by oncologists (57.7%), pulmonologists (31.9%), and thoracic surgeons (6.6%). EGFR testing was more commonly requested for women, younger patients, stage IV disease, non-smokers, and adenocarcinoma histology. Of 586 cases successfully tested for EGFR mutations, 209 (35.7%) were positive, including 118 cases with exon 19 deletions and 62 with L858R mutations. EGFR mutation positive patients were more likely to be female, never-smokers, never-drinkers and to have adenocarcinoma. In a large cancer center in Korea, the proportion of EGFR testing increased from 2007 through 2010. The high frequency of EGFR mutation positive cases warrants the need for generalized testing in Asian NSCLC patients.

Is RNASEL:p.Glu265* a modifier of early-onset breast cancer risk for carriers of high-risk mutations?

PubMed

Nguyen-Dumont, Tú; Teo, Zhi L; Hammet, Fleur; Roberge, Alexis; Mahmoodi, Maryam; Tsimiklis, Helen; Park, Daniel J; Pope, Bernard J; Lonie, Andrew; Kapuscinski, Miroslav K; Mahmood, Khalid; Goldgar, David E; Giles, Graham G; Winship, Ingrid; Hopper, John L; Southey, Melissa C

2018-02-08

Breast cancer risk for BRCA1 and BRCA2 pathogenic mutation carriers is modified by risk factors that cluster in families, including genetic modifiers of risk. We considered genetic modifiers of risk for carriers of high-risk mutations in other breast cancer susceptibility genes. In a family known to carry the high-risk mutation PALB2:c.3113G>A (p.Trp1038*), whole-exome sequencing was performed on germline DNA from four affected women, three of whom were mutation carriers. RNASEL:p.Glu265* was identified in one of the PALB2 carriers who had two primary invasive breast cancer diagnoses before 50 years. Gene-panel testing of BRCA1, BRCA2, PALB2 and RNASEL in the Australian Breast Cancer Family Registry identified five carriers of RNASEL:p.Glu265* in 591 early onset breast cancer cases. Three of the five women (60%) carrying RNASEL:p.Glu265* also carried a pathogenic mutation in a breast cancer susceptibility gene compared with 30 carriers of pathogenic mutations in the 586 non-carriers of RNASEL:p.Glu265* (5%) (p < 0.002). Taqman genotyping demonstrated that the allele frequency of RNASEL:p.Glu265* was similar in affected and unaffected Australian women, consistent with other populations. Our study suggests that RNASEL:p.Glu265* may be a genetic modifier of risk for early-onset breast cancer predisposition in carriers of high-risk mutations. Much larger case-case and case-control studies are warranted to test the association observed in this report.

Competitive amplification of differentially melting amplicons (CADMA) enables sensitive and direct detection of all mutation types by high-resolution melting analysis.

PubMed

Kristensen, Lasse S; Andersen, Gitte B; Hager, Henrik; Hansen, Lise Lotte

2012-01-01

Sensitive and specific mutation detection is of particular importance in cancer diagnostics, prognostics, and individualized patient treatment. However, the majority of molecular methodologies that have been developed with the aim of increasing the sensitivity of mutation testing have drawbacks in terms of specificity, convenience, or costs. Here, we have established a new method, Competitive Amplification of Differentially Melting Amplicons (CADMA), which allows very sensitive and specific detection of all mutation types. The principle of the method is to amplify wild-type and mutated sequences simultaneously using a three-primer system. A mutation-specific primer is designed to introduce melting temperature decreasing mutations in the resulting mutated amplicon, while a second overlapping primer is designed to amplify both wild-type and mutated sequences. When combined with a third common primer very sensitive mutation detection becomes possible, when using high-resolution melting (HRM) as detection platform. The introduction of melting temperature decreasing mutations in the mutated amplicon also allows for further mutation enrichment by fast coamplification at lower denaturation temperature PCR (COLD-PCR). For proof-of-concept, we have designed CADMA assays for clinically relevant BRAF, EGFR, KRAS, and PIK3CA mutations, which are sensitive to, between 0.025% and 0.25%, mutated alleles in a wild-type background. In conclusion, CADMA enables highly sensitive and specific mutation detection by HRM analysis. © 2011 Wiley Periodicals, Inc.

Efficient Detection of Copy Number Mutations in PMS2 Exons with a Close Homolog.

PubMed

Herman, Daniel S; Smith, Christina; Liu, Chang; Vaughn, Cecily P; Palaniappan, Selvi; Pritchard, Colin C; Shirts, Brian H

2018-07-01

Detection of 3' PMS2 copy-number mutations that cause Lynch syndrome is difficult because of highly homologous pseudogenes. To improve the accuracy and efficiency of clinical screening for these mutations, we developed a new method to analyze standard capture-based, next-generation sequencing data to identify deletions and duplications in PMS2 exons 9 to 15. The approach captures sequences using PMS2 targets, maps sequences randomly among regions with equal mapping quality, counts reads aligned to homologous exons and introns, and flags read count ratios outside of empirically derived reference ranges. The method was trained on 1352 samples, including 8 known positives, and tested on 719 samples, including 17 known positives. Clinical implementation of the first version of this method detected new mutations in the training (N = 7) and test (N = 2) sets that had not been identified by our initial clinical testing pipeline. The described final method showed complete sensitivity in both sample sets and false-positive rates of 5% (training) and 7% (test), dramatically decreasing the number of cases needing additional mutation evaluation. This approach leveraged the differences between gene and pseudogene to distinguish between PMS2 and PMS2CL copy-number mutations. These methods enable efficient and sensitive Lynch syndrome screening for 3' PMS2 copy-number mutations and may be applied similarly to other genomic regions with highly homologous pseudogenes. Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Comparison of plasma ctDNA and tissue/cytology-based techniques for the detection of EGFR mutation status in advanced NSCLC: Spanish data subset from ASSESS.

PubMed

Arriola, E; Paredes-Lario, A; García-Gomez, R; Diz-Tain, P; Constenla, M; García-Girón, C; Márquez, G; Reck, M; López-Vivanco, G

2018-04-05

The analysis of epidermal growth factor receptor (EGFR) mutations in many patients with advanced non-small-cell lung cancer (aNSCLC) has provided the opportunity for successful treatment with specific, targeted EGFR tyrosine kinase inhibitors. However, this therapeutic decision may be challenging when insufficient tumor tissue is available for EGFR mutation testing. Therefore, blood surrogate samples for EGFR mutation analysis have been suggested. Data were collected from the Spanish cohort of patients in the large, non-interventional, diagnostic ASSESS study (NCT01785888) evaluating the utility of circulating free tumor-derived DNA from plasma for EGFR mutation testing. The incidence of EGFR mutation in Spain and the level of concordance between matched tissue/cytology and plasma samples were evaluated. In a cohort of 154 eligible patients, EGFR mutations were identified in 15.1 and 11.0% of tumor and plasma samples, respectively. The most commonly used EGFR mutation testing method for the tumor tissue samples was the QIAGEN Therascreen ® EGFR RGQ PCR kit (52.1%). Fragment Length Analysis + PNA LNA Clamp was used for the plasma samples. The concordance rate for EGFR mutation status between the tissue/cytology and plasma samples was 88.8%; the sensitivity was 45.5%, and the specificity was 96.7%. The high concordance between the different DNA sources for EGFR mutation testing supports the use of plasma samples when tumor tissue is unavailable.

Using high-sensitivity sequencing for the detection of mutations in BTK and PLCγ2 genes in cellular and cell-free DNA and correlation with progression in patients treated with BTK inhibitors.

PubMed

Albitar, Adam; Ma, Wanlong; DeDios, Ivan; Estella, Jeffrey; Ahn, Inhye; Farooqui, Mohammed; Wiestner, Adrian; Albitar, Maher

2017-03-14

Patients with chronic lymphocytic leukemia (CLL) that develop resistance to Bruton tyrosine kinase (BTK) inhibitors are typically positive for mutations in BTK or phospholipase c gamma 2 (PLCγ2). We developed a high sensitivity (HS) assay utilizing wild-type blocking polymerase chain reaction achieved via bridged and locked nucleic acids. We used this high sensitivity assay in combination with Sanger sequencing and next generation sequencing (NGS) and tested cellular DNA and cell-free DNA (cfDNA) from patients with CLL treated with the BTK inhibitor, ibrutinib. We also tested ibrutinib-naïve patients with CLL. HS testing achieved 100x greater sensitivity than Sanger. HS Sanger sequencing was capable of detecting < 1 mutant allele in background of 1000 wild-type alleles (1:1000). Similar sensitivity was achieved with HS NGS. No BTK or PLCγ2 mutations were detected in any of the 44 ibrutinib-naïve CLL patients. We demonstrate that without the HS testing 56% of positive samples would have been missed for BTK and 85% of PLCγ2 would have been missed. With the use of HS, we were able to detect multiple mutant clones in the same sample in 37.5% of patients; most would have been missed without HS testing. We also demonstrate that with HS sequencing, plasma cfDNA is more reliable than cellular DNA in detecting mutations. Our studies indicate that wild-type blocking and HS sequencing is necessary for proper and early detection of BTK or PLCγ2 mutations in monitoring patients treated with BTK inhibitors. Furthermore, cfDNA from plasma is very reliable sample-type for testing.

Detection of high-risk thrombophilia with an automated, global test: the Coagulation Inhibitor Potential assay.

PubMed

Réger, Barbara; Losonczy, Hajna; Nagy, Ágnes; Péterfalvi, Ágnes; Mózes, Réka; Pótó, László; Farkas, Nelli; Kovács, Gábor L; Miseta, Attila; Hussain, Alizadeh; Tóth, Orsolya

2018-05-17

: The diagnosis of thrombophilia is a cost-consuming and time-consuming process, as each defect should be separately investigated. The Coagulation Inhibitor Potential (CIP) assay is a promising new global test, sensitive for most of the hereditary thrombophilias, developed for manual methodology. We adapt the original method to an optical coagulation analyser. By this automation, the test will be easier, faster and more precise, and it also allows carrying out 18 measurements simultaneously. The CIP assay was performed in 126 healthy subjects and 193 patients with different types of hereditary thrombophilia conditions. Detected with conventional laboratory tests high-risk thrombophilia was present in 70 patients: deficiencies of antithrombin (AT) (n = 12), protein C (PC) (n = 14), protein S (PS) (n = 6), homozygous factor V Leiden (FVL) mutation (n = 9) and combined types (n = 29). Low-risk thrombophilia was present in 123 patients: heterozygous FVL (n = 115) and FII G2010A mutation (n = 8). Significantly lower median CIP values were found for AT-,PC-, PS deficiencies, homozygous and heterozygous FVL mutations and combined thrombophilias (P < 0.01) as compared with healthy controls. There was no significant difference between the heterozygous FIIG20210A (P = 0.669) thrombophilia group and the healthy controls. The best performance of the test was achieved at the cut-off value of 90.0 U (area: 0.981) with 96% sensitivity and 92% specificity in the high-risk thrombophilia group estimated by receiver operating characteristic analysis. The new method seems to be appropriate and reliable for the detection of AT-, PC- and PS deficiencies, homozygous FVL mutation and also for combined deficiencies. The automated CIP test is insensitive to FII G2010A mutation.

The prevalence of BRCA1 and BRCA2 mutations among young Mexican women with triple-negative breast cancer

PubMed Central

Villarreal-Garza, C.; Weitzel, J. N.; Llacuachaqui, M.; Sifuentes, E.; Magallanes-Hoyos, M. C.; Gallardo, L.; Alvarez-Gómez, R. M.; Herzog, J.; Castillo, D.; Royer, R.; Akbari, Mohammad; Lara-Medina, F.; Herrera, L. A.; Mohar, A.

2015-01-01

Various guidelines recommend that women with triple-negative breast cancer should be tested for BRCA1 mutations, but the prevalence of mutations may vary with ethnic group and with geographic region, and the optimal cutoff age for testing has not been established. We estimated the frequencies of BRCA1 and BRCA2 (BRCA) mutations among 190 women with triple-negative breast cancer, unselected for family history, diagnosed at age 50 or less at a single hospital in Mexico City. Patients were screened for 115 recurrent BRCA mutations, which have been reported previously in women of Hispanic origin, including a common large rearrangement Mexican founder mutation (BRCA1 ex9-12del). A BRCA mutation was detected in 44 of 190 patients with triple-negative breast cancer (23 %). Forty-three mutations were found in BRCA1 and one mutation was found in BRCA2. Seven different mutations accounted for 39 patients (89 % of the total mutations). The Mexican founder mutation (BRCA1 ex9-12del) was found 18 times and accounted for 41 % of all mutations detected. There is a high prevalence of BRCA1 mutations among young triple-negative breast cancer patients in Mexico. Women with triple-negative breast cancer in Mexico should be screened for mutations in BRCA1. PMID:25716084

Cancer Susceptibility Gene Mutations in Individuals With Colorectal Cancer

PubMed Central

Yurgelun, Matthew B.; Kulke, Matthew H.; Fuchs, Charles S.; Allen, Brian A.; Uno, Hajime; Hornick, Jason L.; Ukaegbu, Chinedu I.; Brais, Lauren K.; McNamara, Philip G.; Mayer, Robert J.; Schrag, Deborah; Meyerhardt, Jeffrey A.; Ng, Kimmie; Kidd, John; Singh, Nanda; Hartman, Anne-Renee; Wenstrup, Richard J.

2017-01-01

Purpose Hereditary factors play an important role in colorectal cancer (CRC) risk, yet the prevalence of germline cancer susceptibility gene mutations in patients with CRC unselected for high-risk features (eg, early age at diagnosis, personal/family history of cancer or polyps, tumor microsatellite instability [MSI], mismatch repair [MMR] deficiency) is unknown. Patients and Methods We recruited 1,058 participants who received CRC care in a clinic-based setting without preselection for age at diagnosis, personal/family history, or MSI/MMR results. All participants underwent germline testing for mutations in 25 genes associated with inherited cancer risk. Each gene was categorized as high penetrance or moderate penetrance on the basis of published estimates of the lifetime cancer risks conferred by pathogenic germline mutations in that gene. Results One hundred five (9.9%; 95% CI, 8.2% to 11.9%) of 1,058 participants carried one or more pathogenic mutations, including 33 (3.1%) with Lynch syndrome (LS). Twenty-eight (96.6%) of 29 available LS CRCs demonstrated abnormal MSI/MMR results. Seventy-four (7.0%) of 1,058 participants carried non-LS gene mutations, including 23 (2.2%) with mutations in high-penetrance genes (five APC, three biallelic MUTYH, 11 BRCA1/2, two PALB2, one CDKN2A, and one TP53), 15 of whom lacked clinical histories suggestive of their underlying mutation. Thirty-eight (3.6%) participants had moderate-penetrance CRC risk gene mutations (19 monoallelic MUTYH, 17 APC*I1307K, two CHEK2). Neither proband age at CRC diagnosis, family history of CRC, nor personal history of other cancers significantly predicted the presence of pathogenic mutations in non-LS genes. Conclusion Germline cancer susceptibility gene mutations are carried by 9.9% of patients with CRC. MSI/MMR testing reliably identifies LS probands, although 7.0% of patients with CRC carry non-LS mutations, including 1.0% with BRCA1/2 mutations. PMID:28135145

Germline MLH1 Mutations Are Frequently Identified in Lynch Syndrome Patients With Colorectal and Endometrial Carcinoma Demonstrating Isolated Loss of PMS2 Immunohistochemical Expression.

PubMed

Dudley, Beth; Brand, Randall E; Thull, Darcy; Bahary, Nathan; Nikiforova, Marina N; Pai, Reetesh K

2015-08-01

Current guidelines on germline mutation testing for patients suspected of having Lynch syndrome are not entirely clear in patients with tumors demonstrating isolated loss of PMS2 immunohistochemical expression. We analyzed the clinical and pathologic features of patients with tumors demonstrating isolated loss of PMS2 expression in an attempt to (1) determine the frequency of germline MLH1 and PMS2 mutations and (2) correlate mismatch-repair protein immunohistochemistry and tumor histology with germline mutation results. A total of 3213 consecutive colorectal carcinomas and 215 consecutive endometrial carcinomas were prospectively analyzed for DNA mismatch-repair protein expression by immunohistochemistry. In total, 32 tumors from 31 patients demonstrated isolated loss of PMS2 immunohistochemical expression, including 16 colorectal carcinomas and 16 endometrial carcinomas. Microsatellite instability (MSI) polymerase chain reaction was performed in 29 tumors from 28 patients with the following results: 28 tumors demonstrated high-level MSI, and 1 tumor demonstrated low-level MSI. Twenty of 31 (65%) patients in the study group had tumors demonstrating histopathology associated with high-level MSI. Seventeen patients underwent germline mutation analysis with the following results: 24% with MLH1 mutations, 35% with PMS2 mutations, 12% with PMS2 variants of undetermined significance, and 29% with no mutations in either MLH1 or PMS2. Three of the 4 patients with MLH1 germline mutations had a mutation that results in decreased stability and quantity of the MLH1 protein that compromises the MLH1-PMS2 protein complex, helping to explain the presence of immunogenic but functionally inactive MLH1 protein within the tumor. The high frequency of MLH1 germline mutations identified in our study has important implications for testing strategies in patients suspected of having Lynch syndrome and indicates that patients with tumors demonstrating isolated loss of PMS2 expression without a germline PMS2 mutation must have MLH1 mutation analysis performed.

Molecular Testing of Brain Tumor

PubMed Central

Park, Sung-Hye; Won, Jaekyung; Kim, Seong-Ik; Lee, Yujin; Park, Chul-Kee; Kim, Seung-Ki; Choi, Seung-Hong

2017-01-01

The World Health Organization (WHO) classification of central nervous system (CNS) tumors was revised in 2016 with a basis on the integrated diagnosis of molecular genetics. We herein provide the guidelines for using molecular genetic tests in routine pathological practice for an accurate diagnosis and appropriate management. While astrocytomas and IDH-mutant (secondary) glioblastomas are characterized by the mutational status of IDH, TP53, and ATRX, oligodendrogliomas have a 1p/19q codeletion and mutations in IDH, CIC, FUBP1, and the promoter region of telomerase reverse transcriptase (TERTp). IDH-wildtype (primary) glioblastomas typically lack mutations in IDH, but are characterized by copy number variations of EGFR, PTEN, CDKN2A/B, PDGFRA, and NF1 as well as mutations of TERTp. High-grade pediatric gliomas differ from those of adult gliomas, consisting of mutations in H3F3A, ATRX, and DAXX, but not in IDH genes. In contrast, well-circumscribed low-grade neuroepithelial tumors in children, such as pilocytic astrocytoma, pleomorphic xanthoastrocytoma, and ganglioglioma, often have mutations or activating rearrangements in the BRAF, FGFR1, and MYB genes. Other CNS tumors, such as ependymomas, neuronal and glioneuronal tumors, embryonal tumors, meningothelial, and other mesenchymal tumors have important genetic alterations, many of which are diagnostic, prognostic, and predictive markers and therapeutic targets. Therefore, the neuropathological evaluation of brain tumors is increasingly dependent on molecular genetic tests for proper classification, prediction of biological behavior and patient management. Identifying these gene abnormalities requires cost-effective and high-throughput testing, such as next-generation sequencing. Overall, this paper reviews the global guidelines and diagnostic algorithms for molecular genetic testing of brain tumors. PMID:28535583

The impact of p53 protein core domain structural alteration on ovarian cancer survival.

PubMed

Rose, Stephen L; Robertson, Andrew D; Goodheart, Michael J; Smith, Brian J; DeYoung, Barry R; Buller, Richard E

2003-09-15

Although survival with a p53 missense mutation is highly variable, p53-null mutation is an independent adverse prognostic factor for advanced stage ovarian cancer. By evaluating ovarian cancer survival based upon a structure function analysis of the p53 protein, we tested the hypothesis that not all missense mutations are equivalent. The p53 gene was sequenced from 267 consecutive ovarian cancers. The effect of individual missense mutations on p53 structure was analyzed using the International Agency for Research on Cancer p53 Mutational Database, which specifies the effects of p53 mutations on p53 core domain structure. Mutations in the p53 core domain were classified as either explained or not explained in structural or functional terms by their predicted effects on protein folding, protein-DNA contacts, or mutation in highly conserved residues. Null mutations were classified by their mechanism of origin. Mutations were sequenced from 125 tumors. Effects of 62 of the 82 missense mutations (76%) could be explained by alterations in the p53 protein. Twenty-three (28%) of the explained mutations occurred in highly conserved regions of the p53 core protein. Twenty-two nonsense point mutations and 21 frameshift null mutations were sequenced. Survival was independent of missense mutation type and mechanism of null mutation. The hypothesis that not all missense mutations are equivalent is, therefore, rejected. Furthermore, p53 core domain structural alteration secondary to missense point mutation is not functionally equivalent to a p53-null mutation. The poor prognosis associated with p53-null mutation is independent of the mutation mechanism.

MutScan: fast detection and visualization of target mutations by scanning FASTQ data.

PubMed

Chen, Shifu; Huang, Tanxiao; Wen, Tiexiang; Li, Hong; Xu, Mingyan; Gu, Jia

2018-01-22

Some types of clinical genetic tests, such as cancer testing using circulating tumor DNA (ctDNA), require sensitive detection of known target mutations. However, conventional next-generation sequencing (NGS) data analysis pipelines typically involve different steps of filtering, which may cause miss-detection of key mutations with low frequencies. Variant validation is also indicated for key mutations detected by bioinformatics pipelines. Typically, this process can be executed using alignment visualization tools such as IGV or GenomeBrowse. However, these tools are too heavy and therefore unsuitable for validating mutations in ultra-deep sequencing data. We developed MutScan to address problems of sensitive detection and efficient validation for target mutations. MutScan involves highly optimized string-searching algorithms, which can scan input FASTQ files to grab all reads that support target mutations. The collected supporting reads for each target mutation will be piled up and visualized using web technologies such as HTML and JavaScript. Algorithms such as rolling hash and bloom filter are applied to accelerate scanning and make MutScan applicable to detect or visualize target mutations in a very fast way. MutScan is a tool for the detection and visualization of target mutations by only scanning FASTQ raw data directly. Compared to conventional pipelines, this offers a very high performance, executing about 20 times faster, and offering maximal sensitivity since it can grab mutations with even one single supporting read. MutScan visualizes detected mutations by generating interactive pile-ups using web technologies. These can serve to validate target mutations, thus avoiding false positives. Furthermore, MutScan can visualize all mutation records in a VCF file to HTML pages for cloud-friendly VCF validation. MutScan is an open source tool available at GitHub: https://github.com/OpenGene/MutScan.

Performance of amplicon-based next generation DNA sequencing for diagnostic gene mutation profiling in oncopathology.

PubMed

Sie, Daoud; Snijders, Peter J F; Meijer, Gerrit A; Doeleman, Marije W; van Moorsel, Marinda I H; van Essen, Hendrik F; Eijk, Paul P; Grünberg, Katrien; van Grieken, Nicole C T; Thunnissen, Erik; Verheul, Henk M; Smit, Egbert F; Ylstra, Bauke; Heideman, Daniëlle A M

2014-10-01

Next generation DNA sequencing (NGS) holds promise for diagnostic applications, yet implementation in routine molecular pathology practice requires performance evaluation on DNA derived from routine formalin-fixed paraffin-embedded (FFPE) tissue specimens. The current study presents a comprehensive analysis of TruSeq Amplicon Cancer Panel-based NGS using a MiSeq Personal sequencer (TSACP-MiSeq-NGS) for somatic mutation profiling. TSACP-MiSeq-NGS (testing 212 hotspot mutation amplicons of 48 genes) and a data analysis pipeline were evaluated in a retrospective learning/test set approach (n = 58/n = 45 FFPE-tumor DNA samples) against 'gold standard' high-resolution-melting (HRM)-sequencing for the genes KRAS, EGFR, BRAF and PIK3CA. Next, the performance of the validated test algorithm was assessed in an independent, prospective cohort of FFPE-tumor DNA samples (n = 75). In the learning set, a number of minimum parameter settings was defined to decide whether a FFPE-DNA sample is qualified for TSACP-MiSeq-NGS and for calling mutations. The resulting test algorithm revealed 82% (37/45) compliance to the quality criteria and 95% (35/37) concordant assay findings for KRAS, EGFR, BRAF and PIK3CA with HRM-sequencing (kappa = 0.92; 95% CI = 0.81-1.03) in the test set. Subsequent application of the validated test algorithm to the prospective cohort yielded a success rate of 84% (63/75), and a high concordance with HRM-sequencing (95% (60/63); kappa = 0.92; 95% CI = 0.84-1.01). TSACP-MiSeq-NGS detected 77 mutations in 29 additional genes. TSACP-MiSeq-NGS is suitable for diagnostic gene mutation profiling in oncopathology.

Efficient IDUA Gene Mutation Detection with Combined Use of dHPLC and Dried Blood Samples

PubMed Central

Duarte, Ana Joana; Vieira, Luis

2013-01-01

Objectives. Development of a simple mutation directed method in order to allow lowering the cost of mutation testing using an easily obtainable biological material. Assessment of the feasibility of such method was tested using a GC-rich amplicon. Design and Methods. A method of denaturing high-performance liquid chromatography (dHPLC) was improved and implemented as a technique for the detection of variants in exon 9 of the IDUA gene. The optimized method was tested in 500 genomic DNA samples obtained from dried blood spots (DBS). Results. With this dHPLC approach it was possible to detect different variants, including the common p.Trp402Ter mutation in the IDUA gene. The high GC content did not interfere with the resolution and reliability of this technique, and discrimination of G-C transversions was also achieved. Conclusion. This PCR-based dHPLC method is proved to be a rapid, a sensitive, and an excellent option for screening numerous samples obtained from DBS. Furthermore, it resulted in the consistent detection of clearly distinguishable profiles of the common p.Trp402Ter IDUA mutation with an advantageous balance of cost and technical requirements. PMID:27335677

Mutation Testing for Effective Verification of Digital Components of Physical Systems

NASA Astrophysics Data System (ADS)

Kushik, N. G.; Evtushenko, N. V.; Torgaev, S. N.

2015-12-01

Digital components of modern physical systems are often designed applying circuitry solutions based on the field programmable gate array technology (FPGA). Such (embedded) digital components should be carefully tested. In this paper, an approach for the verification of digital physical system components based on mutation testing is proposed. The reference description of the behavior of a digital component in the hardware description language (HDL) is mutated by introducing into it the most probable errors and, unlike mutants in high-level programming languages, the corresponding test case is effectively derived based on a comparison of special scalable representations of the specification and the constructed mutant using various logic synthesis and verification systems.

A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens

PubMed Central

Besaratinia, Ahmad; Li, Haiqing; Yoon, Jae-In; Zheng, Albert; Gao, Hanlin; Tommasi, Stella

2012-01-01

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue® mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents. PMID:22735701

A high-throughput next-generation sequencing-based method for detecting the mutational fingerprint of carcinogens.

PubMed

Besaratinia, Ahmad; Li, Haiqing; Yoon, Jae-In; Zheng, Albert; Gao, Hanlin; Tommasi, Stella

2012-08-01

Many carcinogens leave a unique mutational fingerprint in the human genome. These mutational fingerprints manifest as specific types of mutations often clustering at certain genomic loci in tumor genomes from carcinogen-exposed individuals. To develop a high-throughput method for detecting the mutational fingerprint of carcinogens, we have devised a cost-, time- and labor-effective strategy, in which the widely used transgenic Big Blue mouse mutation detection assay is made compatible with the Roche/454 Genome Sequencer FLX Titanium next-generation sequencing technology. As proof of principle, we have used this novel method to establish the mutational fingerprints of three prominent carcinogens with varying mutagenic potencies, including sunlight ultraviolet radiation, 4-aminobiphenyl and secondhand smoke that are known to be strong, moderate and weak mutagens, respectively. For verification purposes, we have compared the mutational fingerprints of these carcinogens obtained by our newly developed method with those obtained by parallel analyses using the conventional low-throughput approach, that is, standard mutation detection assay followed by direct DNA sequencing using a capillary DNA sequencer. We demonstrate that this high-throughput next-generation sequencing-based method is highly specific and sensitive to detect the mutational fingerprints of the tested carcinogens. The method is reproducible, and its accuracy is comparable with that of the currently available low-throughput method. In conclusion, this novel method has the potential to move the field of carcinogenesis forward by allowing high-throughput analysis of mutations induced by endogenous and/or exogenous genotoxic agents.

Comparison of plasma and tissue samples in epidermal growth factor receptor mutation by ARMS in advanced non-small cell lung cancer.

PubMed

Ma, MeiLi; Shi, ChunLei; Qian, JiaLin; Teng, JiaJun; Zhong, Hua; Han, BaoHui

2016-10-10

The aim of this study was to assess the effectiveness and accuracy of blood-based circulating-free tumor DNA on testing epidermal growth factor receptor (EGFR) gene mutations. In total, 219 non-small cell lung cancer patients in stages III-IV were enrolled into this study. All patients had tissue samples and matched plasma DNA samples. EGFR gene mutations were detected by the Amplification Refractory Mutation System (ARMS). We compared the mutations in tumor tissue samples with matched plasma samples and determined the correlation between EGFR mutation status and clinical pathologic characteristics. The overall concordance rate of EGFR mutation status between the 219 matched plasma and tissue samples was 82% (179/219). The sensitivity and specificity for the ARMS EGFR mutation test in the plasma compared with tumor tissue were 60% (54/90) and 97% (125/129), respectively. The positive predictive value was 93% (54/58) and the negative predictive value was 78% (125/161). The median overall survival was longer for those with EGFR mutations than for those without EGFR mutations both in tissue samples (23.98 vs. 12.16months; P<0.001) and in plasma (19.96 vs. 13.63months; P=0.009). For the 68 patients treated with EGFR- tyrosine kinase inhibitors (TKIs), the median progression-free survival (PFS) was significantly prolonged in the EGFR mutant group compared to the non-mutation group in tumor tissue samples (12.26months vs. 2.40months, P<0.001). In plasma samples, the PFS of the mutant group was longer than that of the non-mutant group. However, there was no significant difference between the two groups (10.88months vs. 9.89months, P=0.411). The detection of EGFR mutations in plasma using ARMS is relatively sensitive and highly specific. However, EGFR mutation status tested by ARMS in plasma cannot replace a tumor tissue biopsy. Positive EGFR mutation results detected in plasma are fairly reliable, but negative results are hampered by a high rate of false negatives. Copyright © 2016. Published by Elsevier B.V.

Frequency of CDH1 germline mutations in gastric carcinoma coming from high- and low-risk areas: metanalysis and systematic review of the literature

PubMed Central

2012-01-01

Background The frequency of E-cadherin germline mutations in countries with different incidence rates for gastric carcinoma has not been well established. The goal of this study was to assess the worldwide frequency of CDH1 germline mutations in gastric cancers coming from low- and high-risk areas. Methods English articles using MEDLINE access (from 1998 to 2011). Search terms included CDH1, E-cadherin, germline mutation, gastric cancer, hereditary, familial and diffuse histotype. The study included all E-cadherin germline mutations identified in gastric cancer patients; somatic mutations and germline mutations reported in other tumors were excluded. The method of this study was scheduled in accordance with the "PRISMA statement for reporting systematic reviews and meta-analyses". Countries were classified as low- or middle/high risk-areas for gastric carcinoma incidence. Statistical analysis was performed to correlate the CDH1 mutation frequency with gastric cancer incidence areas. Results A total of 122 E-cadherin germline mutations have been identified; the majority (87.5%) occurred in gastric cancers coming from low-risk areas. In high-risk areas, we identified 16 mutations in which missense mutations were predominant. (68.8%). We verified a significant association between the mutation frequency and the gastric cancer risk area (p < 0.001: overall identified mutations in low- vs. middle/high-risk areas). Conclusions E-cadherin genetic screenings performed in low-risk areas for gastric cancer identified a higher frequency of CDH1 germline mutations. This data could open new approaches in the gastric cancer prevention test; before proposing a proband candidate for the CDH1 genetic screening, geographic variability, alongside the family history should be considered. PMID:22225527

Mutational spectrum in a worldwide study of 29,700 families with BRCA1 or BRCA2 mutations.

PubMed

Rebbeck, Timothy R; Friebel, Tara M; Friedman, Eitan; Hamann, Ute; Huo, Dezheng; Kwong, Ava; Olah, Edith; Olopade, Olufunmilayo I; Solano, Angela R; Teo, Soo-Hwang; Thomassen, Mads; Weitzel, Jeffrey N; Chan, T L; Couch, Fergus J; Goldgar, David E; Kruse, Torben A; Palmero, Edenir Inêz; Park, Sue Kyung; Torres, Diana; van Rensburg, Elizabeth J; McGuffog, Lesley; Parsons, Michael T; Leslie, Goska; Aalfs, Cora M; Abugattas, Julio; Adlard, Julian; Agata, Simona; Aittomäki, Kristiina; Andrews, Lesley; Andrulis, Irene L; Arason, Adalgeir; Arnold, Norbert; Arun, Banu K; Asseryanis, Ella; Auerbach, Leo; Azzollini, Jacopo; Balmaña, Judith; Barile, Monica; Barkardottir, Rosa B; Barrowdale, Daniel; Benitez, Javier; Berger, Andreas; Berger, Raanan; Blanco, Amie M; Blazer, Kathleen R; Blok, Marinus J; Bonadona, Valérie; Bonanni, Bernardo; Bradbury, Angela R; Brewer, Carole; Buecher, Bruno; Buys, Saundra S; Caldes, Trinidad; Caliebe, Almuth; Caligo, Maria A; Campbell, Ian; Caputo, Sandrine M; Chiquette, Jocelyne; Chung, Wendy K; Claes, Kathleen B M; Collée, J Margriet; Cook, Jackie; Davidson, Rosemarie; de la Hoya, Miguel; De Leeneer, Kim; de Pauw, Antoine; Delnatte, Capucine; Diez, Orland; Ding, Yuan Chun; Ditsch, Nina; Domchek, Susan M; Dorfling, Cecilia M; Velazquez, Carolina; Dworniczak, Bernd; Eason, Jacqueline; Easton, Douglas F; Eeles, Ros; Ehrencrona, Hans; Ejlertsen, Bent; Engel, Christoph; Engert, Stefanie; Evans, D Gareth; Faivre, Laurence; Feliubadaló, Lidia; Ferrer, Sandra Fert; Foretova, Lenka; Fowler, Jeffrey; Frost, Debra; Galvão, Henrique C R; Ganz, Patricia A; Garber, Judy; Gauthier-Villars, Marion; Gehrig, Andrea; Gerdes, Anne-Marie; Gesta, Paul; Giannini, Giuseppe; Giraud, Sophie; Glendon, Gord; Godwin, Andrew K; Greene, Mark H; Gronwald, Jacek; Gutierrez-Barrera, Angelica; Hahnen, Eric; Hauke, Jan; Henderson, Alex; Hentschel, Julia; Hogervorst, Frans B L; Honisch, Ellen; Imyanitov, Evgeny N; Isaacs, Claudine; Izatt, Louise; Izquierdo, Angel; Jakubowska, Anna; James, Paul; Janavicius, Ramunas; Jensen, Uffe Birk; John, Esther M; Vijai, Joseph; Kaczmarek, Katarzyna; Karlan, Beth Y; Kast, Karin; Investigators, KConFab; Kim, Sung-Won; Konstantopoulou, Irene; Korach, Jacob; Laitman, Yael; Lasa, Adriana; Lasset, Christine; Lázaro, Conxi; Lee, Annette; Lee, Min Hyuk; Lester, Jenny; Lesueur, Fabienne; Liljegren, Annelie; Lindor, Noralane M; Longy, Michel; Loud, Jennifer T; Lu, Karen H; Lubinski, Jan; Machackova, Eva; Manoukian, Siranoush; Mari, Véronique; Martínez-Bouzas, Cristina; Matrai, Zoltan; Mebirouk, Noura; Meijers-Heijboer, Hanne E J; Meindl, Alfons; Mensenkamp, Arjen R; Mickys, Ugnius; Miller, Austin; Montagna, Marco; Moysich, Kirsten B; Mulligan, Anna Marie; Musinsky, Jacob; Neuhausen, Susan L; Nevanlinna, Heli; Ngeow, Joanne; Nguyen, Huu Phuc; Niederacher, Dieter; Nielsen, Henriette Roed; Nielsen, Finn Cilius; Nussbaum, Robert L; Offit, Kenneth; Öfverholm, Anna; Ong, Kai-Ren; Osorio, Ana; Papi, Laura; Papp, Janos; Pasini, Barbara; Pedersen, Inge Sokilde; Peixoto, Ana; Peruga, Nina; Peterlongo, Paolo; Pohl, Esther; Pradhan, Nisha; Prajzendanc, Karolina; Prieur, Fabienne; Pujol, Pascal; Radice, Paolo; Ramus, Susan J; Rantala, Johanna; Rashid, Muhammad Usman; Rhiem, Kerstin; Robson, Mark; Rodriguez, Gustavo C; Rogers, Mark T; Rudaitis, Vilius; Schmidt, Ane Y; Schmutzler, Rita Katharina; Senter, Leigha; Shah, Payal D; Sharma, Priyanka; Side, Lucy E; Simard, Jacques; Singer, Christian F; Skytte, Anne-Bine; Slavin, Thomas P; Snape, Katie; Sobol, Hagay; Southey, Melissa; Steele, Linda; Steinemann, Doris; Sukiennicki, Grzegorz; Sutter, Christian; Szabo, Csilla I; Tan, Yen Y; Teixeira, Manuel R; Terry, Mary Beth; Teulé, Alex; Thomas, Abigail; Thull, Darcy L; Tischkowitz, Marc; Tognazzo, Silvia; Toland, Amanda Ewart; Topka, Sabine; Trainer, Alison H; Tung, Nadine; van Asperen, Christi J; van der Hout, Annemieke H; van der Kolk, Lizet E; van der Luijt, Rob B; Van Heetvelde, Mattias; Varesco, Liliana; Varon-Mateeva, Raymonda; Vega, Ana; Villarreal-Garza, Cynthia; von Wachenfeldt, Anna; Walker, Lisa; Wang-Gohrke, Shan; Wappenschmidt, Barbara; Weber, Bernhard H F; Yannoukakos, Drakoulis; Yoon, Sook-Yee; Zanzottera, Cristina; Zidan, Jamal; Zorn, Kristin K; Hutten Selkirk, Christina G; Hulick, Peter J; Chenevix-Trench, Georgia; Spurdle, Amanda B; Antoniou, Antonis C; Nathanson, Katherine L

2018-05-01

The prevalence and spectrum of germline mutations in BRCA1 and BRCA2 have been reported in single populations, with the majority of reports focused on White in Europe and North America. The Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) has assembled data on 18,435 families with BRCA1 mutations and 11,351 families with BRCA2 mutations ascertained from 69 centers in 49 countries on six continents. This study comprehensively describes the characteristics of the 1,650 unique BRCA1 and 1,731 unique BRCA2 deleterious (disease-associated) mutations identified in the CIMBA database. We observed substantial variation in mutation type and frequency by geographical region and race/ethnicity. In addition to known founder mutations, mutations of relatively high frequency were identified in specific racial/ethnic or geographic groups that may reflect founder mutations and which could be used in targeted (panel) first pass genotyping for specific populations. Knowledge of the population-specific mutational spectrum in BRCA1 and BRCA2 could inform efficient strategies for genetic testing and may justify a more broad-based oncogenetic testing in some populations. © 2018 Wiley Periodicals, Inc.

Pathologic Findings at Risk-Reducing Salpingo-Oophorectomy: Primary Results From Gynecologic Oncology Group Trial GOG-0199

PubMed Central

Sherman, Mark E.; Piedmonte, Marion; Mai, Phuong L.; Ioffe, Olga B.; Ronnett, Brigitte M.; Van Le, Linda; Ivanov, Iouri; Bell, Maria C.; Blank, Stephanie V.; DiSilvestro, Paul; Hamilton, Chad A.; Tewari, Krishnansu S.; Wakeley, Katie; Kauff, Noah D.; Yamada, S. Diane; Rodriguez, Gustavo; Skates, Steven J.; Alberts, David S.; Walker, Joan L.; Minasian, Lori; Lu, Karen; Greene, Mark H.

2014-01-01

Purpose Risk-reducing salpingo-oophorectomy (RRSO) lowers mortality from ovarian/tubal and breast cancers among BRCA1/2 mutation carriers. Uncertainties persist regarding potential benefits of RRSO among high-risk noncarriers, optimal surgical age, and anatomic origin of clinically occult cancers detected at surgery. To address these topics, we analyzed surgical treatment arm results from Gynecologic Oncology Group Protocol-0199 (GOG-0199), the National Ovarian Cancer Prevention and Early Detection Study. Participants and Methods This analysis included asymptomatic high-risk women age ≥ 30 years who elected RRSO at enrollment. Women provided risk factor data and underwent preoperative cancer antigen 125 (CA-125) serum testing and transvaginal ultrasound (TVU). RRSO specimens were processed according to a standardized tissue processing protocol and underwent central pathology panel review. Research-based BRCA1/2 mutation testing was performed when a participant's mutation status was unknown at enrollment. Relationships between participant characteristics and diagnostic findings were assessed using univariable statistics and multivariable logistic regression. Results Invasive or intraepithelial ovarian/tubal/peritoneal neoplasms were detected in 25 (2.6%) of 966 RRSOs (BRCA1 mutation carriers, 4.6%; BRCA2 carriers, 3.5%; and noncarriers, 0.5%; P < .001). In multivariable models, positive BRCA1/2 mutation status (P = .0056), postmenopausal status (P = .0023), and abnormal CA-125 levels and/or TVU examinations (P < .001) were associated with detection of clinically occult neoplasms at RRSO. For 387 women with negative BRCA1/2 mutation testing and normal CA-125 levels, findings at RRSO were benign. Conclusion Clinically occult cancer was detected among 2.6% of high-risk women undergoing RRSO. BRCA1/2 mutation, postmenopausal status, and abnormal preoperative CA-125 and/or TVU were associated with cancer detection at RRSO. These data can inform management decisions among women at high risk of ovarian/tubal cancer. PMID:25199754

Cytology smears as diagnostic material for EGFR gene testing in non-small cell lung cancer.

PubMed

Powrózek, Tomasz; Krawczyk, Paweł; Pankowski, Juliusz; Reszka, Katarzyna; Jakubiak, Magdalena; Obrochta, Anna; Wojas-Krawczyk, Kamila; Buczkowski, Jarosław; Milanowski, Janusz

2015-11-14

Cytology smears can be effectively used for EGFR mutation testing in the qualification of NSCLC patients for EGFR tyrosine kinase inhibitor therapy. However, tissue specimens are preferred for EGFR mutation analysis. The aim of this study was to estimate the effectiveness of the real-time PCR method for EGFR testing in histology and cytology materials obtained simultaneously from NSCLC patients. Fourteen adenocarcinoma patients with EGFR-mutation-positive primary tumor tissues were included in the study. Corresponding cytological smears of metastatic lymph nodes obtained by EBUS-TBNA were examined. EGFR Mutation Analysis Kit (EntroGen, USA) and real-time PCR (m2000rt system, Abbott, USA) were used for EGFR mutation analysis in both types of material. In primary tumor tissues, 12 deletions in exon 19 and 2 substitutions in exon 21 (L858R mutation) of the EGFR gene were found. Except for 1 deletion in exon 19, the same EGFR gene mutations were detected in all corresponding cytology samples. The percentage of tumor cells, DNA concentration, percentage of mutated DNA as well as ΔCt values were similar in cytology slides and histology material. In both types of materials, no significant correlations were found between the percentage of tumor cells and the percentage of mutated DNA nor between the DNA concentration and the percentage of mutated DNA. We demonstrated the high effectiveness of a sensitive real-time PCR method in EGFR gene mutation detection in cytology smears.

Metastatic Pheochromocytoma/Paraganglioma Related to Primary Tumor Development in Childhood or Adolescence: Significant Link to SDHB Mutations

PubMed Central

King, Kathryn S.; Prodanov, Tamara; Kantorovich, Vitaly; Fojo, Tito; Hewitt, Jacqueline K.; Zacharin, Margaret; Wesley, Robert; Lodish, Maya; Raygada, Margarita; Gimenez-Roqueplo, Anne-Paule; McCormack, Shana; Eisenhofer, Graeme; Milosevic, Dragana; Kebebew, Electron; Stratakis, Constantine A.; Pacak, Karel

2011-01-01

Purpose To present data on the high rate of SDHB mutations in patients with metastatic pheochromocytoma/paraganglioma whose initial tumor presentation began in childhood or adolescence. Patients and Methods From 2000 to 2010, 263 patients with pheochromocytoma/paraganglioma were evaluated through the National Institutes of Health (NIH), Bethesda, MD. Of the 263 patients, 125 patients were found to have metastatic disease; of these 125 patients, 32 patients presented with a tumor before 20 years of age. An additional 17 patients presented with a tumor before 20 years of age but demonstrated no development of metastatic disease. Genetic testing for mutations in the VHL, MEN, and SDHB/C/D genes was performed on patients without previously identified genetic mutations. Results Of the 32 patients who presented with metastatic disease and had their primary tumor in childhood or adolescence, sequence analysis of germline DNA showed SDHB mutations in 23 patients (71.9%), SDHD mutations in three patients (9.4%), VHL mutations in two patients (6.3%), and an absence of a known mutation in four patients (12.5%). The majority of these 32 patients (78.1%) presented with primary tumors in an extra-adrenal location. Conclusion The majority of patients with metastatic pheochromocytoma/paraganglioma who presented with a primary tumor in childhood/adolescence had primary extra-adrenal tumors and harbored SDHB mutations. Except for primary tumors located in the head and neck where SDHD genetic testing is advised, we recommend that patients who present with metastatic pheochromocytoma/paraganglioma with primary tumor development in childhood or adolescence undergo SDHB genetic testing before they undergo testing for other gene mutations, unless clinical presentation or family history suggests a different mutation. PMID:21969497

Multigene panels in Ashkenazi Jewish patients yield high rates of actionable mutations in multiple non-BRCA cancer-associated genes.

PubMed

Frey, Melissa K; Sandler, Gabriella; Sobolev, Rachel; Kim, Sarah H; Chambers, Rachelle; Bassett, Rebecca Y; Martineau, Jessica; Sapra, Katherine J; Boyd, Leslie; Curtin, John P; Pothuri, Bhavana; Blank, Stephanie V

2017-07-01

To evaluate the results of multigene panel testing among Ashkenazi Jewish compared with non-Ashkenazi Jewish patients. We reviewed the medical records for all patients who underwent multigene panel testing and targeted BRCA1/2 testing at a single institution between 6/2013-1/2015. Clinical actionability for identified pathogenic mutations was characterized based on the National Comprehensive Cancer Network (NCCN) guidelines and consensus statements and expert opinion for genes not addressed by these guidelines. Four hundred and fifty-four patients underwent multigene panel screening, including 138 Ashkenazi Jewish patients. The median patient age was fifty-two years. Three hundred and fifty-four patients (78%) had a personal history of cancer. Two hundred and fifty-one patients had breast cancer, 49, ovarian cancer, 26, uterine cancer and 20, colorectal cancer. We identified 62 mutations in 56 patients and 291 variants of uncertain significance in 196 patients. Among the 56 patients with mutations, 51 (91%) had actionable mutations. Twenty mutations were identified by multigene panels among Ashkenazi Jewish patients, 18 of which were in genes other than BRCA1/2. A review of targeted BRCA1/2 testing performed over the same study period included 103 patients and identified six mutations in BRCA1/2, all of which occurred in Ashkenazi Jewish patients. Among all Ashkenazi Jewish patients undergoing genetic testing, 25/183 (14%) had a mutation, 24/25 of which were actionable (96%) and 17/25 patients (68%) had mutations in non BRCA1/2 genes. With the rapid acceptance of multigene panels there is a pressing need to understand how this testing will affect patient management. While traditionally many Ashkenazi Jewish patients have undergone targeted BRCA1/2 testing, our data suggest consideration of multigene panels in this population as the majority of the results are clinically actionable and often in genes other than BRCA1/2. Copyright © 2017 Elsevier Inc. All rights reserved.

Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor.

PubMed

Dong, Chongmei; Vincent, Kate; Sharp, Peter

2009-12-04

TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM) analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor software, aimed at simultaneous detection of mutations in three homoeologous genes. We demonstrate that High Resolution Melting (HRM) analysis can be used in mutation scans in mixed PCR amplicons containing three homoeologous gene fragments. Combining HRM scanning with sequence analysis using Mutation Surveyor is sensitive enough to detect a single nucleotide mutation in the heterozygous state in a mixed PCR amplicon containing three homoeoloci. The method was tested and validated in an EMS (ethylmethane sulfonate)-treated wheat TILLING population, screening mutations in the carboxyl terminal domain of the Starch Synthase II (SSII) gene. Selected identified mutations of interest can be further analysed by cloning to confirm the mutation and determine the genomic origin of the mutation. Polyploidy is common in plants. Conserved regions of a gene often represent functional domains and have high sequence similarity between homoeologous loci. The method described here is a useful alternative to locus-specific based methods for screening mutations in conserved functional domains of homoeologous genes. This method can also be used for SNP (single nucleotide polymorphism) marker development and eco-TILLING in polyploid species.

Assessment of cellularity, genomic DNA yields, and technical platforms for BRAF mutational testing in thyroid fine-needle aspirate samples.

PubMed

Dyhdalo, Kathryn; Macnamara, Stephen; Brainard, Jennifer; Underwood, Dawn; Tubbs, Raymond; Yang, Bin

2014-02-01

BRAF mutation V600E (substitution Val600Glu) is a molecular signature for papillary thyroid carcinoma (PTC). Testing for BRAF mutation is clinically useful in providing prognostic prediction and facilitating accurate diagnosis of PTC in thyroid fine-needle aspirate (FNA) samples. This study assessed the correlation of cellularity with DNA yield and compared 2 technical platforms with different sensitivities in detection of BRAF mutation in cytologic specimens. Cellularity was evaluated based on groups of 10+ cells on a ThinPrep slide: 1+ (1-5 groups), 2+ (6-10 groups), 3+ (11-20 groups), and 4+ (> 20 groups). Genomic DNA was extracted from residual materials of thyroid FNAs after cytologic diagnosis. Approximately 49% of thyroid FNA samples had low cellularity (1-2+). DNA yield is proportionate with increased cellularity and increased nearly 4-fold from 1+ to 4+ cellularity in cytologic samples. When applied to BRAF mutational assay, using a cutoff of 6 groups of follicular cells with 10+ cells per group, 96.7% of cases yielded enough DNA for at least one testing for BRAF mutation. Five specimens (11.6%) with lower cellularity did not yield sufficient DNA for duplicate testing. Comparison of Sanger sequencing to allele-specific polymerase chain reaction methods shows the latter confers better sensitivity in detection of BRAF mutation, especially in limited cytologic specimens with a lower percentage of malignant cells. This study demonstrates that by using 6 groups of 10+ follicular cells as a cutoff, nearly 97% of thyroid FNA samples contain enough DNA for BRAF mutational assay. Careful selection of a molecular testing system with high sensitivity facilitates the successful conduction of molecular testing in limited cytologic specimens. Cancer (Cancer Cytopathol) 2014;122:114-22 © 2013 American Cancer Society. © 2013 American Cancer Society.

Mutations of the Imprinted CDKN1C Gene as a Cause of the Overgrowth Beckwith-Wiedemann Syndrome: Clinical Spectrum and Functional Characterization.

PubMed

Brioude, Frederic; Netchine, Irène; Praz, Francoise; Le Jule, Marilyne; Calmel, Claire; Lacombe, Didier; Edery, Patrick; Catala, Martin; Odent, Sylvie; Isidor, Bertrand; Lyonnet, Stanislas; Sigaudy, Sabine; Leheup, Bruno; Audebert-Bellanger, Séverine; Burglen, Lydie; Giuliano, Fabienne; Alessandri, Jean-Luc; Cormier-Daire, Valérie; Laffargue, Fanny; Blesson, Sophie; Coupier, Isabelle; Lespinasse, James; Blanchet, Patricia; Boute, Odile; Baumann, Clarisse; Polak, Michel; Doray, Berenice; Verloes, Alain; Viot, Géraldine; Le Bouc, Yves; Rossignol, Sylvie

2015-09-01

Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder associating macroglossia, abdominal wall defects, visceromegaly, and a high risk of childhood tumor. Molecular anomalies are mostly epigenetic; however, mutations of CDKN1C are implicated in 8% of cases, including both sporadic and familial forms. We aimed to describe the phenotype of BWS patients with CDKN1C mutations and develop a functional test for CDKN1C mutations. For each propositus, we sequenced the three exons and intron-exon boundaries of CDKN1C in patients presenting a BWS phenotype, including abdominal wall defects, without 11p15 methylation defects. We developed a functional test based on flow cytometry. We identified 37 mutations in 38 pedigrees (50 patients and seven fetuses). Analysis of parental samples when available showed that all mutations tested but one was inherited from the mother. The four missense mutations led to a less severe phenotype (lower frequency of exomphalos) than the other 33 mutations. The following four tumors occurred: one neuroblastoma, one ganglioneuroblastoma, one melanoma, and one acute lymphoid leukemia. Cases of BWS caused by CDKN1C mutations are not rare. CDKN1C sequencing should be performed for BWS patients presenting with abdominal wall defects or cleft palate without 11p15 methylation defects or body asymmetry, or in familial cases of BWS. © 2015 WILEY PERIODICALS, INC.

A universal array-based multiplexed test for cystic fibrosis carrier screening.

PubMed

Amos, Jean A; Bridge-Cook, Philippa; Ponek, Victor; Jarvis, Michael R

2006-01-01

Cystic fibrosis is a multisystem autosomal recessive disorder with high carrier frequencies in caucasians and significant, but lower, carrier frequencies in other ethnicities. Based on technology that allows high detection of mutations in caucasians and significant detection in other ethnic groups, the American College of Medical Genetics (ACMG) and American College of Obstetricians and Gynecologists (ACOG) have recommended pan-ethnic cystic fibrosis carrier screening for all reproductive couples. This paper discusses carrier screening using the Tag-It multiplex mutation platform and the Cystic Fibrosis Mutation Detection Kit. The Tag-It cystic fibrosis assay is a multiplexed genotyping assay that detects a panel of 40 cystic fibrosis transmembrane conductance regulator mutations including the 23 mutations recommended by the ACMG and ACOG for population screening. A total of 16 additional mutations detected by the Tag-It cystic fibrosis assay may also be common. The assay method is described in detail, and its performance in a genetics reference laboratory performing high-volume cystic fibrosis carrier screening is assessed.

THE FUNDUS PHENOTYPE ASSOCIATED WITH THE p.Ala243Val BEST1 MUTATION.

PubMed

Khan, Kamron N; Islam, Farrah; Moore, Anthony T; Michaelides, Michel

2018-03-01

To describe a highly recognizable and reproducible retinal phenotype associated with a specific BEST1 mutation-p.Ala243Val. Retrospective review of consecutive cases where genetic testing has identified p.Ala243Val BEST1 as the cause of disease. Electronic patient records were used to extract demographic, as well as functional and anatomical data. These data were compared with those observed with the most common BEST1 genotype, p.Arg218Cys. Eight individuals (six families) were identified with the p.Ala243Val BEST1 mutation and seven patients with the pathologic variant p.Arg218Cys. No patients with mutation of codon 243 knowingly had a family history of retinal disease, whereas all patients with the p.Arg218Cys variant did. The maculopathy was bilateral in all cases. The p.Ala243Val mutation was associated with a pattern dystrophy-type appearance, most visible with near-infrared reflectance and fundus autofluorescence imaging. This phenotype was never observed with any other genotype. This mutation was associated with an older median age of symptom onset (median = 42, interquartile range = 22) compared with those harboring the p.Arg218Cys mutation (median = 18, interquartile range = 12; Mann-Whitney U test; P < 0.05). Despite their older age, the final recorded acuity seemed to be better in the p.Ala243Val group (median = 0.55, interquartile range = 0.6475; median = 0.33, interquartile range = 0.358), although this did not reach statistical significance (Mann-Whitney U test; P > 0.05). The mutation p.Ala243Val is associated with highly recognizable and reproducible pattern dystrophy-like phenotype. Patients develop symptoms at a later age and tend to have better preservation of electrooculogram amplitudes.

Validity of Models for Predicting BRCA1 and BRCA2 Mutations

PubMed Central

Parmigiani, Giovanni; Chen, Sining; Iversen, Edwin S.; Friebel, Tara M.; Finkelstein, Dianne M.; Anton-Culver, Hoda; Ziogas, Argyrios; Weber, Barbara L.; Eisen, Andrea; Malone, Kathleen E.; Daling, Janet R.; Hsu, Li; Ostrander, Elaine A.; Peterson, Leif E.; Schildkraut, Joellen M.; Isaacs, Claudine; Corio, Camille; Leondaridis, Leoni; Tomlinson, Gail; Amos, Christopher I.; Strong, Louise C.; Berry, Donald A.; Weitzel, Jeffrey N.; Sand, Sharon; Dutson, Debra; Kerber, Rich; Peshkin, Beth N.; Euhus, David M.

2008-01-01

Background Deleterious mutations of the BRCA1 and BRCA2 genes confer susceptibility to breast and ovarian cancer. At least 7 models for estimating the probabilities of having a mutation are used widely in clinical and scientific activities; however, the merits and limitations of these models are not fully understood. Objective To systematically quantify the accuracy of the following publicly available models to predict mutation carrier status: BRCAPRO, family history assessment tool, Finnish, Myriad, National Cancer Institute, University of Pennsylvania, and Yale University. Design Cross-sectional validation study, using model predictions and BRCA1 or BRCA2 mutation status of patients different from those used to develop the models. Setting Multicenter study across Cancer Genetics Network participating centers. Patients 3 population-based samples of participants in research studies and 8 samples from genetic counseling clinics. Measurements Discrimination between individuals testing positive for a mutation in BRCA1 or BRCA2 from those testing negative, as measured by the c-statistic, and sensitivity and specificity of model predictions. Results The 7 models differ in their predictions. The better-performing models have a c-statistic around 80%. BRCAPRO has the largest c-statistic overall and in all but 2 patient subgroups, although the margin over other models is narrow in many strata. Outside of high-risk populations, all models have high false-negative and false-positive rates across a range of probability thresholds used to refer for mutation testing. Limitation Three recently published models were not included. Conclusions All models identify women who probably carry a deleterious mutation of BRCA1 or BRCA2 with adequate discrimination to support individualized genetic counseling, although discrimination varies across models and populations. PMID:17909205

A multisite blinded study for the detection of BRAF mutations in formalin-fixed, paraffin-embedded malignant melanoma

PubMed Central

Richter, Anna; Grieu, Fabienne; Carrello, Amerigo; Amanuel, Benhur; Namdarian, Kateh; Rynska, Aleksandra; Lucas, Amanda; Michael, Victoria; Bell, Anthony; Fox, Stephen B.; Hewitt, Chelsee A.; Do, Hongdo; McArthur, Grant A.; Wong, Stephen Q.; Dobrovic, Alexander; Iacopetta, Barry

2013-01-01

Melanoma patients with BRAF mutations respond to treatment with vemurafenib, thus creating a need for accurate testing of BRAF mutation status. We carried out a blinded study to evaluate various BRAF mutation testing methodologies in the clinical setting. Formalin-fixed, paraffin-embedded melanoma samples were macrodissected before screening for mutations using Sanger sequencing, single-strand conformation analysis (SSCA), high resolution melting analysis (HRM) and competitive allele-specific TaqMan® PCR (CAST-PCR). Concordance of 100% was observed between the Sanger sequencing, SSCA and HRM techniques. CAST-PCR gave rapid and accurate results for the common V600E and V600K mutations, however additional assays are required to detect rarer BRAF mutation types found in 3–4% of melanomas. HRM and SSCA followed by Sanger sequencing are effective two-step strategies for the detection of BRAF mutations in the clinical setting. CAST-PCR was useful for samples with low tumour purity and may also be a cost-effective and robust method for routine diagnostics. PMID:23584600

Four novel germline mutations in the MLH1 and PMS2 mismatch repair genes in patients with hereditary nonpolyposis colorectal cancer.

PubMed

Montazer Haghighi, Mahdi; Radpour, Ramin; Aghajani, Katayoun; Zali, Narges; Molaei, Mahsa; Zali, Mohammad Reza

2009-08-01

Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common cause of early onset hereditary colorectal cancer. In the majority of HNPCC families, microsatellite instability (MSI) and germline mutation in one of the DNA mismatch repair (MMR) genes are found. The entire coding sequence of MMR genes (MLH1, MLH2, MLH6, and PMS2) was analyzed using direct sequencing. Also, tumor tests were done as MSI and immunohistochemistry testing. We were able to find three novel MLH1 and one novel PMS2 germline mutations in three Iranian HNPCC patients. The first was a transversion mutation c.346A>C (T116P) and happened in the highly conserved HATPase-c region of MLH1 protein. The second was a transversion mutation c.736A>T (I246L), which caused an amino acid change of isoleucine to leucine. The third mutation (c.2145,6 delTG) was frameshift and resulted in an immature stop codon in five codons downstream. All of these three mutations were detected in the MLH1 gene. The other mutation was a transition mutation, c.676G>A (G207E), which has been found in exon six of the PMS2 gene and caused an amino acid change of glycine to glutamic acid. MSI assay revealed high instability in microsatellite for two patients and microsatellite stable for one patient. In all patients, an abnormal expression of the MMR proteins in HNPCC was related to the above novel mutations.

Multisite analytic performance studies of a real-time polymerase chain reaction assay for the detection of BRAF V600E mutations in formalin-fixed, paraffin-embedded tissue specimens of malignant melanoma.

PubMed

Anderson, Steven; Bloom, Kenneth J; Vallera, Dino U; Rueschoff, Josef; Meldrum, Cliff; Schilling, Robert; Kovach, Barbara; Lee, Ju Ruey-Jiuan; Ochoa, Pam; Langland, Rachel; Halait, Harkanwal; Lawrence, H Jeffrey; Dugan, Michael C

2012-11-01

A polymerase chain reaction-based companion diagnostic (cobas 4800 BRAF V600 Mutation Test) was recently approved by the US Food and Drug Administration to select patients with BRAF-mutant metastatic melanoma for treatment with the BRAF inhibitor vemurafenib. (1) To compare the analytic performance of the cobas test to Sanger sequencing by using screening specimens from phase II and phase III trials of vemurafenib, and (2) to assess the reproducibility of the cobas test at different testing sites. Specimens from 477 patients were used to determine positive and negative percent agreements between the cobas test and Sanger sequencing for detecting V600E (1799T>A) mutations. Specimens were evaluated with a massively parallel pyrosequencing method (454) to resolve discordances between polymerase chain reaction and Sanger results. Reproducibility of the cobas test was assessed at 3 sites by using 3 reagent lots and an 8-member panel of melanoma samples. A valid cobas result was obtained for all eligible patients. Sanger sequencing had a failure rate of 9.2% (44 of 477). For the remaining 433 specimens, positive percent agreement was 96.4% (215 of 223) and negative percent agreement, 80% (168 of 210). Among 42 cobas mutation-positive/Sanger V600E-negative specimens, 17 were V600E positive and 24 were V600K positive by 454. The cobas test detected 70% of V600K mutations. In the reproducibility study, a correct interpretation was made for 100% of wild-type specimens and specimens with greater than 5% mutant alleles; V600E mutations were detected in 90% of specimens with less than 5% mutant alleles. The cobas test (1) had a lower assay failure rate than that of Sanger, (2) was more sensitive in detecting V600E mutations, (3) detected most V600K mutations, and (4) was highly reproducible.

Neuropathy Tests

MedlinePlus

... Mutation Mycophenolic Acid Mycoplasma Myoglobin Nicotine and Cotinine Non-High Density Lipoprotein Cholesterol Opioid Testing Osmolality Ova ... that make neuropathy worse Detect and evaluate complications Non-laboratory tests The diagnostic workup for neuropathy begins ...

Is High Resolution Melting Analysis (HRMA) Accurate for Detection of Human Disease-Associated Mutations? A Meta Analysis

PubMed Central

Ma, Feng-Li; Jiang, Bo; Song, Xiao-Xiao; Xu, An-Gao

2011-01-01

Background High Resolution Melting Analysis (HRMA) is becoming the preferred method for mutation detection. However, its accuracy in the individual clinical diagnostic setting is variable. To assess the diagnostic accuracy of HRMA for human mutations in comparison to DNA sequencing in different routine clinical settings, we have conducted a meta-analysis of published reports. Methodology/Principal Findings Out of 195 publications obtained from the initial search criteria, thirty-four studies assessing the accuracy of HRMA were included in the meta-analysis. We found that HRMA was a highly sensitive test for detecting disease-associated mutations in humans. Overall, the summary sensitivity was 97.5% (95% confidence interval (CI): 96.8–98.5; I2 = 27.0%). Subgroup analysis showed even higher sensitivity for non-HR-1 instruments (sensitivity 98.7% (95%CI: 97.7–99.3; I2 = 0.0%)) and an eligible sample size subgroup (sensitivity 99.3% (95%CI: 98.1–99.8; I2 = 0.0%)). HRMA specificity showed considerable heterogeneity between studies. Sensitivity of the techniques was influenced by sample size and instrument type but by not sample source or dye type. Conclusions/Significance These findings show that HRMA is a highly sensitive, simple and low-cost test to detect human disease-associated mutations, especially for samples with mutations of low incidence. The burden on DNA sequencing could be significantly reduced by the implementation of HRMA, but it should be recognized that its sensitivity varies according to the number of samples with/without mutations, and positive results require DNA sequencing for confirmation. PMID:22194806

Economic Evaluation of Companion Diagnostic Testing for EGFR Mutations and First-Line Targeted Therapy in Advanced Non-Small Cell Lung Cancer Patients in South Korea.

PubMed

Lim, Eun-A; Lee, Haeyoung; Bae, Eunmi; Lim, Jaeok; Shin, Young Kee; Choi, Sang-Eun

2016-01-01

As targeted therapy becomes increasingly important, diagnostic techniques for identifying targeted biomarkers have also become an emerging issue. The study aims to evaluate the cost-effectiveness of treating patients as guided by epidermal growth factor receptor (EGFR) mutation status compared with a no-testing strategy that is the current clinical practice in South Korea. A cost-utility analysis was conducted to compare an EGFR mutation testing strategy with a no-testing strategy from the Korean healthcare payer's perspective. The study population consisted of patients with stage 3b and 4 lung adenocarcinoma. A decision tree model was employed to select the appropriate treatment regimen according to the results of EGFR mutation testing and a Markov model was constructed to simulate disease progression of advanced non-small cell lung cancer. The length of a Markov cycle was one month, and the time horizon was five years (60 cycles). In the base case analysis, the testing strategy was a dominant option. Quality-adjusted life-years gained (QALYs) were 0.556 and 0.635, and total costs were $23,952 USD and $23,334 USD in the no-testing and testing strategy respectively. The sensitivity analyses showed overall robust results. The incremental cost-effectiveness ratios (ICERs) increased when the number of patients to be treated with erlotinib increased, due to the high cost of erlotinib. Treating advanced adenocarcinoma based on EGFR mutation status has beneficial effects and saves the cost compared to no testing strategy in South Korea. However, the cost-effectiveness of EGFR mutation testing was heavily affected by the cost-effectiveness of the targeted therapy.

Juvenile retinoschisis: a model for molecular diagnostic testing of X-linked ophthalmic disease.

PubMed

Sieving, P A; Yashar, B M; Ayyagari, R

1999-01-01

X-linked juvenile retinoschisis (RS) provides a starting point to define clinical paradigms and understand the limitations of diagnostic molecular testing. The RS phenotype is specific, but the broad severity range is clinically confusing. Molecular diagnostic testing obviates unnecessary examinations for boys at-risk and identifies carrier females who otherwise show no clinical signs. The XLRS1 gene has 6 exons of 26-196 base-pair size. Each exon is amplified by a single polymerase chain reaction and then sequenced, starting with exons 4 through 6, which contain mutation "hot spots." The 6 XLRS1 exons are sequenced serially. If alterations are found, they are compared with mutations in our > 120 XLRS families and with the > 300 mutations reported worldwide. Point mutations, small deletions, or rearrangements are identified in nearly 90% of males with a clinical diagnosis of RS. XLRS1 has very few sequence polymorphisms. Carrier-state testing produces 1 of 3 results: (1) positive, in which the woman has the same mutation as an affected male relative or known in other RS families; (2) negative, in which she lacks the mutation of her affected male relative; and (3) uninformative, in which no known mutation is identified or no information exists about the familial mutation. Molecular RS screening is an effective diagnostic tool that complements the clinician's skills for early detection of at-risk males. Useful outcomes of carrier testing depend on several factors: (1) a male relative with a clear clinical diagnosis; (2) a well-defined inheritance pattern; (3) high disease penetrance; (4) size and organization of the gene; and (5) the types of disease-associated mutations. Ethical questions include molecular diagnostic testing of young at-risk females before the age of consent, the impact of this information on the emotional health of the patient and family, and issues of employability and insurance coverage.

Juvenile retinoschisis: a model for molecular diagnostic testing of X-linked ophthalmic disease.

PubMed Central

Sieving, P A; Yashar, B M; Ayyagari, R

1999-01-01

BACKGROUND AND PURPOSE: X-linked juvenile retinoschisis (RS) provides a starting point to define clinical paradigms and understand the limitations of diagnostic molecular testing. The RS phenotype is specific, but the broad severity range is clinically confusing. Molecular diagnostic testing obviates unnecessary examinations for boys at-risk and identifies carrier females who otherwise show no clinical signs. METHODS: The XLRS1 gene has 6 exons of 26-196 base-pair size. Each exon is amplified by a single polymerase chain reaction and then sequenced, starting with exons 4 through 6, which contain mutation "hot spots." RESULTS: The 6 XLRS1 exons are sequenced serially. If alterations are found, they are compared with mutations in our > 120 XLRS families and with the > 300 mutations reported worldwide. Point mutations, small deletions, or rearrangements are identified in nearly 90% of males with a clinical diagnosis of RS. XLRS1 has very few sequence polymorphisms. Carrier-state testing produces 1 of 3 results: (1) positive, in which the woman has the same mutation as an affected male relative or known in other RS families; (2) negative, in which she lacks the mutation of her affected male relative; and (3) uninformative, in which no known mutation is identified or no information exists about the familial mutation. CONCLUSIONS: Molecular RS screening is an effective diagnostic tool that complements the clinician's skills for early detection of at-risk males. Useful outcomes of carrier testing depend on several factors: (1) a male relative with a clear clinical diagnosis; (2) a well-defined inheritance pattern; (3) high disease penetrance; (4) size and organization of the gene; and (5) the types of disease-associated mutations. Ethical questions include molecular diagnostic testing of young at-risk females before the age of consent, the impact of this information on the emotional health of the patient and family, and issues of employability and insurance coverage. Images FIGURE 2A FIGURE 2B PMID:10703138

Familial Hypercholesterolemia: Cascade Screening in Children and Relatives of the Affected.

PubMed

Setia, Nitika; Saxena, Renu; Sawhney, J P S; Verma, Ishwar C

2018-05-01

Familial Hypercholesterolemia (FH) is an inherited disorder of lipid metabolism characterized by very high low density lipoprotein (LDL) cholesterol since birth, resulting in premature atherosclerosis and coronary artery disease (CAD). Cascade screening of children and family members of proven FH individuals can identify more subjects who have high LDL cholesterol or the family mutation and appropriate intervention can reduce their risk of atherosclerosis and prevent its complications. Cascade screening by molecular testing, was carried out in 133 family members, comprising 24 children, of 31 probands with FH having a pathogenic mutation in LDLR/ApoB gene. Lipid profiles were obtained in 44 family members including 11 children. Of 133 family members tested, 88 (66.1%) were identified to carry the family mutation. Twelve of these were children below 18 y of age and 76 were adults. CAD was present in 15 (11.2%) family members and 63(47.4%) family members, including nine children, were already on Lipid Lowering Therapy. Cascade screening led to identification of 88 new cases, with a pathogenic mutation, who were at a very high risk of developing premature CAD. The authors identified 12 children with family specific mutation, out of which 9 were initiated on low dose statin therapy. Four homozygous children were treated with high dose statins because of substantially increased risk of CAD. Cascade screening, therefore, proved to be a successful initiative towards primary prevention of CAD in India.

Detection of BRAF mutations from solid tumors using Tumorplex™ technology

PubMed Central

Yo, Jacob; Hay, Katie S.L.; Vinayagamoorthy, Dilanthi; Maryanski, Danielle; Carter, Mark; Wiegel, Joseph; Vinayagamoorthy, Thuraiayah

2015-01-01

Allele specific multiplex sequencing (Tumorplex™) is a new molecular platform for the detection of single base mutation in tumor biopsies with high sensitivity for clinical testing. Tumorplex™ is a novel modification of Sanger sequencing technology that generates both mutant and wild type nucleotide sequences simultaneously in the same electropherogram. The molecular weight of the two sequencing primers are different such that the two sequences generated are separated, thus eliminating possible suppression of mutant signal by the more abundant wild type signal. Tumorplex™ platform technology was tested using BRAF mutation V600E. These studies were performed with cloned BRAF mutations and genomic DNA extracted from tumor cells carrying 50% mutant allele. The lower limit of detection for BRAF V600E was found to be 20 genome equivalents (GE) using genomic DNA extracted from mutation specific cell lines. Sensitivity of the assay was tested by challenging the mutant allele with wild type allele at 20 GE, and was able to detect BRAF mutant signal at a GE ration of 20:1 × 107 (mutant to wild-type). This level of sensitivity can detect low abundance of clonal mutations in tumor biopsies and eliminate the need for cell enrichment. • Tumorplex™ is a single tube assay that permits the recognition of mutant allele without suppression by wildtype signal. • Tumorplex™ provides a high level of sensitivity. • Tumorplex™ can be used with small sample size with mixed population of cells carrying heterogeneous gDNA. PMID:26258049

Hereditary breast/ovarian cancer--pitfalls in genetic counseling.

PubMed

Dagan, E; Gershoni-Baruch, R

2001-10-01

Genetic counseling and risk assessment, given to women with a family history of breast/ovarian cancer, are regularly based on pedigree analysis. In the Ashkenazi Jewish population, hereditary breast/ovarian cancer is mainly attributed to three founder mutations, namely, 185delAG, 5382insC, and 6174delT, in BRCA1/2 genes. The overall frequency of these mutations, in the Jewish Ashkenazi population, is as high as 2.5%. Based on clinical and family history data, the results of BRCA molecular testing, in Ashkenazi individuals at risk, are appropriately anticipated in most cases. Here we report on five families, in which the segregation of BRCA1/2 mutations, in affected and unaffected family members, was unexpected, emphasizing the need to test, for founder mutations, every Ashkenazi individual at risk, irrespective of the genotype of affected family members. Ultimately, risk assessments and recommendations, in Ashkenazi women, should be invariably based on the results of genetic testing.

LDL Particle Testing

MedlinePlus

... Mutation Mycophenolic Acid Mycoplasma Myoglobin Nicotine and Cotinine Non-High Density Lipoprotein Cholesterol Opioid Testing Osmolality Ova ... the group concluded that both tests were nearly equivalent in their ability to assess CVD risk and ...

Monoclonal origin of peritoneal implants and lymph node deposits in serous borderline ovarian tumors (s-BOT) with high intratumoral homogeneity.

PubMed

Horn, Lars-Christian; Höhn, Anne K; Einenkel, Jens; Siebolts, Udo

2014-11-01

Molecular studies have shown that the most prevalent mutations in serous ovarian borderline tumors (s-BOT) are BRAF and/or KRAS alterations. About one third of s-BOT represent peritoneal implants and/or lymph node involvement. These extraovarian deposits may be monoclonal or polyclonal in origin. To test both the hypotheses, mutational analyses using pyrosequencing for BRAF codon 600 and KRAS codon 12/13 and 61 of microdissected tissue was performed in 15 s-BOT and their invasive and noninvasive peritoneal implants. Two to 6 implants from different peritoneal sites were examined in 13 cases. Lymph node deposits were available for the analysis in 3 cases. Six s-BOT showed mutation in exon 2 codon 12 of the KRAS proto-oncogen. Five additional cases showed BRAF p.V600E mutation representing an overall mutation rate of 73.3%. Multiple (2-6) peritoneal implants were analyzed after microdissection in 13 of 15 cases. All showed identical mutational results when compared with the ovarian site of the disease. All lymph node deposits, including those with multiple deposits in different nodes, showed identical results, suggesting high intratumoral mutational homogeneity. The evidence presented in this study and the majority of data reported in the literature support the hypothesis that s-BOT with their peritoneal implants and lymph node deposits show identical mutational status of BRAF and KRAS suggesting a monoclonal rather than a polyclonal disease regarding these both tested genetic loci. In addition, a high intratumoral genetic homogeneity can be suggested. In conclusion, the results of the present study support the monoclonal origin of s-BOT and their peritoneal implants and lymph node deposits.

High-resolution melting analysis of gyrA codon 84 and grlA codon 80 mutations conferring resistance to fluoroquinolones in Staphylococcus pseudintermedius isolates from canine clinical samples.

PubMed

Loiacono, Monica; Martino, Piera A; Albonico, Francesca; Dell'Orco, Francesca; Ferretti, Manuela; Zanzani, Sergio; Mortarino, Michele

2017-09-01

Staphylococcus pseudintermedius is an opportunistic pathogen of dogs and cats. A high-resolution melting analysis (HRMA) protocol was designed and tested on 42 clinical isolates with known fluoroquinolone (FQ) susceptibility and gyrA codon 84 and grlA codon 80 mutation status. The HRMA approach was able to discriminate between FQ-sensitive and FQ-resistant strains and confirmed previous reports that the main mutation site associated with FQ resistance in S. pseudintermedius is located at position 251 (Ser84Leu) of gyrA. Routine, HRMA-based FQ susceptibility profiles may be a valuable tool to guide therapy. The FQ resistance-predictive power of the assay should be tested in a significantly larger number of isolates.

Germline mutations in 40 cancer susceptibility genes among Chinese patients with high hereditary risk breast cancer.

PubMed

Li, Junyan; Jing, Ruilin; Wei, Hongyi; Wang, Minghao; Qi, Xiaowei; Liu, Haoxi; Liu, Jian; Ou, Jianghua; Jiang, Weihua; Tian, Fuguo; Sheng, Yuan; Li, Hengyu; Xu, Hong; Zhang, Ruishan; Guan, Aihua; Liu, Ke; Jiang, Hongchuan; Ren, Yu; He, Jianjun; Huang, Weiwei; Liao, Ning; Cai, Xiangjun; Ming, Jia; Ling, Rui; Xu, Yan; Hu, Chunyan; Zhang, Jianguo; Guo, Baoliang; Ouyang, Lizhi; Shuai, Ping; Liu, Zhenzhen; Zhong, Ling; Zeng, Zhen; Zhang, Ting; Xuan, Zhaoling; Tan, Xuanni; Liang, Junbin; Pan, Qinwen; Chen, Li; Zhang, Fan; Fan, Linjun; Zhang, Yi; Yang, Xinhua; Li, Jingbo; Chen, Chongjian; Jiang, Jun

2018-05-12

Multigene panel testing of breast cancer predisposition genes have been extensively conducted in Europe and America, which is relatively rare in Asia however. In this study, we assessed the frequency of germline mutations in 40 cancer predisposition genes, including BRCA1 and BRCA2, among a large cohort of Chinese patients with high hereditary risk of BC. From 2015 to 2016, consecutive BC patients from 26 centers of China with high hereditary risk were recruited (n=937). Clinical information was collected and next-generation sequencing (NGS) was performed using blood samples of participants to identify germline mutations. In total, we acquired 223 patients with putative germline mutations, including 159 in BRCA1/2, 61 in 15 other BC susceptibility genes and 3 in both BRCA1/2 and non-BRCA1/2 gene. Major mutant non-BRCA1/2 genes were TP53 (n=18), PALB2 (n=11), CHEK2 (n=6), ATM (n=6), and BARD1 (n=5). No factors predicted pathologic mutations in non-BRCA1/2 genes when treated as a whole. TP53 mutations were associated with HER-2 positive BC and younger age at diagnosis; and CHEK2 and PALB2 mutations were enriched in patients with luminal BC. Among high hereditary risk Chinese BC patients, 23.8% contained germline mutations, including 6.8% in non-BRCA1/2 genes. TP53 and PALB2 had a relatively high mutation rates (1.9% and 1.2%). Although no factors predicted for detrimental mutations in non-BRCA1/2 genes, some clinical features were associated with mutations of several particular genes. This article is protected by copyright. All rights reserved. © 2018 UICC.

Inherited mutations in 17 breast cancer susceptibility genes among a large triple-negative breast cancer cohort unselected for family history of breast cancer.

PubMed

Couch, Fergus J; Hart, Steven N; Sharma, Priyanka; Toland, Amanda Ewart; Wang, Xianshu; Miron, Penelope; Olson, Janet E; Godwin, Andrew K; Pankratz, V Shane; Olswold, Curtis; Slettedahl, Seth; Hallberg, Emily; Guidugli, Lucia; Davila, Jaime I; Beckmann, Matthias W; Janni, Wolfgang; Rack, Brigitte; Ekici, Arif B; Slamon, Dennis J; Konstantopoulou, Irene; Fostira, Florentia; Vratimos, Athanassios; Fountzilas, George; Pelttari, Liisa M; Tapper, William J; Durcan, Lorraine; Cross, Simon S; Pilarski, Robert; Shapiro, Charles L; Klemp, Jennifer; Yao, Song; Garber, Judy; Cox, Angela; Brauch, Hiltrud; Ambrosone, Christine; Nevanlinna, Heli; Yannoukakos, Drakoulis; Slager, Susan L; Vachon, Celine M; Eccles, Diana M; Fasching, Peter A

2015-02-01

Recent advances in DNA sequencing have led to the development of breast cancer susceptibility gene panels for germline genetic testing of patients. We assessed the frequency of mutations in 17 predisposition genes, including BRCA1 and BRCA2, in a large cohort of patients with triple-negative breast cancer (TNBC) unselected for family history of breast or ovarian cancer to determine the utility of germline genetic testing for those with TNBC. Patients with TNBC (N = 1,824) unselected for family history of breast or ovarian cancer were recruited through 12 studies, and germline DNA was sequenced to identify mutations. Deleterious mutations were identified in 14.6% of all patients. Of these, 11.2% had mutations in the BRCA1 (8.5%) and BRCA2 (2.7%) genes. Deleterious mutations in 15 other predisposition genes were detected in 3.7% of patients, with the majority observed in genes involved in homologous recombination, including PALB2 (1.2%) and BARD1, RAD51D, RAD51C, and BRIP1 (0.3% to 0.5%). Patients with TNBC with mutations were diagnosed at an earlier age (P < .001) and had higher-grade tumors (P = .01) than those without mutations. Deleterious mutations in predisposition genes are present at high frequency in patients with TNBC unselected for family history of cancer. Mutation prevalence estimates suggest that patients with TNBC, regardless of age at diagnosis or family history of cancer, should be considered for germline genetic testing of BRCA1 and BRCA2. Although mutations in other predisposition genes are observed among patients with TNBC, better cancer risk estimates are needed before these mutations are used for clinical risk assessment in relatives. © 2014 by American Society of Clinical Oncology.

Inherited Mutations in 17 Breast Cancer Susceptibility Genes Among a Large Triple-Negative Breast Cancer Cohort Unselected for Family History of Breast Cancer

PubMed Central

Couch, Fergus J.; Hart, Steven N.; Sharma, Priyanka; Toland, Amanda Ewart; Wang, Xianshu; Miron, Penelope; Olson, Janet E.; Godwin, Andrew K.; Pankratz, V. Shane; Olswold, Curtis; Slettedahl, Seth; Hallberg, Emily; Guidugli, Lucia; Davila, Jaime I.; Beckmann, Matthias W.; Janni, Wolfgang; Rack, Brigitte; Ekici, Arif B.; Slamon, Dennis J.; Konstantopoulou, Irene; Fostira, Florentia; Vratimos, Athanassios; Fountzilas, George; Pelttari, Liisa M.; Tapper, William J.; Durcan, Lorraine; Cross, Simon S.; Pilarski, Robert; Shapiro, Charles L.; Klemp, Jennifer; Yao, Song; Garber, Judy; Cox, Angela; Brauch, Hiltrud; Ambrosone, Christine; Nevanlinna, Heli; Yannoukakos, Drakoulis; Slager, Susan L.; Vachon, Celine M.; Eccles, Diana M.; Fasching, Peter A.

2015-01-01

Purpose Recent advances in DNA sequencing have led to the development of breast cancer susceptibility gene panels for germline genetic testing of patients. We assessed the frequency of mutations in 17 predisposition genes, including BRCA1 and BRCA2, in a large cohort of patients with triple-negative breast cancer (TNBC) unselected for family history of breast or ovarian cancer to determine the utility of germline genetic testing for those with TNBC. Patients and Methods Patients with TNBC (N = 1,824) unselected for family history of breast or ovarian cancer were recruited through 12 studies, and germline DNA was sequenced to identify mutations. Results Deleterious mutations were identified in 14.6% of all patients. Of these, 11.2% had mutations in the BRCA1 (8.5%) and BRCA2 (2.7%) genes. Deleterious mutations in 15 other predisposition genes were detected in 3.7% of patients, with the majority observed in genes involved in homologous recombination, including PALB2 (1.2%) and BARD1, RAD51D, RAD51C, and BRIP1 (0.3% to 0.5%). Patients with TNBC with mutations were diagnosed at an earlier age (P < .001) and had higher-grade tumors (P = .01) than those without mutations. Conclusion Deleterious mutations in predisposition genes are present at high frequency in patients with TNBC unselected for family history of cancer. Mutation prevalence estimates suggest that patients with TNBC, regardless of age at diagnosis or family history of cancer, should be considered for germline genetic testing of BRCA1 and BRCA2. Although mutations in other predisposition genes are observed among patients with TNBC, better cancer risk estimates are needed before these mutations are used for clinical risk assessment in relatives. PMID:25452441

Screening for BRCA1, BRCA2, CHEK2, PALB2, BRIP1, RAD50, and CDH1 mutations in high-risk Finnish BRCA1/2-founder mutation-negative breast and/or ovarian cancer individuals.

PubMed

Kuusisto, Kirsi M; Bebel, Aleksandra; Vihinen, Mauno; Schleutker, Johanna; Sallinen, Satu-Leena

2011-02-28

Two major high-penetrance breast cancer genes, BRCA1 and BRCA2, are responsible for approximately 20% of hereditary breast cancer (HBC) cases in Finland. Additionally, rare mutations in several other genes that interact with BRCA1 and BRCA2 increase the risk of HBC. Still, a majority of HBC cases remain unexplained which is challenging for genetic counseling. We aimed to analyze additional mutations in HBC-associated genes and to define the sensitivity of our current BRCA1/2 mutation analysis protocol used in genetic counseling. Eighty-two well-characterized, high-risk hereditary breast and/or ovarian cancer (HBOC) BRCA1/2-founder mutation-negative Finnish individuals, were screened for germline alterations in seven breast cancer susceptibility genes, BRCA1, BRCA2, CHEK2, PALB2, BRIP1, RAD50, and CDH1. BRCA1/2 were analyzed by multiplex ligation-dependent probe amplification (MLPA) and direct sequencing. CHEK2 was analyzed by the high resolution melt (HRM) method and PALB2, RAD50, BRIP1 and CDH1 were analyzed by direct sequencing. Carrier frequencies between 82 (HBOC) BRCA1/2-founder mutation-negative Finnish individuals and 384 healthy Finnish population controls were compared by using Fisher's exact test. In silico prediction for novel missense variants effects was carried out by using Pathogenic-Or-Not -Pipeline (PON-P). Three previously reported breast cancer-associated variants, BRCA1 c.5095C > T, CHEK2 c.470T > C, and CHEK2 c.1100delC, were observed in eleven (13.4%) individuals. Ten of these individuals (12.2%) had CHEK2 variants, c.470T > C and/or c.1100delC. Fourteen novel sequence alterations and nine individuals with more than one non-synonymous variant were identified. One of the novel variants, BRCA2 c.72A > T (Leu24Phe) was predicted to be likely pathogenic in silico. No large genomic rearrangements were detected in BRCA1/2 by multiplex ligation-dependent probe amplification (MLPA). In this study, mutations in previously known breast cancer susceptibility genes can explain 13.4% of the analyzed high-risk BRCA1/2-negative HBOC individuals. CHEK2 mutations, c.470T > C and c.1100delC, make a considerable contribution (12.2%) to these high-risk individuals but further segregation analysis is needed to evaluate the clinical significance of these mutations before applying them in clinical use. Additionally, we identified novel variants that warrant additional studies. Our current genetic testing protocol for 28 Finnish BRCA1/2-founder mutations and protein truncation test (PTT) of the largest exons is sensitive enough for clinical use as a primary screening tool.

Screening for BRCA1, BRCA2, CHEK2, PALB2, BRIP1, RAD50, and CDH1 mutations in high-risk Finnish BRCA1/2-founder mutation-negative breast and/or ovarian cancer individuals

PubMed Central

2011-01-01

Introduction Two major high-penetrance breast cancer genes, BRCA1 and BRCA2, are responsible for approximately 20% of hereditary breast cancer (HBC) cases in Finland. Additionally, rare mutations in several other genes that interact with BRCA1 and BRCA2 increase the risk of HBC. Still, a majority of HBC cases remain unexplained which is challenging for genetic counseling. We aimed to analyze additional mutations in HBC-associated genes and to define the sensitivity of our current BRCA1/2 mutation analysis protocol used in genetic counseling. Methods Eighty-two well-characterized, high-risk hereditary breast and/or ovarian cancer (HBOC) BRCA1/2-founder mutation-negative Finnish individuals, were screened for germline alterations in seven breast cancer susceptibility genes, BRCA1, BRCA2, CHEK2, PALB2, BRIP1, RAD50, and CDH1. BRCA1/2 were analyzed by multiplex ligation-dependent probe amplification (MLPA) and direct sequencing. CHEK2 was analyzed by the high resolution melt (HRM) method and PALB2, RAD50, BRIP1 and CDH1 were analyzed by direct sequencing. Carrier frequencies between 82 (HBOC) BRCA1/2-founder mutation-negative Finnish individuals and 384 healthy Finnish population controls were compared by using Fisher's exact test. In silico prediction for novel missense variants effects was carried out by using Pathogenic-Or-Not -Pipeline (PON-P). Results Three previously reported breast cancer-associated variants, BRCA1 c.5095C > T, CHEK2 c.470T > C, and CHEK2 c.1100delC, were observed in eleven (13.4%) individuals. Ten of these individuals (12.2%) had CHEK2 variants, c.470T > C and/or c.1100delC. Fourteen novel sequence alterations and nine individuals with more than one non-synonymous variant were identified. One of the novel variants, BRCA2 c.72A > T (Leu24Phe) was predicted to be likely pathogenic in silico. No large genomic rearrangements were detected in BRCA1/2 by multiplex ligation-dependent probe amplification (MLPA). Conclusions In this study, mutations in previously known breast cancer susceptibility genes can explain 13.4% of the analyzed high-risk BRCA1/2-negative HBOC individuals. CHEK2 mutations, c.470T > C and c.1100delC, make a considerable contribution (12.2%) to these high-risk individuals but further segregation analysis is needed to evaluate the clinical significance of these mutations before applying them in clinical use. Additionally, we identified novel variants that warrant additional studies. Our current genetic testing protocol for 28 Finnish BRCA1/2-founder mutations and protein truncation test (PTT) of the largest exons is sensitive enough for clinical use as a primary screening tool. PMID:21356067

A1C Test

MedlinePlus

... Mutation Mycophenolic Acid Mycoplasma Myoglobin Nicotine and Cotinine Non-High Density Lipoprotein Cholesterol Opioid Testing Osmolality Ova ... as older RBCs die and younger RBCs (with non-glycated hemoglobin) take their place. This test may ...

High frequency of the recurrent c.1310_1313delAAGA BRCA2 mutation in the North-East of Morocco and implication for hereditary breast-ovarian cancer prevention and control.

PubMed

Laarabi, Fatima-Zahra; Ratbi, Ilham; Elalaoui, Siham Chafai; Mezzouar, Loubna; Doubaj, Yassamine; Bouguenouch, Laila; Ouldim, Karim; Benjaafar, Noureddine; Sefiani, Abdelaziz

2017-06-02

To date, a limited number of BRCA1/2 germline mutations have been reported in hereditary breast and/or ovarian cancer in the Moroccan population. Less than 20 different mutations of these two genes have been identified in Moroccan patients, and recently we reported a further BRCA2 mutation (c.1310_1313delAAGA; p.Lys437IlefsX22) in three unrelated patients, all from the North-East of the country. We aimed in this study to evaluate the frequency and geographic distribution of this BRCA2 frameshift mutation, in order to access its use as the first-line BRCA genetic testing strategy for Moroccan patients. We enrolled in this study 122 patients from different regions of Morocco, with suggestive inherited predisposition to breast and ovarian cancers. All subjects gave written informed consent to BRCA1/2 genetic testing. According to available resources of our lab and enrolled families, 51 patients were analyzed by the conventional individual exon-by-exon Sanger sequencing, 23 patients were able to benefit from a BRCA next generation sequencing and a target screening for exon 10 of BRCA2 gene was performed in 48 patients. Overall, and among the 122 patients analyzed for at least the exon 10 of the BRCA2 gene, the c.1310_1313delAAGA frameshift mutation was found in 14 patients. Genealogic investigation revealed that all carriers of this mutation shared the same geographic origin and were descendants of the North-East of Morocco. In this study, we highlighted that c.1310_1313delAAGA mutation of BRCA2 gene is recurrent with high frequency in patients from the North-East region of Morocco. Therefore, we propose to use, in public health strategies, the detection of this mutation as the first-line screening tests in patients with breast and ovarian cancer originated from this region.

Novel AVPR2 mutation causing partial nephrogenic diabetes insipidus in a Japanese family.

PubMed

Yamashita, Sumie; Hata, Astuko; Usui, Takeshi; Oda, Hirotsugu; Hijikata, Atsushi; Shirai, Tsuyoshi; Kaneko, Naoto; Hata, Daisuke

2016-05-01

X-linked recessive congenital nephrogenic diabetes insipidus (NDI) is caused by mutations of the arginine vasopressin type 2 receptor gene (AVPR2). More than 200 mutations of the AVPR2 gene with complete NDI have been reported although only 15 mutations with partial NDI has been reported to date. We herein report a Japanese kindred with partial NDI. The proband is an 8-year-old boy who was referred to our hospital for nocturnal enuresis. Water deprivation test and hypertonic saline test suggested partial renal antidiuretic hormone arginine vasopressin (AVP) resistance. Analysis of genomic DNA revealed a novel missense mutation (p.L161P) in the patient. The patient's mother was heterozygous for the mutation. Three-dimensional (3-D) modeling study showed that L161P possibly destabilizes the transmembrane domain of the V2 receptor, resulting in its misfolding or mislocalization. Distinguishing partial NDI from nocturnal enuresis is important. A clinical clue for diagnosis of partial NDI is an incompatibly high level of AVP despite normal serum osmolality.

Integrating surgery and genetic testing for the modern surgeon.

PubMed

Caso, Raul; Beamer, Matthew; Lofthus, Alexander D; Sosin, Michael

2017-10-01

The field of cancer genetics is rapidly evolving and several genetic mutations have been identified in hereditary cancer syndromes. These mutations can be diagnosed via routine genetic testing allowing prompt intervention. This is especially true for certain variants of colorectal, breast, and thyroid cancers where genetic testing may guide surgical therapy. Ultimately, surgical intervention may drastically diminish disease manifestation or progression in individuals deemed as high-risk based on their genetic makeup. Understanding the concepts of gene-based testing and integrating into current surgical practice is crucial. This review addresses common genetic syndromes, tests, and interventions salient to the current surgeon.

HIV type-1 genotypic resistance profiles in vertically infected patients from Argentina reveal an association between K103N+L100I and L74V mutations.

PubMed

Aulicino, Paula C; Rocco, Carlos A; Mecikovsky, Debora; Bologna, Rosa; Mangano, Andrea; Sen, Luisa

2010-01-01

Patterns and pathways of HIV type-1 (HIV-1) antiretroviral (ARV) drug resistance-associated mutations in clinical isolates are conditioned by ARV history and factors such as viral subtype and fitness. Our aim was to analyse the frequency and association of ARV drug resistance mutations in a group of long-term vertically infected patients from Argentina. Plasma samples from 71 patients (38 children and 33 adolescents) were collected for genotypic HIV-1 ARV resistance testing during the period between February 2006 and October 2008. Statistically significant pairwise associations between ARV resistance mutations in pol, as well as associations between mutations and drug exposure, were identified using Fisher's exact tests with Bonferroni and false discovery rate corrections. Phylogenetic analyses were performed for subtype assignment. In protease (PR), resistance-associated mutations M46I/L, I54M/L/V/A/S and V82A/F/T/S/M/I were associated with each other and with minor mutations at codons 10, 24 and 71. Mutations V82A/F/T/S/M/I were primarily selected by the administration of ritonavir (RTV) in an historical ARV regimen. In reverse transcriptase, thymidine analogue mutation (TAM)1 profile was more common than TAM2. The non-nucleoside K103N+L100I mutations were observed at high frequency (15.5%) and were significantly associated with the nucleoside mutation L74V in BF recombinants. Associations of mutations at PR sites reflect the frequent use of RTV at an early time in this group of patients and convergent resistance mechanisms driven by the high exposure to protease inhibitors, as well as local HIV-1 diversity. The results provide clinical evidence of a molecular interaction between K103N+L100I and L74V mutations at the reverse transcriptase gene in vivo, limiting the future use of second-generation non-nucleoside reverse transcriptase inhibitors such as etravirine.

Germline stem cell competition, mutation hot spots, genetic disorders and older dads

PubMed Central

Arnheim, Norman; Calabrese, Peter

2016-01-01

Some de novo human mutations arise at frequencies far exceeding the genome average mutation rate. Examples are the common mutations at one or a few sites in the genes causing achondroplasia, Noonan syndrome, multiple endocrine neoplasia 2B and Apert syndrome. These mutations are recurrent, provide a gain of function, are paternally derived and are more likely transmitted as the father ages. Recent experiments tested whether the high mutation frequencies are due to an elevated mutation rate per cell division, as expected, or an advantage of the mutant spermatogonial stem cells over wild-type stem cells. The evidence, which includes the surprising discovery of testis mutation clusters, rejects the former model but not the latter. We propose how the mutations might alter spermatogonial stem cell function and discuss how germline selection contributes to the paternal age effect, the human mutational load and adaptive evolution. PMID:27070266

[Clinical significance and distribution of BRCA genes mutation in sporadic high grade serous ovarian cancer].

PubMed

Liu, W L; Wang, Z Z; Zhao, J Z; Hou, Y Y; Wu, X X; Li, W; Dong, B; Tong, T T; Guo, Y J

2017-01-25

Objective: To investigate the mutations of BRCA genes in sporadic high grade serous ovarian cancer (HGSOC) and study its clinical significance. Methods: Sixty-eight patients between January 2015 and January 2016 from the Affiliated Cancer Hospital of Zhengzhou University were collected who were based on pathological diagnosis of ovarian cancer and had no reported family history, and all patients firstly hospitalized were untreated in other hospitals before. (1) The BRCA genes were detected by next-generation sequencing (NGS) method. (2) The serum tumor markers included carcinoembryonic antigen (CEA), CA(125), CA(199), and human epididymis protein 4 (HE4) were detected by the chemiluminescence methods, and their correlation was analyzed by Pearson linear correlation. Descriptive statistics and comparisons were performed using two-tailed t -tests, Pearson's chi square test, Fisher's exact tests or logistic regression analysis as appropriate to research the clinicopathologic features associated with BRCA mutations, including age, International Federation of Gynecology and Obstetrics (FIGO) stage, platinum-based chemotherapy sensitivity, distant metastases, serum tumor markers (STM) . Results: (1) Fifteen cases (22%, 15/68) BRCA mutations were identified (BRCA1: 11 cases; BRCA2: 4 cases), and four novel mutations were observed. (2) The levels of CEA, CA(199), and HE4 were lower in BRCA mutations compared to that in control group, while no significant differences were found ( P >0.05), but the level of CA(125) was much higher in BRCA mutation group than that in controls ( t =-3.536, P =0.003). Further linear regression analysis found that there was a significant linear correlation between CA(125) and HE4 group ( r =0.494, P <0.01), and the same correlation as CEA and CA(199) group ( r =0.897, P <0.01). (3) Single factor analysis showed that no significant differences were observed in onset age, FIGO stage, distant metastasis, and STM between BRCA(+) and BRCA(-) group ( P >0.05), while significant differences were found in CA(125) and sensitivity to platinum-based chemotherapy between the patients with BRCA mutation and wild type ( P <0.05). The multiple factors analysis showed that the high level of CA(125) was a independent risk factor of BRCA mutations in sporadic HGSOC ( P =0.007). Conclusion: The combination of CA(125) with BRCA have great clinical significance, the mutation of BRCA gene could guild the clinical chemotherapy regiments.

Tumor genome analysis includes germline genome: Are we ready for surprises?

PubMed Central

Catenacci, Daniel VT; Amico, Andrea L; Nielsen, Sarah M; Geynisman, Daniel M; Rambo, Brittany; Carey, George B; Gulden, Cassandra; Fackenthal, Jim; Marsh, Robert D; Kindler, Hedy L; Olopade, Olufunmilayo I

2015-01-01

We sought to describe the spectrum of potential and confirmed germline genomic events incidentally identified during routine medium-throughput somatic tumor DNA sequencing, and to provide a framework for pre- and post-test consent and counseling for patients and families. Targeted tumor-only next-generation sequencing (NGS) had been used to evaluate for possible druggable genomic events obtained from consecutive new patients with metastatic gastroesophageal, hepatobiliary or colorectal cancer seen at the University of Chicago. A panel of medical oncologists, cancer geneticists and genetic counselors retrospectively grouped these patients (N = 111) based on probability of possessing a potentially inherited mutation in a cancer susceptibility gene, both prior to and after incorporating tumor-only NGS results. High-risk patients (determined from NGS results) were contacted and counseled in person by a genetic counselor (N = 21). When possible and indicated, germline genetic testing was offered. Of 8 evaluable high-risk patients, 7 underwent germline testing. Three (37.5%) had confirmed actionable germline mutations (all in the BRCA2 gene). NGS offers promise, but poses significant challenges for oncologists who are ill prepared to handle incidental findings that have clinical implications for at risk family members. In this relatively small cohort of patients undergoing tumor genomic testing for gastrointestinal malignancies, we incidentally identified 3 BRCA2 mutations carriers. This report underscores the need for oncologists to develop a framework for pre- and post-test communication of risks to patients undergoing routine tumor-only sequencing. What's new? High-throughput, ‘next-generation sequencing’ (NGS) allows millions of DNA strands to be sequenced in parallel. NGS is increasingly used to test tumors for mutations that may guide therapy. Sometimes, however, this testing can reveal mutations that are known to be inherited, which means that family members are also at increased risk for cancer. How should this information be presented? This article underscores the need for oncologists to develop a framework for pre- and post-test communication and counseling regarding risk for patients undergoing tumor-only sequencing. PMID:25123297

Genetic Diagnosis before Surgery has an Impact on Surgical Decision in BRCA Mutation Carriers with Breast Cancer.

PubMed

Park, Sungmin; Lee, Jeong Eon; Ryu, Jai Min; Kim, Issac; Bae, Soo Youn; Lee, Se Kyung; Yu, Jonghan; Kim, Seok Won; Nam, Seok Jin

2018-05-01

The first aim of our study was to evaluate surgical decision-making by BRCA mutation carriers with breast cancer based on the timing of knowledge of their BRCA mutation status. The second aim was to evaluate breast cancer outcome following surgical treatment. This was a retrospective study of 164 patients diagnosed with invasive breast cancer, tested for BRCA mutation, and treated with primary surgery between 2004 and 2015 at Samsung Medical Center in Seoul, Korea. We reviewed types of surgery and timing of the BRCA test result. We compared surgical decision- making of BRCA carriers with breast cancer based on the timing of knowledge of their BRCA mutation status. Only 15 (9.1%) patients knew their BRCA test results before their surgery, and 149 (90.9%) knew the results after surgery. In patients with unilateral cancer, there was a significant difference between groups whose BRCA mutation status known before surgery and groups whose BRCA status unknown before surgery regarding the choice of surgery (p = 0.017). No significant difference was observed across surgery types of risk of ipsilateral breast tumor recurrence (p = 0.765) and contralateral breast cancer (p = 0.69). Genetic diagnosis before surgery has an impact on surgical decision choosing unilateral mastectomy or bilateral mastectomy in BRCA mutation carriers with breast cancer. Knowledge about BRCA mutation status after initial surgery led to additional surgeries for patients with BCS. Thus, providing genetic counseling and genetic testing before surgical choice and developing treatment strategies for patients with a high risk of breast cancer are important.

Rapid and accurate detection of KRAS mutations in colorectal cancers using the isothermal-based optical sensor for companion diagnostics

PubMed Central

Koo, Bonhan; Lee, Tae Yoon; Lee, Jeong Hoon; Shin, Yong; Lim, Seok-Byung

2017-01-01

Although KRAS mutational status testing is becoming a companion diagnostic tool for managing patients with colorectal cancer (CRC), there are still several difficulties when analyzing KRAS mutations using the existing assays, particularly with regard to low sensitivity, its time-consuming, and the need for large instruments. We developed a rapid, sensitive, and specific mutation detection assay based on the bio-photonic sensor termed ISAD (isothermal solid-phase amplification/detection), and used it to analyze KRAS gene mutations in human clinical samples. To validate the ISAD-KRAS assay for use in clinical diagnostics, we examined for hotspot KRAS mutations (codon 12 and codon 13) in 70 CRC specimens using PCR and direct sequencing methods. In a serial dilution study, ISAD-KRAS could detect mutations in a sample containing only 1% of the mutant allele in a mixture of wild-type DNA, whereas both PCR and direct sequencing methods could detect mutations in a sample containing approximately 30% of mutant cells. The results of the ISAD-KRAS assay from 70 clinical samples matched those from PCR and direct sequencing, except in 5 cases, wherein ISAD-KRAS could detect mutations that were not detected by PCR and direct sequencing. We also found that the sensitivity and specificity of ISAD-KRAS were 100% within 30 min. The ISAD-KRAS assay provides a rapid, highly sensitive, and label-free method for KRAS mutation testing, and can serve as a robust and near patient testing approach for the rapid detection of patients most likely to respond to anti-EGFR drugs. PMID:29137388

Significance of KRAS, NRAS, BRAF and PIK3CA mutations in metastatic colorectal cancer patients receiving Bevacizumab: a single institution experience

PubMed Central

Baltruškevičienė, Edita; Mickys, Ugnius; Žvirblis, Tadas; Stulpinas, Rokas; Pipirienė Želvienė, Teresė; Aleknavičius, Eduardas

2016-01-01

Background. KRAS mutation is an important predictive and prognostic factor for patients receiving anti-EGFR therapy. An expanded KRAS, NRAS, BRAF, PIK3CA mutation analysis provides additional prognostic information, but its role in predicting bevacizumab efficacy is unclear. The aim of our study was to evaluate the incidence of KRAS, NRAS, BRAF and PIK3CA mutations in metastatic colorectal cancer patients receiving first line oxaliplatin based chemotherapy with or without bevacizumab and to evaluate their prognostic and predictive significance. Methods. 55 patients with the first-time diagnosed CRC receiving FOLFOX ± bevacizumab were involved in the study. Tumour blocks were tested for KRAS mutations in exons 2, 3 and 4, NRAS mutations in exons 2, 3 and 4, BRAF mutation in exon 15 and PIK3CA mutations in exons 9 and 20. The association between mutations and clinico-pathological factors, treatment outcomes and survival was analyzed. Results. KRAS mutations were detected in 67.3% of the patients, BRAF in 1.8%, PIK3CA in 5.5% and there were no NRAS mutations. A significant association between the high CA 19–9 level and KRAS mutation was detected (mean CA 19–9 levels were 276 and 87 kIU/l, respectively, p = 0.019). There was a significantly higher response rate in the KRAS, NRAS, BRAF and PIK3CA wild type cohort receiving bevacizumab compared to any gene mutant type (100 and 60%, respectively, p = 0.030). The univariate Cox regression analysis did not confirm KRAS and other tested mutations as prognostic factors for PFS or OS. Conclusions. Our study revealed higher KRAS and lower NRAS, BRAF and PIK3CA mutation rates in the Lithuanian population than those reported in the literature. KRAS mutation was associated with the high CA 19–9 level and mucinous histology type, but did not show any predictive or prognostic significance. The expanded KRAS, NRAS, BRAF and PIK3CA mutation analysis provided additional significant predictive information. PMID:28356789

High resolution melting analysis for epidermal growth factor receptor mutations in formalin-fixed paraffin-embedded tissue and plasma free DNA from non-small cell lung cancer patients.

PubMed

Jing, Chang-Wen; Wang, Zhuo; Cao, Hai-Xia; Ma, Rong; Wu, Jian-Zhong

2014-01-01

The aim of the research was to explore a cost effective, fast, easy to perform, and sensitive method for epidermal growth factor receptor (EGFR) mutation testing. High resolution melting analysis (HRM) was introduced to evaluate the efficacy of the analysis for dectecting EGFR mutations in exons 18 to 21 using formalin-fixed paraffin-embedded (FFPE) tissues and plasma free DNA from 120 patients. The total EGFR mutation rate was 37.5% (45/120) detected by direct sequencing. There were 48 mutations in 120 FFPE tissues assessed by HRM. For plasma free DNA, the EGFR mutation rate was 25.8% (31/120). The sensitivity of HRM assays in FFPE samples was 100% by HRM. There was a low false-positive mutation rate but a high false-negative rate in plasma free DNA detected by HRM. Our results show that HRM analysis has the advantage of small tumor sample need. HRM applied with plasma free DNA showed a high false-negative rate but a low false-positive rate. Further research into appropriate methods and analysis needs to be performed before HRM for plasma free DNA could be accepted as an option in diagnostic or screening settings.

Detection of KatG/inhA Mutation to Guide Isoniazid and Ethionamide Use for Drug-resistant Tuberculosis

PubMed Central

Namburete, Evangelina

2017-01-01

Abstract Background Both Mozambique and Brazil are countries with a high burden of tuberculosis. Isoniazid (INH) is one of the cornerstones of tuberculosis treatment and, depending on the mutated gene (katG or inhA), the organism may be susceptible to high doses of INH (inhA mutation) or to ethionamide (-Eth-KatG mutation). Methods To analyze isoniazid genotypic resistance profile in Mycobacterium tuberculosis to guide decision making about management of resistant tuberculosis. Descriptive study of 311 M. tuberculosis isolated from Ribeirão Preto, Brazil (2011–2014) and 155 isolates from Beira, Mozambique (2014–2015). Drug resistance patterns and the specific genes mutations were determined using Genotype MTBDRplus (Hain Lifescience GmbH, Germany). Results Mutations in katG gene were detected in 13/22 (59%) of Brazilian and in 32/38 (84.2%) of Mozambican isolates. Unique inhA mutations were observed in 8/22 (36%) isolates in Brazil and 4/38 (10.5%) in Mozambique. Both katG and inhA mutations where detected in 1/22 (5%) and 2/38(5.2%), respectively. katG mutations were more frequent among INH previously treated patients. Conclusion There is a geographical variation of INH mutations and the new molecular tests can be used to guide and accelerate decision making towards the use of ETH or high doses of INH based on detected mutations. Disclosures All authors: No reported disclosures.

Mutational analysis using Sanger and next generation sequencing in sporadic spindle cell hemangiomas: A study of 19 cases.

PubMed

Ten Broek, Roel W; Bekers, Elise M; de Leng, Wendy W J; Strengman, Eric; Tops, Bastiaan B J; Kutzner, Heinz; Leeuwis, Jan Willem; van Gorp, Joost M; Creytens, David H; Mentzel, Thomas; van Diest, Paul J; Eijkelenboom, Astrid; Flucke, Uta

2017-12-01

Spindle cell hemangioma (SCH) is a distinct vascular soft-tissue lesion characterized by cavernous blood vessels and a spindle cell component mainly occurring in the distal extremities of young adults. The majority of cases harbor heterozygous mutations in IDH1/2 sporadically or rarely in association with Maffucci syndrome. However, based on mosaicism and accordingly a low percentage of lesional cells harboring a mutant allele, detection can be challenging. We tested 19 sporadic SCHs by Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), conventional next generation sequencing (NGS), and NGS using a single molecule molecular inversion probes (smMIP)-based library preparation to compare their diagnostic value. Out of 10 cases tested by Sanger sequencing and 2 analyzed using MLPA, 4 and 1, respectively, revealed a mutation in IDH1 (p.R132C). The 7 remaining negative cases and additional 6 cases were investigated using smMIP/NGS, showing hot spot mutations in IDH1 (p.R132C) (8 cases) and IDH2 (3 cases; twice p.R172S and once p.R172G, respectively). One case was negative. Owing to insufficient DNA quality and insufficient coverage, 2 cases were excluded. In total, in 16 out of 17 cases successfully tested, an IDH1/2 mutation was found. Given that IDH1/2 mutations were absent in 161 other vascular lesions tested by smMIP/NGS, the mutation can be considered as highly specific for SCH. © 2017 Wiley Periodicals, Inc.

Multi-institutional oncogenic driver mutation analysis in lung adenocarcinoma: The Lung Cancer Mutation Consortium experience

PubMed Central

Dias-Santagata, Dora; Wistuba, Ignacio I.; Chen, Heidi; Fujimoto, Junya; Kugler, Kelly; Franklin, Wilbur A.; Iafrate, A. John; Ladanyi, Marc; Kris, Mark G.; Johnson, Bruce E.; Bunn, Paul A.; Minna, John D.; Kwiatkowski, David J.

2015-01-01

Introduction Molecular genetic analyses of lung adenocarcinoma have recently become standard of care for treatment selection. The Lung Cancer Mutation Consortium was formed to enable collaborative multi-institutional analyses of 10 potential oncogenic driver mutations. Technical aspects of testing, and clinicopathologic correlations are presented. Methods Mutation testing in at least one of 8 genes (EGFR, KRAS, ERBB2, AKT1, BRAF, MEK1, NRAS, PIK3CA) using SNaPshot, mass spectrometry, Sanger sequencing +/− PNA and/or sizing assays, along with ALK and/or MET FISH were performed in 6 labs on 1007 patients from 14 institutions. Results 1007 specimens had mutation analysis performed, and 733 specimens had all 10 genes analyzed. Mutation identification rates did not vary by analytic method. Biopsy and cytology specimens were inadequate for testing in 26% and 35% of cases compared to 5% of surgical specimens. Among the 1007 cases with mutation analysis performed, EGFR, KRAS, ALK, and ERBB2 alterations were detected in 22, 25, 8.5, and 2.4% of cases, respectively. EGFR mutations were highly associated with female sex, Asian race, and never smoking status; and less strongly associated with stage IV disease, presence of bone metastases, and absence of adrenal metastases. ALK rearrangements were strongly associated with never smoking status, and more weakly associated with presence of liver metastases. ERBB2 mutations were strongly associated with Asian race and never smoking status. Two mutations were seen in 2.7% of samples, all but one of which involved one or more of PIK3CA, ALK or MET. Conclusion Multi-institutional molecular analysis across multiple platforms, sample types, and institutions can yield consistent results and novel clinicopathological observations. PMID:25738220

Epidermal growth factor receptor mutations in lung adenocarcinoma in Malaysian patients.

PubMed

Liam, Chong-Kin; Wahid, Mohamed Ibrahim A; Rajadurai, Pathmanathan; Cheah, Yoke-Kqueen; Ng, Tiffany Shi-Yeen

2013-06-01

Despite available data from other Asian countries, the prevalence of epidermal growth factor receptor (EGFR) mutations among lung adenocarcinoma patients has not been reported in Malaysia. This study sought to determine the frequency of EGFR mutations among multiethnic Malaysian patients diagnosed with lung adenocarcinoma. Demographic and clinical information of patients whose lung adenocarcinoma biopsy specimens were submitted for EGFR mutation testing at Sime Darby Medical Center from 2009 to 2011 were analyzed. EGFR mutations at exons 18, 19, 20, and 21 were detected either through bidirectional sequencing or real-time polymerase chain reaction. Among 812 patients in the study, 49% were female, 63.7% were ethnic Chinese, 29.4% Malay, 4.8% Indian, and 2.1% other ethnic groups. Mutations were present in the tumors of 321 patients (39.5%), with mutations at exons 19 (23.5%) and 21 (14.9%) being the most common. Mutations were significantly more frequent among women than in men (52.5% versus 27.8%, p < 0.001). Although mutations were more common among Chinese (40.8%) compared with Malay (37.2%) or Indian (33.3%) patients, the difference was not statistically significant (p = 0.591). Of 211 patients with smoking history records, never-smokers had a higher mutation rate compared with ever-smokers (54.8% versus 20.7%, p < 0.001). EGFR mutations were present in 39.5% of patients. Mutations were more common in women and never-smokers with no differences in mutation frequency between different ethnicities. Because of the high mutation rates, reflex testing for EGFR mutation should be a routine practice for advanced lung adenocarcinoma patients in Malaysia.

High-throughput discovery of mutations in tef semi-dwarfing genes by next-generation sequencing analysis.

PubMed

Zhu, Qihui; Smith, Shavannor M; Ayele, Mulu; Yang, Lixing; Jogi, Ansuya; Chaluvadi, Srinivasa R; Bennetzen, Jeffrey L

2012-11-01

Tef (Eragrostis tef) is a major cereal crop in Ethiopia. Lodging is the primary constraint to increasing productivity in this allotetraploid species, accounting for losses of ∼15-45% in yield each year. As a first step toward identifying semi-dwarf varieties that might have improved lodging resistance, an ∼6× fosmid library was constructed and used to identify both homeologues of the dw3 semi-dwarfing gene of Sorghum bicolor. An EMS mutagenized population, consisting of ∼21,210 tef plants, was planted and leaf materials were collected into 23 superpools. Two dwarfing candidate genes, homeologues of dw3 of sorghum and rht1 of wheat, were sequenced directly from each superpool with 454 technology, and 120 candidate mutations were identified. Out of 10 candidates tested, six independent mutations were validated by Sanger sequencing, including two predicted detrimental mutations in both dw3 homeologues with a potential to improve lodging resistance in tef through further breeding. This study demonstrates that high-throughput sequencing can identify potentially valuable mutations in under-studied plant species like tef and has provided mutant lines that can now be combined and tested in breeding programs for improved lodging resistance.

The high frequency of GJB2 gene mutation c.313_326del14 suggests its possible origin in ancestors of Lithuanian population.

PubMed

Mikstiene, Violeta; Jakaitiene, Audrone; Byckova, Jekaterina; Gradauskiene, Egle; Preiksaitiene, Egle; Burnyte, Birute; Tumiene, Birute; Matuleviciene, Ausra; Ambrozaityte, Laima; Uktveryte, Ingrida; Domarkiene, Ingrida; Rancelis, Tautvydas; Cimbalistiene, Loreta; Lesinskas, Eugenijus; Kucinskas, Vaidutis; Utkus, Algirdas

2016-02-19

Congenital hearing loss (CHL) is diagnosed in 1 - 2 newborns in 1000, genetic factors contribute to two thirds of CHL cases in industrialised countries. Mutations of the GJB2 gene located in the DFNB1 locus (13q11-12) are a major cause of CHL worldwide. The aim of this cross-sectional study was to assess the contribution of the DFNB1 locus containing the GJB2 and GJB6 genes in the development of early onset hearing loss in the affected group of participants, to determine the population-specific mutational profile and DFNB1-related HL burden in Lithuanian population. Clinical data were obtained from a collection of 158 affected participants (146 unrelated probands) with early onset non-syndromic HL. GJB2 and GJB6 gene sequencing and GJB6 gene deletion testing were performed. The data of GJB2 and GJB6 gene sequencing in 98 participants in group of self-reported healthy Lithuanian inhabitants were analysed. Statistic summary, homogeneity tests, and logistic regression analysis were used for the assessment of genotype-phenotype correlation. Our findings show 57.5% of affected participants with two pathogenic GJB2 gene mutations identified. The most prevalent GJB2 mutations were c.35delG, p. (Gly12Valfs*2) (rs80338939) and c.313_326del14, p. (Lys105Glyfs*5) (rs111033253) with allele frequencies 64.7% and 28.3% respectively. GJB6 gene mutations were not identified in the affected group of participants. The statistical analysis revealed significant differences between GJB2(-) and GJB2(+) groups in disease severity (p = 0.001), and family history (p = 0.01). The probability of identification of GJB2 mutations in patients with various HL characteristics was estimated. The carrier rate of GJB2 gene mutations - 7.1% (~1 in 14) was identified in the group of healthy participants and a high frequency of GJB2-related hearing loss was estimated in our population. The results show a very high proportion of GJB2-positive individuals in the research group affected with sensorineural HL. The allele frequency of c.35delG mutation (64.7 %) is consistent with many previously published studies in groups of affected individuals of Caucasian populations. The high frequency of the c.313_326del14 (28.3 % of pathogenic alleles) mutation in affected group of participants was an unexpected finding in our study suggesting not only a high frequency of carriers of this mutation in our population but also its possible origin in Lithuanian ancestors. The high frequency of carriers of the c.313_326del14 mutation in the entire Lithuanian population is supported by it being identified twice in the ethnic Lithuanian group of healthy participants (a frequency 2.0 % of carriers in the study group). Analysis of the allele frequency of GJB2 gene mutations revealed a high proportion of c. 313_326del14 (rs111033253) mutations in the GJB2-positive group suggesting its possible origin in Lithuanian forebears. The high frequency of carriers of GJB2 gene mutations in the group of healthy participants corresponds to the substantial frequency of GJB2-associated HL in Lithuania. The observations of the study indicate the significant contribution of GJB2 gene mutations to the pathogenesis of the disorder in the Lithuanian population and will contribute to introducing principles to predict the characteristics of the disease in patients.

BRAF V600 mutation detection in melanoma: a comparison of two laboratory testing methods.

PubMed

O'Brien, Odharnaith; Lyons, Tomas; Murphy, Sandra; Feeley, Linda; Power, Derek; Heffron, Cynthia C B B

2017-11-01

The assessment of B-raf proto-oncogene, serine/threonine kinase ( BRAF ) gene status is now standard practice in patients diagnosed with metastatic melanoma with its presence predicting a clinical response to treatment with BRAF inhibitors. The gold standard in determining BRAF status is currently by DNA-based methods. More recently, a BRAF V600E antibody has been developed. We aim to investigate whether immunohistochemical detection of BRAF mutation is a suitable alternative to molecular testing by polymerase chain reaction (PCR). We assessed the incidence of BRAF mutation in our cohort of 132 patients, as determined by PCR, as well as examining clinical and histopathological features. We investigated the sensitivity and specificity of the anti-BRAF V600E VE1 clone antibody in detecting the presence of the BRAF V600E mutation in 122 cases deemed suitable for testing. The incidence of BRAF mutation in our cohort was 28.8% (38/132). Patients with the BRAF mutation were found to be significantly younger at age of diagnosis. BRAF-mutated melanomas tended to be thinner and more mitotically active. The antibody showed a sensitivity of 86.1% with a specificity of 96.9%. The positive predictive value was 96.9%; the negative predictive value was 94.4%. The concordance rate between PCR and immunohistochemical BRAF status was 95.1% (116/122). The rate of BRAF mutation in our cohort (28.8%) was lower than international published rates of 40%-60%. This may reflect ethnic or geographic differences within population cohorts. The high concordance rate of PCR and immunohistochemical methods in determining BRAF status suggests that immunohistochemistry is potentially a viable, cost-effective alternative to PCR testing and suitable as a screening test for the BRAF mutation. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Clinical utility of TERT promoter mutations and ALK rearrangement in thyroid cancer patients with a high prevalence of the BRAF V600E mutation.

PubMed

Bae, Ja Seong; Kim, Yourha; Jeon, Sora; Kim, Se Hee; Kim, Tae Jung; Lee, Sohee; Kim, Min-Hee; Lim, Dong Jun; Lee, Youn Soo; Jung, Chan Kwon

2016-02-09

Mutations in the TERT promoter, ALK rearrangement, and the BRAF V600E mutation are associated with aggressive clinicopathologic features in thyroid cancers. However, little is known about the impact of TERT promoter mutations and ALK rearrangement in thyroid cancer patients with a high prevalence of BRAF mutations. We performed Sanger sequencing to detect BRAF V600E and TERT promoter mutations and both immunohistochemistry and fluorescence in situ hybridization to identify ALK rearrangement on 243 thyroid cancers. TERT promoter mutations were not present in 192 well-differentiated thyroid carcinomas (WDTC) without distant metastasis or in 9 medullary carcinomas. However, the mutations did occur in 40 % (12/30) of WDTC with distant metastasis, 29 % (2/7) of poorly differentiated carcinomas and 60 % (3/5) of anaplastic carcinomas. ALK rearrangement was not present in all thyroid cancers. The BRAF V600E mutation was more frequently found in WDTC without distant metastasis than in WDTC with distant metastasis (p = 0.007). In the cohort of WDTC with distant metastasis, patients with wild-type BRAF and TERT promoter had a significantly higher response rate after radioiodine therapy (p = 0.024), whereas the BRAF V600E mutation was significantly correlated with progressive disease (p = 0.025). The TERT promoter mutation is an independent predictor for distant metastasis of WDTC, but ALK testing is not useful for clinical decision-making in Korean patients with a high prevalence of the BRAF V600E mutation. Radioiodine therapy for distant metastasis of WDTC is most effective in patients without BRAF V600E and TERT promoter mutations.

Uptake of genetic testing and long-term tumor surveillance in von Hippel-Lindau disease

PubMed Central

2010-01-01

Background von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germline mutations in the VHL gene. Patients have significant morbidity and mortality secondary to vascular tumors. Disease management is centered on tumor surveillance that allows early detection and treatment. Presymptomatic genetic testing is therefore recommended, including in at-risk children. Methods We tested 17 families (n = 109 individuals) for VHL mutations including 43 children under the age of 18. Personalized genetic counseling was provided pre and post-test and the individuals undergoing presymptomatic testing filled out questionnaires gathering socio-demographic, psychological and psychiatric data. Mutation analysis was performed by direct sequencing of the VHL gene. Mutation-carriers were screened for VHL disease-related tumors and were offered follow-up annual examinations. Results Mutations were identified in 36 patients, 17 of whom were asymptomatic. In the initial screening, we identified at least one tumor in five of 17 previously asymptomatic individuals. At the end of five years, only 38.9% of the mutation-carriers continued participating in our tumor surveillance program. During this time, 14 mutation carriers developed a total of 32 new tumors, three of whom died of complications. Gender, education, income, marital status and religiosity were not found to be associated with adherence to the surveillance protocol. Follow-up adherence was also independent of pre-test depression, severity of disease, or number of affected family members. The only statistically significant predictor of adherence was being symptomatic at the time of testing (OR = 5; 95% CI 1.2 - 20.3; p = 0.02). Pre-test anxiety was more commonly observed in patients that discontinued follow-up (64.7% vs. 35.3%; p = 0.01). Conclusions The high initial uptake rate of genetic testing for VHL disease, including in minors, allowed the discontinuation of unnecessary screening procedures in non mutation-carriers. However, mutation-carriers showed poor adherence to long-term tumor surveillance. Therefore, many of them did not obtain the full benefit of early detection and treatment, which is central to the reduction of morbidity and mortality in VHL disease. Studies designed to improve adherence to vigilance protocols will be necessary to improve treatment and quality of life in patients with hereditary cancer syndromes. PMID:20064270

Filaggrin haploinsufficiency is highly penetrant and is associated with increased severity of eczema: further delineation of the skin phenotype in a prospective epidemiological study of 792 school children

PubMed Central

Brown, SJ; Relton, CL; Liao, H; Zhao, Y; Sandilands, A; McLean, WHI; Cordell, HJ; Reynolds, NJ

2009-01-01

Background Null mutations within the filaggrin gene (FLG) cause ichthyosis vulgaris and are associated with atopic eczema. However, the dermatological features of filaggrin haploinsufficiency have not been clearly defined. Objectives This study investigated the genotype–phenotype association between detailed skin phenotype and FLG genotype data in a population-based cohort of children. Methods Children (n= 792) aged 7–9 years were examined by a dermatologist. Features of ichthyosis vulgaris, atopic eczema and xerosis were recorded and eczema severity graded using the Three Item Severity score. Each child was genotyped for the six most prevalent FLG null mutations (R501X, 2282del4, R2447X, S3247X, 3702delG, 3673delC). Fisher’s exact test was used to compare genotype frequencies in phenotype groups; logistic regression analysis was used to estimate odds ratios and penetrance of the FLG null genotype and a permutation test performed to investigate eczema severity in different genotype groups. Results Ten children in this cohort had ichthyosis vulgaris, of whom five had mild–moderate eczema. The penetrance of FLG null mutations with respect to flexural eczema was 55·6% in individuals with two mutations, 16·3% in individuals with one mutation and 14·2% in wild-type individuals. Summating skin features known to be associated with FLG null mutations (ichthyosis, keratosis pilaris, palmar hyperlinearity and flexural eczema) showed a penetrance of 100% in children with two FLG mutations, 87·8% in children with one FLG mutation and 46·5% in wild-type individuals (P< 0·0001, Fisher exact test). FLG null mutations were associated with more severe eczema (P= 0·0042) but the mean difference was only 1–2 points in severity score. Three distinct patterns of palmar hyperlinearity were observed and these are reported for the first time. Conclusions Filaggrin haploinsufficiency appears to be highly penetrant when all relevant skin features are included in the analysis. FLG null mutations are associated with more severe eczema, but the effect size is small in a population setting. PMID:19681860

Mutation Spectra of Common Cancer-Associated Genes in Different Phenotypes of Colorectal Carcinoma Without Distant Metastasis.

PubMed

Chang, Shih-Ching; Lin, Pei-Ching; Lin, Jen-Kou; Lin, Chien-Hsing; Yang, Shung-Haur; Liang, Wen-Yi; Chen, Wei-Shone; Jiang, Jeng-Kai

2016-03-01

Colorectal cancer (CRC) is a heterogeneous disease caused by genetic and epigenetic alterations. This study aimed to describe the mutation frequency of 12 genes in different CRC phenotypes. Patients who underwent surgery at the Taipei Veterans General Hospital during 2000-2010 for CRC (n = 1249) were enrolled. The endpoint was overall survival. The prognostic value was determined with the log-rank test and Cox regression analysis. We found 1836 mutations of 12 genes in 997 (79.8%) tumors. Mutations were most frequently in KRAS (485, 38.8%), TP53 (373, 29.9%), APC (363, 29.0%), and PIK3CA (179, 14.3%); 137 (11.0%) cancers had high microsatellite instability (MSI). Women had significantly higher high MSI (14.3%) and BRAF mutation (6.3%) frequencies. The abnormal MSI (21.7%) and KRAS (44.6%), BRAF (8.6%), PIK3CA (19.4%), AKT1 (2.2%), and TGF - βR (9.6%) mutation frequencies were significantly higher in proximal colon cancer. The high MSI (35.6%) and BRAF (20.3%), TGF - βR (18.6%), PTEN (5.1%), and AKT1 (3.4%) mutation frequencies were significantly higher in 59 (4.7%) poorly differentiated tumors. The high MSI (21.3%) and KRAS (51.9%), BRAF (8.3%), PIK3CA (25.0%), AKT1 (4.6%), and SMAD4 (8.3%) mutation frequencies were significantly higher in 108 mucinous tumors. TNM stage, lymphovascular invasion, and mucinous histology were significantly associated with patient outcomes in univariate and multivariate analyses. Only NRAS mutation (hazard ratio 1.59, 95% confidence interval 1.06-2.38) affected patient survival. Mutational spectra differ significantly between CRC subtypes, implying diverse carcinogenetic pathways. The NRAS mutation is important, despite its low frequency.

Ovarian cancer patients at high risk of BRCA mutation: the constitutional genetic characterization does not change prognosis.

PubMed

Sabatier, Renaud; Lavit, Elise; Moretta, Jessica; Lambaudie, Eric; Noguchi, Tetsuro; Eisinger, François; Cherau, Elisabeth; Provansal, Magali; Livon, Doriane; Rabayrol, Laetitia; Popovici, Cornel; Charaffe-Jauffret, Emmanuelle; Sobol, Hagay; Viens, Patrice

2016-10-01

Ovarian neoplasms secondary to germline BRCA mutations had been described to have a more favourable survival. There is only few data concerning the prognosis of non mutated patients presenting clinical features evocative of BRCA alterations. We retrospectively collected data from patients treated in our institution for an invasive ovarian carcinoma between 1995 and 2011. Patients considered at high risk of BRCA mutation were tested for BRCA1/2 germline mutations. We described clinical, pathological and therapeutic features and compared prognosis of BRCA mutation carriers and non-mutated patients. Out of 617 ovarian cancer patients, we identified 104 patients who were considered at high risk of mutation. The 33 mutated patients were more likely to present a personal (33 vs. 10 %, p = 0.003) or a family (42 vs. 24 %, p = 0.06) history of breast/ovarian cancers. BRCA1/2 mutation carriers and wild type patients displayed similar prognosis: median progression-free survival (PFS) of 20.9 versus 37.7 months (p = 0.21); median overall survival (OS) of 151.2 versus 122.5 months (p = 0.52). Personal history of breast cancer increased both PFS [HR = 0.45 (95CI 0.25-0.81)] and OS [HR = 0.35 (95CI 0.16-0.75)]. In multivariate analysis, this parameter was an independent prognostic feature, whereas the identification of a BRCA1/2 mutation was not. In our cohort, all patients at high risk of BRCA mutation share a similar prognosis, whatever is their germline mutation status. Prognosis seems to be more influenced by clinical history than by germline mutations identification. If it is confirmed in larger and independent series, this result suggests that the hypothesis of other BRCA pathway alterations (BRCAness phenotype) deserves to be deeply explored.

With Home Testing, Consumers Take Charge of Their Health

MedlinePlus

... Mutation Mycophenolic Acid Mycoplasma Myoglobin Nicotine and Cotinine Non-High Density Lipoprotein Cholesterol Opioid Testing Osmolality Ova ... at the bedside," Nichols says, "but not necessarily equivalent to the quality of testing performed in a ...

Weaver syndrome and EZH2 mutations: Clarifying the clinical phenotype.

PubMed

Tatton-Brown, Katrina; Murray, Anne; Hanks, Sandra; Douglas, Jenny; Armstrong, Ruth; Banka, Siddharth; Bird, Lynne M; Clericuzio, Carol L; Cormier-Daire, Valerie; Cushing, Tom; Flinter, Frances; Jacquemont, Marie-Line; Joss, Shelagh; Kinning, Esther; Lynch, Sally Ann; Magee, Alex; McConnell, Vivienne; Medeira, Ana; Ozono, Keiichi; Patton, Michael; Rankin, Julia; Shears, Debbie; Simon, Marleen; Splitt, Miranda; Strenger, Volker; Stuurman, Kyra; Taylor, Clare; Titheradge, Hannah; Van Maldergem, Lionel; Temple, I Karen; Cole, Trevor; Seal, Sheila; Rahman, Nazneen

2013-12-01

Weaver syndrome, first described in 1974, is characterized by tall stature, a typical facial appearance, and variable intellectual disability. In 2011, mutations in the histone methyltransferase, EZH2, were shown to cause Weaver syndrome. To date, we have identified 48 individuals with EZH2 mutations. The mutations were primarily missense mutations occurring throughout the gene, with some clustering in the SET domain (12/48). Truncating mutations were uncommon (4/48) and only identified in the final exon, after the SET domain. Through analyses of clinical data and facial photographs of EZH2 mutation-positive individuals, we have shown that the facial features can be subtle and the clinical diagnosis of Weaver syndrome is thus challenging, especially in older individuals. However, tall stature is very common, reported in >90% of affected individuals. Intellectual disability is also common, present in ~80%, but is highly variable and frequently mild. Additional clinical features which may help in stratifying individuals to EZH2 mutation testing include camptodactyly, soft, doughy skin, umbilical hernia, and a low, hoarse cry. Considerable phenotypic overlap between Sotos and Weaver syndromes is also evident. The identification of an EZH2 mutation can therefore provide an objective means of confirming a subtle presentation of Weaver syndrome and/or distinguishing Weaver and Sotos syndromes. As mutation testing becomes increasingly accessible and larger numbers of EZH2 mutation-positive individuals are identified, knowledge of the clinical spectrum and prognostic implications of EZH2 mutations should improve. © 2013 Wiley Periodicals, Inc.

Four case reports of Chinese cystic fibrosis patients and literature review.

PubMed

Xu, Juan; Yin, Yong; Zhang, Lei; Zhang, Jing; Yuan, Shuhua; Zhang, Hao

2017-08-01

Cystic fibrosis (CF) is an extremely rare disease in Asians. Here, we report four Chinese children with CF and review the literature about Chinese CF patients. The cystic fibrosis transmembrane conductance regulator (CFTR) gene testing was performed on four suspected patients for CF screening. We also reviewed the literature about Chinese CF patients from 1970s. The clinical data of all these CF patients were summarized. We diagnosed four CF patients who had mutations in the CFTR gene. We identified six different mutations in the four patients. The c.1766+5G>T, c.595C>T, c.2909G>A, and c.4056G>C had been reported already. The two splicing mutations of c.579+1_579+2insACAT and c.1117-1G>C were novel mutations. There have been 46 Chinese CF patients reported in literature from 1974 up to present (2016.12). The clinical manifestations of CF involved several systems. The most common symptom was recurrent pulmonary infections. Thirty-three different mutations were identified; c.1766 + 5G>T was the most common mutation among Chinese CF patients. Only one of these mutations (R553X) was in the Caucasian CF screening panel. The spectrum of CFTR mutations in Chinese was highly different from that of Caucasian. There was a high risk of misdiagnosis or delayed diagnosis of CF even in suspected cases in China. It is necessary to educate Chinese clinicians about the signs, symptoms, and diagnosis of cystic fibrosis and promote the implementation of the sweat chloride test. © 2017 Wiley Periodicals, Inc.

[A report of atypical hypomyelinating leukodystrophy with atrophy of the basal ganglia and cerebellum caused by a de novo mutation in tubulin beta 4A (TUBB4A) gene and literature review].

PubMed

Du, Y; Li, C; Guo, J; Guo, P; Li, Z Y; Zhang, W

2017-06-01

Objective: To explore the clinical symptoms and neuroimaging features of a patient with atypical hypomyelinating leukodystrophy with atrophy of the basal ganglia and cerebellum (H-ABC) caused by a novel TUBB4A mutation. Methods: We analyzed the clinical data, imaging features and the result of genetic testing of a case diagnosed as atypical H-ABC. Results: The initial symptoms were progressive spasticity, mild cerebellar ataxia and mild cognitive impairment. MRI showed regional blurring of slight high signal on T(2)-weight and FLAIR image in white matter of the bilateral midbrain ventral, internal capsule, posteior horn of lateral ventricle and centrum semiovale, with normal bilateral cerebellar and caudoputamen nucleus. Compared with normal subjects of the same age and gender, hypometabolism was found by (18)F-FDG-PET in brainstem, cerebellar and caudoputamen nucleus in the patient. Genetic testing revealed a de novo pathogenic exome missense heterozygous mutations c. 70G>A in TUBB4A, which was not reported in the human gene mutation database (HGMDpro) and was assessed to be a pathogenic mutation by pathogenic mutation prediction software. Conclusions: The diversity of TUBB4A gene mutations may cause different functional and/or structural impairment in subcortical white matter, cerebellar and caudoputamen nucleus, leading to atypical symptoms and neuroimaging features. Genetic testing for pathogenic mutation in TUBB 4A gene is a key for the diagnosis of H - ABC .

Clinical relevance of KRAS mutation detection in metastatic colorectal cancer treated by Cetuximab plus chemotherapy

PubMed Central

Di Fiore, F; Blanchard, F; Charbonnier, F; Le Pessot, F; Lamy, A; Galais, M P; Bastit, L; Killian, A; Sesboüé, R; Tuech, J J; Queuniet, A M; Paillot, B; Sabourin, J C; Michot, F; Michel, P; Frebourg, T

2007-01-01

The predictive value of KRAS mutation in metastatic colorectal cancer (MCRC) patients treated with cetuximab plus chemotherapy has recently been suggested. In our study, 59 patients with a chemotherapy-refractory MCRC treated with cetuximab plus chemotherapy were included and clinical response was evaluated according to response evaluation criteria in solid tumours (RECIST). Tumours were screened for KRAS mutations using first direct sequencing, then two sensitive methods based on SNaPshot and PCR-ligase chain reaction (LCR) assays. Clinical response was evaluated according to gene mutations using the Fisher exact test. Times to progression (TTP) were calculated using the Kaplan–Meier method and compared with log-rank test. A KRAS mutation was detected in 22 out of 59 tumours and, in six cases, was missed by sequencing analysis but detected using the SNaPshot and PCR-LCR assays. Remarkably, no KRAS mutation was found in the 12 patients with clinical response. KRAS mutation was associated with disease progression (P=0.0005) and TTP was significantly decreased in mutated KRAS patients (3 vs 5.5 months, P=0.015). Our study confirms that KRAS mutation is highly predictive of a non-response to cetuximab plus chemotherapy in MCRC and highlights the need to use sensitive molecular methods, such as SNaPshot or PCR-LCR assays, to ensure an efficient mutation detection. PMID:17375050

High-risk individuals' perceptions of reproductive genetic testing for CDH1 mutations.

PubMed

Hallowell, Nina; Badger, Shirlene; Richardson, Sue; Caldas, Carlos; Hardwick, Richard H; Fitzgerald, Rebecca C; Lawton, Julia

2017-10-01

Reproductive genetic testing- PreNatal Diagnosis (PND) and Preimplantation Genetic Diagnosis (PGD)-for CDH1 mutations associated with Hereditary Diffuse Gastric Cancer (HDGC)is available in the UK. This qualitative interview study examined high-risk individuals' (n = 35) views of CDH1 reproductive genetic testing. Interviewees generally regarded reproductive genetic testing as an acceptable form of HDGC risk management. However, some were concerned that their genetic risks required them to plan reproduction and anticipated difficulties communicating this to reproductive partners. Individuals had a preference for PGD over PND because it avoided the need for a termination of pregnancy. However, those who had not yet had children expressed concerns about having to undergo IVF procedures and worries about their effectiveness and the need for embryo selection in PGD. It is suggested that high-risk individuals are provided with access to reproductive genetic counselling.

Heterogeneity and frequency of BRAF mutations in primary melanoma: Comparison between molecular methods and immunohistochemistry

PubMed Central

Bruno, William; Martinuzzi, Claudia; Andreotti, Virginia; Pastorino, Lorenza; Spagnolo, Francesco; Dalmasso, Bruna; Cabiddu, Francesco; Gualco, Marina; Ballestrero, Alberto; Bianchi-Scarrà, Giovanna; Queirolo, Paola

2017-01-01

Finding the best technique to identify BRAF mutations with a high sensitivity and specificity is mandatory for accurate patient selection for target therapy. BRAF mutation frequency ranges from 40 to 60% depending on melanoma clinical characteristics and detection technique used. Intertumoral heterogeneity could lead to misinterpretation of BRAF mutational status; this is especially important if testing is performed on primary specimens, when metastatic lesions are unavailable. Aim of this study was to identify the best combination of methods for detecting BRAF mutations (among peptide nucleic acid – PNA-clamping real-time PCR, immunohistochemistry and capillary sequencing) and investigate BRAF mutation heterogeneity in a series of 100 primary melanomas and a subset of 25 matched metastatic samples. Overall, we obtained a BRAF mutation frequency of 62%, based on the combination of at least two techniques. Concordance between mutation status in primary and metastatic tumor was good but not complete (67%), when agreement of at least two techniques were considered. Next generation sequencing was used to quantify the threshold of detected mutant alleles in discordant samples. Combining different methods excludes that the observed heterogeneity is technique-based. We propose an algorithm for BRAF mutation testing based on agreement between immunohistochemistry and PNA; a third molecular method could be added in case of discordance of the results. Testing the primary tumor when the metastatic sample is unavailable is a good option if at least two methods of detection are used, however the presence of intertumoral heterogeneity or the occurrence of additional primaries should be carefully considered. PMID:28039443

Gene panel sequencing in familial breast/ovarian cancer patients identifies multiple novel mutations also in genes others than BRCA1/2.

PubMed

Kraus, Cornelia; Hoyer, Juliane; Vasileiou, Georgia; Wunderle, Marius; Lux, Michael P; Fasching, Peter A; Krumbiegel, Mandy; Uebe, Steffen; Reuter, Miriam; Beckmann, Matthias W; Reis, André

2017-01-01

Breast and ovarian cancer (BC/OC) predisposition has been attributed to a number of high- and moderate to low-penetrance susceptibility genes. With the advent of next generation sequencing (NGS) simultaneous testing of these genes has become feasible. In this monocentric study, we report results of panel-based screening of 14 BC/OC susceptibility genes (BRCA1, BRCA2, RAD51C, RAD51D, CHEK2, PALB2, ATM, NBN, CDH1, TP53, MLH1, MSH2, MSH6 and PMS2) in a group of 581 consecutive individuals from a German population with BC and/or OC fulfilling diagnostic criteria for BRCA1 and BRCA2 testing including 179 with a triple-negative tumor. Altogether we identified 106 deleterious mutations in 105 (18%) patients in 10 different genes, including seven different exon deletions. Of these 106 mutations, 16 (15%) were novel and only six were found in BRCA1/2. To further characterize mutations located in or nearby splicing consensus sites we performed RT-PCR analysis which allowed confirmation of pathogenicity in 7 of 9 mutations analyzed. In PALB2, we identified a deleterious variant in six cases. All but one were associated with early onset BC and a positive family history indicating that penetrance for PALB2 mutations is comparable to BRCA2. Overall, extended testing beyond BRCA1/2 identified a deleterious mutation in further 6% of patients. As a downside, 89 variants of uncertain significance were identified highlighting the need for comprehensive variant databases. In conclusion, panel testing yields more accurate information on genetic cancer risk than assessing BRCA1/2 alone and wide-spread testing will help improve penetrance assessment of variants in these risk genes. © 2016 UICC.

Cost-effectiveness of Population Screening for BRCA Mutations in Ashkenazi Jewish Women Compared With Family History–Based Testing

PubMed Central

Manchanda, Ranjit; Legood, Rosa; Burnell, Matthew; McGuire, Alistair; Raikou, Maria; Loggenberg, Kelly; Wardle, Jane; Sanderson, Saskia; Gessler, Sue; Side, Lucy; Balogun, Nyala; Desai, Rakshit; Kumar, Ajith; Dorkins, Huw; Wallis, Yvonne; Chapman, Cyril; Taylor, Rohan; Jacobs, Chris; Tomlinson, Ian; Beller, Uziel; Menon, Usha

2015-01-01

Background: Population-based testing for BRCA1/2 mutations detects the high proportion of carriers not identified by cancer family history (FH)–based testing. We compared the cost-effectiveness of population-based BRCA testing with the standard FH-based approach in Ashkenazi Jewish (AJ) women. Methods: A decision-analytic model was developed to compare lifetime costs and effects amongst AJ women in the UK of BRCA founder-mutation testing amongst: 1) all women in the population age 30 years or older and 2) just those with a strong FH (≥10% mutation risk). The model assumes that BRCA carriers are offered risk-reducing salpingo-oophorectomy and annual MRI/mammography screening or risk-reducing mastectomy. Model probabilities utilize the Genetic Cancer Prediction through Population Screening trial/published literature to estimate total costs, effects in terms of quality-adjusted life-years (QALYs), cancer incidence, incremental cost-effectiveness ratio (ICER), and population impact. Costs are reported at 2010 prices. Costs/outcomes were discounted at 3.5%. We used deterministic/probabilistic sensitivity analysis (PSA) to evaluate model uncertainty. Results: Compared with FH-based testing, population-screening saved 0.090 more life-years and 0.101 more QALYs resulting in 33 days’ gain in life expectancy. Population screening was found to be cost saving with a baseline-discounted ICER of -£2079/QALY. Population-based screening lowered ovarian and breast cancer incidence by 0.34% and 0.62%. Assuming 71% testing uptake, this leads to 276 fewer ovarian and 508 fewer breast cancer cases. Overall, reduction in treatment costs led to a discounted cost savings of £3.7 million. Deterministic sensitivity analysis and 94% of simulations on PSA (threshold £20000) indicated that population screening is cost-effective, compared with current NHS policy. Conclusion: Population-based screening for BRCA mutations is highly cost-effective compared with an FH-based approach in AJ women age 30 years and older. PMID:25435542

Attitudes and compliance of clinical management after genetic testing for hereditary breast and ovarian cancer among high-risk Southern Chinese females with breast cancer history.

PubMed

Kwong, Ava; Chu, Annie Tsz-Wai; Wu, Christine Teen-Sum; Tse, Desiree Man-Sik

2014-09-01

Western studies have shown that the uptake rates of surveillance and prophylaxis may vary among BRCA mutation carriers between ethnicities. The present study is the first to investigate the behavioural impact and subjective attitudes in Southern Chinese high-risk families who had undergone BRCA1 and BRCA2 genetic testing up to 2.5 years post-testing. Individuals who had such genetic testing and have consented to participate in the prospective database of Hong Kong Hereditary Breast Cancer Family Registry were recruited and surveyed by a face-to-face or telephone interview. Sociodemographic information, genetic test results, pre- and post-testing surveillance, medical regimes, and attitudes towards the choice of clinical management were obtained by interviews and retrieval of medical records using this prospective database. 69 females with breast cancer history were recruited into the study. Twenty-nine female carriers (15 BRCA1 mutated gene-carriers and 14 BRCA2 mutated gene-carriers) and 40 non-carriers of a BRCA 1/2 mutations were interviewed. The uptake rate of high risk breast screening i.e. clinical breast examination, mammography, and breast MRI is significantly higher among female carriers (48.3 %) after knowing genetic testing results than before (p < 0.01). A strong significant relationship between any increase or decrease of ovarian ultrasound screening (OS) and genetic status is found (p < .001), with more females did OS and with a higher frequency after knowing genetic testing results among both carriers (22.7 % → 86.4 %) and non-carriers (37.5 % → 50.0 %). Among carriers, very few opted for prophylactic surgeries. The present cohort might see prophylaxis as last resort and would use traditional Chinese medicine in cancer risk management.

Value of transmission electron microscopy for primary ciliary dyskinesia diagnosis in the era of molecular medicine: Genetic defects with normal and non-diagnostic ciliary ultrastructure.

PubMed

Shapiro, Adam J; Leigh, Margaret W

2017-01-01

Primary ciliary dyskinesia (PCD) is a genetic disorder causing chronic oto-sino-pulmonary disease. No single diagnostic test will detect all PCD cases. Transmission electron microscopy (TEM) of respiratory cilia was previously considered the gold standard diagnostic test for PCD, but 30% of all PCD cases have either normal ciliary ultrastructure or subtle changes which are non-diagnostic. These cases are identified through alternate diagnostic tests, including nasal nitric oxide measurement, high-speed videomicroscopy analysis, immunofluorescent staining of axonemal proteins, and/or mutation analysis of various PCD causing genes. Autosomal recessive mutations in DNAH11 and HYDIN produce normal TEM ciliary ultrastructure, while mutations in genes encoding for radial spoke head proteins result in some cross-sections with non-diagnostic alterations in the central apparatus interspersed with normal ciliary cross-sections. Mutations in nexin link and dynein regulatory complex genes lead to a collection of different ciliary ultrastructures; mutations in CCDC65, CCDC164, and GAS8 produce normal ciliary ultrastructure, while mutations in CCDC39 and CCDC40 cause absent inner dynein arms and microtubule disorganization in some ciliary cross-sections. Mutations in CCNO and MCIDAS cause near complete absence of respiratory cilia due to defects in generation of multiple cellular basal bodies; however, the scant cilia generated may have normal ultrastructure. Lastly, a syndromic form of PCD with retinal degeneration results in normal ciliary ultrastructure through mutations in the RPGR gene. Clinicians must be aware of these genetic causes of PCD resulting in non-diagnostic TEM ciliary ultrastructure and refrain from using TEM of respiratory cilia as a test to rule out PCD.

Integration of next-generation sequencing in clinical diagnostic molecular pathology laboratories for analysis of solid tumours; an expert opinion on behalf of IQN Path ASBL.

PubMed

Deans, Zandra C; Costa, Jose Luis; Cree, Ian; Dequeker, Els; Edsjö, Anders; Henderson, Shirley; Hummel, Michael; Ligtenberg, Marjolijn Jl; Loddo, Marco; Machado, Jose Carlos; Marchetti, Antonio; Marquis, Katherine; Mason, Joanne; Normanno, Nicola; Rouleau, Etienne; Schuuring, Ed; Snelson, Keeda-Marie; Thunnissen, Erik; Tops, Bastiaan; Williams, Gareth; van Krieken, Han; Hall, Jacqueline A

2017-01-01

The clinical demand for mutation detection within multiple genes from a single tumour sample requires molecular diagnostic laboratories to develop rapid, high-throughput, highly sensitive, accurate and parallel testing within tight budget constraints. To meet this demand, many laboratories employ next-generation sequencing (NGS) based on small amplicons. Building on existing publications and general guidance for the clinical use of NGS and learnings from germline testing, the following guidelines establish consensus standards for somatic diagnostic testing, specifically for identifying and reporting mutations in solid tumours. These guidelines cover the testing strategy, implementation of testing within clinical service, sample requirements, data analysis and reporting of results. In conjunction with appropriate staff training and international standards for laboratory testing, these consensus standards for the use of NGS in molecular pathology of solid tumours will assist laboratories in implementing NGS in clinical services.

Clinic and Functional Analysis of p73R1 Mutations in Prostate Cancer

DTIC Science & Technology

2006-02-01

sequence variants in Marfan syndrome and related connective tissue disorders. Genet Test 1:237-42. Matsuoka S, Huang M, Elledge SJ. 1998. Linkage of ATM... Syndrome (LFS; MIM# 151623), a highly penetrant familial cancer phenotype, usually associated with inherited mutations in TP53 (Bell, et al., 1999...TP53 (Table 1). Mutually exclusive mutations of CHEK2 and TP53 have been reported in patients with Li-Fraumeni syndrome (LFS) and also in patients

Hereditary medullary thyroid carcinoma: the management dilemma.

PubMed

Zhou, Ping; Liu, Jian; Cheng, Shao-Wen; Wang, Bing; Yang, Rong; Peng, Ling

2012-06-01

Hereditary medullary thyroid carcinoma (hereditary MTC) is a rare malignancy, accounting for 25-30% of all MTC. It occurs as part of multiple endocrine neoplasia type 2 (MEN 2). Autosomal dominant gain-of-function mutations in the RET proto-oncogene is the cause of the disease, in which the common mutations are codons 609, 611, 618, 620, 630, 634 and 918. In recent years, the spectrum of RET gene mutations has changed. The classical mutations reduced, whereas the less aggressive mutations increased. Hereditary MTC is a time-dependent disease. Stages of the disorder at diagnosis can significantly influence survival rates. Based on the genotype-phenotype, RET mutations have been classified into four risk levels by American Thyroid Association (ATA) at 2009. The classification system guides the hereditary MTC management, including risk assessment, biochemical screenings and surgical intervention. Though the application of genetic testing and codon-specific phenotypes in hereditary MTC diagnosis is effective with high accuracy, there are some difficulties in implementing RET gene testing as a routine for MTC diagnosis. And most of carriers with RET mutations did not undergo thyroidectomy at the age recommended by the ATA guidelines. The aim of the study is to review the hereditary MTC and discuss the management dilemma.

Flexible Lab-Tailored Cut-Offs for Suitability of Formalin-Fixed Tumor Samples for Diagnostic Mutational Analyses

PubMed Central

Mariani, Sara; Tondat, Fabrizio; Pacchioni, Donatella; Molinaro, Luca; Barreca, Antonella; Macrì, Luigia; Chiusa, Luigi; di Celle, Paola Francia; Cassoni, Paola; Sapino, Anna

2015-01-01

The selection of proper tissues from formalin-fixed and paraffin-embedded tumors before diagnostic molecular testing is responsibility of the pathologist and represents a crucial step to produce reliable test results. The international guidelines suggest two cut-offs, one for the percentage and one for the number of tumor cells, in order to enrich the tumor content before DNA extraction. The aim of the present work was two-fold: to evaluate to what extent a low percentage or absolute number of tumor cells can be qualified for somatic mutation testing; and to determine how assay sensitivities can guide pathologists towards a better definition of morphology-based adequacy cut-offs. We tested 1797 tumor specimens from melanomas, colorectal and lung adenocarcinomas. Respectively, their BRAF, K-RAS and EGFR genes were analyzed at specific exons by mutation-enriched PCR, pyrosequencing, direct sequencing and real-time PCR methods. We demonstrate that poorly cellular specimens do not modify the frequency distribution of either mutated or wild-type DNA samples nor that of specific mutations. This observation suggests that currently recommended cut-offs for adequacy of specimens to be processed for molecular assays seem to be too much stringent in a laboratory context that performs highly sensitive routine analytical methods. In conclusion, new cut-offs are needed based on test sensitivities and documented tumor heterogeneity. PMID:25844806

Hereditary cancer genes are highly susceptible to splicing mutations

PubMed Central

Soemedi, Rachel; Maguire, Samantha; Murray, Michael F.; Monaghan, Sean F.

2018-01-01

Substitutions that disrupt pre-mRNA splicing are a common cause of genetic disease. On average, 13.4% of all hereditary disease alleles are classified as splicing mutations mapping to the canonical 5′ and 3′ splice sites. However, splicing mutations present in exons and deeper intronic positions are vastly underreported. A recent re-analysis of coding mutations in exon 10 of the Lynch Syndrome gene, MLH1, revealed an extremely high rate (77%) of mutations that lead to defective splicing. This finding is confirmed by extending the sampling to five other exons in the MLH1 gene. Further analysis suggests a more general phenomenon of defective splicing driving Lynch Syndrome. Of the 36 mutations tested, 11 disrupted splicing. Furthermore, analyzing past reports suggest that MLH1 mutations in canonical splice sites also occupy a much higher fraction (36%) of total mutations than expected. When performing a comprehensive analysis of splicing mutations in human disease genes, we found that three main causal genes of Lynch Syndrome, MLH1, MSH2, and PMS2, belonged to a class of 86 disease genes which are enriched for splicing mutations. Other cancer genes were also enriched in the 86 susceptible genes. The enrichment of splicing mutations in hereditary cancers strongly argues for additional priority in interpreting clinical sequencing data in relation to cancer and splicing. PMID:29505604

Mutagenicity of edible palm oil on the Ghanaian market before and after repeated heating.

PubMed

Asare, George A; Okyere, Genevieve O; Asante, Matilda; Brown, Charles A; Santa, Sheila; Asiedu, Bernice

2013-12-01

Red palm oil produced in Ghana largely by village folks has never been tested for its mutagenic potential. The study aimed at determining the mutagenicity of high-energy heated red palm oil (RRPO) and refined, bleached imported palm oil (PO) on the Ghanaian market. Samples of RRPO and PO were 1× and 5× heated for 10 min at 180 °C with a cooling period of 5 h in-between. Unheated, together with heated samples, were tested for mutagenicity using Salmonella typhimurium TA 98 and TA 100 tester stains. Unheated PO was negative for the Ames mutagenicity test with TA 98 strain. However, 1× and 5× heated PO were mutagenic (P = 0.05, each). Testing PO, using TA 100 strain was negative. RRPO was mutagenic with TA 98 strain for heated oils (P = 0.05, each). Assays with TA 100 strain showed highly significant mutations (P = 0.001, each) that increased with increasing heating frequency. PO 1× and 5× heated samples caused significant frameshift mutation in the S. typhimurium TA 98 strain. RRPO caused highly significant point and frameshift mutations in heated samples. Furthermore, unheated RRPO mutagenic potential has serious health implications. © 2013 Institute of Food Technologists®

High frequency of AML1/RUNX1 point mutations in radiation-associated myelodysplastic syndrome around Semipalatinsk nuclear test site.

PubMed

Zharlyganova, Dinara; Harada, Hironori; Harada, Yuka; Shinkarev, Sergey; Zhumadilov, Zhaxybay; Zhunusova, Aigul; Tchaizhunusova, Naylya J; Apsalikov, Kazbek N; Kemaikin, Vadim; Zhumadilov, Kassym; Kawano, Noriyuki; Kimura, Akiro; Hoshi, Masaharu

2008-09-01

It is known that bone marrow is a sensitive organ to ionizing radiation, and many patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) have been diagnosed in radiation-treated cases and atomic bomb survivors in Hiroshima and Nagasaki. The AML1/RUNX1 gene has been known to be frequently mutated in MDS/AML patients among atomic bomb survivors and radiation therapy-related MDS/AML patients. In this study, we investigated the AML1 mutations in radiation-exposed patients with MDS/AML among the residents near the Semipalatinsk Nuclear Test Site (SNTS), where the risk of solid cancers and leukemias was increased due to the radiation effects. AML1 mutations were identified in 7 (39%) of 18 radiation-exposed MDS/AML patients. In contrast, no AML1 mutation was found in 13 unexposed MDS/AML cases. The frequency of AML1 mutations in radiation-exposed patients with MDS/AML was significantly higher compared with unexposed patients (p < 0.05).We also found a significant correlation between individual estimated doses and AML1 mutations (p < 0.05). Considering these results, AML1 point mutations might be a useful biomarker that differentiates radio-induced MDS/AML from spontaneous MDS/AML.

Optimizing high-resolution melting analysis for the detection of mutations of GPR30/GPER-1 in breast cancer.

PubMed

Aihara, Masamune; Yamamoto, Shigeru; Nishioka, Hiroko; Inoue, Yutaro; Hamano, Kimikazu; Oka, Masaaki; Mizukami, Yoichi

2012-06-15

G protein-coupled receptor 30/G protein estrogen receptor-1 (GPR30/GPER-1) is a novel membrane receptor for estrogen whose mRNA is expressed at high levels in estrogen-dependent cells such as breast cancer cell lines. However, mutations in GRP30 related to diseases remain unreported. To detect unknown mutations in the GPR30 open reading frame (ORF) quickly, the experimental conditions for high-resolution melting (HRM) analysis were examined for PCR primers, Taq polymerases, saturation DNA binding dyes, Mg(2+) concentration, and normalized temperatures. Nine known SNPs and 13 artificial point mutations within the GPR30 ORF, as well as single nucleotide variants in DNA extracted from subjects with breast cancers were tested under the optimal experimental conditions. The combination of Expand High Fidelity(PLUS) and SYTO9 in the presence of 2.0 mM MgCl(2) produced the best separation in melting curves of mutations in all regions of the GPR30 ORF. Under these experimental conditions, the mutations were clearly detected in both heterozygotes and homozygotes. HRM analysis of GPR30 using genomic DNA from subjects with breast cancers showed a novel single nucleotide variant, 111C>T in GPR30 and 4 known SNPs. The experimental conditions determined in this study for HRM analysis are useful for high throughput assays to detect unknown mutations within the GPR30 ORF. Copyright © 2012 Elsevier B.V. All rights reserved.

Sample features associated with success rates in population-based EGFR mutation testing.

PubMed

Shiau, Carolyn J; Babwah, Jesse P; da Cunha Santos, Gilda; Sykes, Jenna R; Boerner, Scott L; Geddie, William R; Leighl, Natasha B; Wei, Cuihong; Kamel-Reid, Suzanne; Hwang, David M; Tsao, Ming-Sound

2014-07-01

Epidermal growth factor receptor (EGFR) mutation testing has become critical in the treatment of patients with advanced non-small-cell lung cancer. This study involves a large cohort and epidemiologically unselected series of EGFR mutation testing for patients with nonsquamous non-small-cell lung cancer in a North American population to determine sample-related factors that influence success in clinical EGFR testing. Data from consecutive cases of Canadian province-wide testing at a centralized diagnostic laboratory for a 24-month period were reviewed. Samples were tested for exon-19 deletion and exon-21 L858R mutations using a validated polymerase chain reaction method with 1% to 5% detection sensitivity. From 2651 samples submitted, 2404 samples were tested with 2293 samples eligible for analysis (1780 histology and 513 cytology specimens). The overall test-failure rate was 5.4% with overall mutation rate of 20.6%. No significant differences in the failure rate, mutation rate, or mutation type were found between histology and cytology samples. Although tumor cellularity was significantly associated with test-success or mutation rates in histology and cytology specimens, respectively, mutations could be detected in all specimen types. Significant rates of EGFR mutation were detected in cases with thyroid transcription factor (TTF)-1-negative immunohistochemistry (6.7%) and mucinous component (9.0%). EGFR mutation testing should be attempted in any specimen, whether histologic or cytologic. Samples should not be excluded from testing based on TTF-1 status or histologic features. Pathologists should report the amount of available tumor for testing. However, suboptimal samples with a negative EGFR mutation result should be considered for repeat testing with an alternate sample.

40 CFR 799.9510 - TSCA bacterial reverse mutation test.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 32 2014-07-01 2014-07-01 false TSCA bacterial reverse mutation test... REQUIREMENTS Health Effects Test Guidelines § 799.9510 TSCA bacterial reverse mutation test. (a) Scope. This... mutation test uses amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect...

Anosmia predicts hypogonadotropic hypogonadism in CHARGE syndrome.

PubMed

Bergman, Jorieke E H; Bocca, Gianni; Hoefsloot, Lies H; Meiners, Linda C; van Ravenswaaij-Arts, Conny M A

2011-03-01

To test the hypothesis that a smell test could predict the occurrence of hypogonadotropic hypogonadism (HH) in patients with CHARGE syndrome, which is a variable combination of ocular coloboma, heart defects, choanal atresia, retardation of growth/development, genital hypoplasia, and ear anomalies or hearing loss caused by mutations in the CHD7 (chromodomain helicase DNA binding protein 7) gene. We performed endocrine studies and smell testing (University of Pennsylvania Smell Identification Test) in 35 adolescent patients with molecularly confirmed CHARGE syndrome. Complete data on smell and puberty were available for 15 patients; 11 patients had both anosmia and HH, whereas 4 patients had normosmia/hyposmia and spontaneous puberty. In addition, 7 boys were highly suspected of having HH (they were too young for definite HH diagnosis, but all had cryptorchidism, micropenis, or both) and had anosmia. The type of CHD7 mutation could not predict HH because a father and daughter with the same CHD7 mutation were discordant for HH and anosmia. Anosmia and HH were highly correlated in our cohort, and therefore smell testing seems to be an attractive method for predicting the occurrence of HH in patients with CHARGE syndrome. The use of this test could prevent delay of hormonal pubertal induction, resulting in an age-appropriate puberty. Copyright © 2011 Mosby, Inc. All rights reserved.

Multiplex single-tube screening for mutations in the Nijmegen Breakage Syndrome (NBS1) gene in Hodgkin's and non-Hodgkin's lymphoma patients of Slavic origin.

PubMed

Soucek, Pavel; Gut, Ivan; Trneny, Marek; Skovlund, Eva; Grenaker Alnaes, Grethe; Kristensen, Tom; Børresen-Dale, Anne-Lise; Kristensen, Vessela N

2003-05-01

Patients with Nijmegen Breakage Syndrome (NBS) have a high risk to develop malignant diseases, most frequently B-cell lymphomas. It has been demonstrated that this chromosomal breakage syndrome results from mutations in the NBS1 gene that cause either a loss of full-length protein expression or expression of a variant protein. A large proportion of the known NBS patients are of Slavic origin who carry a major founder mutation 657del5 in exon 6 of the NBS1 gene. The prevalence of this mutation in Slav populations is reported to be high, possibly contributing to higher cancer risk in these populations. Therefore, if mutations in NBS1 are associated with higher risk of developing lymphoid cancers it would be most likely to be observed in these populations. A multiplex assay for four of the most frequent NBS1 mutations was designed and a series of 119 lymphoma patients from Slavic origin as well as 177 healthy controls were tested. One of the patients was a heterozygote carrier of the ACAAA deletion mutation in exon 6 (1/119). No mutation was observed in the control group, despite the reported high frequency (1/177). The power of this study was 30% to detect a relative risk of 2.0.

[Prognostic value of JAK2, MPL and CALR mutations in Chinese patients with primary myelofibrosis].

PubMed

Xu, Z F; Li, B; Liu, J Q; Li, Y; Ai, X F; Zhang, P H; Qin, T J; Zhang, Y; Wang, J Y; Xu, J Q; Zhang, H L; Fang, L W; Pan, L J; Hu, N B; Qu, S Q; Xiao, Z J

2016-07-01

To evaluate the prognostic value of JAK2, MPL and CALR mutations in Chinese patients with primary myelofibrosis (PMF). Four hundred and two Chinese patients with PMF were retrospectively analyzed. The Kaplan-Meier method, the Log-rank test, the likelihood ratio test and the Cox proportional hazards regression model were used to evaluate the prognostic scoring system. This cohort of patients included 209 males and 193 females with a median age of 55 years (range: 15- 89). JAK2V617F mutations were detected in 189 subjects (47.0% ), MPLW515 mutations in 13 (3.2%) and CALR mutations in 81 (20.1%) [There were 30 (37.0%) type-1, 48 (59.3%) type-2 and 3 (3.7%) less common CALR mutations], respectively. 119 subjects (29.6%) had no detectable mutation in JAK2, MPL or CALR. Univariate analysis indicated that patients with CALR type-2 mutations or no detectable mutations had inferior survival compared to those with JAK2, MPL or CALR type- 1 or other less common CALR mutations (the median survival was 74vs 168 months, respectively [HR 2.990 (95% CI 1.935-4.619),P<0.001]. Therefore, patients were categorized into the high-risk with CALR type- 2 mutations or no detectable driver mutations and the low- risk without aforementioned mutations status. The DIPSS-Chinese molecular prognostic model was proposed by adopting mutation categories and DIPSS-Chinese risk group. The median survival of patients classified in low risk (132 subjects, 32.8% ), intermediate- 1 risk (143 subjects, 35.6%), intermediate- 2 risk (106 subjects, 26.4%) and high risk (21 subjects, 5.2%) were not reached, 156 (95% CI 117- 194), 60 (95% CI 28- 91) and 22 (95% CI 10- 33) months, respectively, and there was a statistically significant difference in overall survival among the four risk groups (P<0.001). There was significantly higher predictive power for survival according to the DIPSS-Chinese molecular prognostic model compared with the DIPSS-Chinese model (P=0.005, -2 log-likelihood ratios of 855.6 and 869.7, respectively). The impact of the CALR type- 2 mutations or no detectable driver mutation on survival was independent of current prognostic scoring systems. The DIPSS- Chinese molecular prognostic model based on the molecular features of Chinese patients was proposed and worked well for prognostic indication.

[Identification of a HPGD mutation in three families affected with primary hypertrophic osteoarthropathy].

PubMed

Zhang, Wanying; Wang, Tao; Huang, Shuaiwu; Zhao, Xiuli

2018-04-10

To detect mutation of HPGD gene among three pedigrees affected with primary hypertrophic osteoarthropathy (PHO) by DNA sequencing and high-resolution melting (HRM) analysis. Genomic DNA was extracted from peripheral blood samples collected from the pedigrees. PCR and direct sequencing were carried out to identify potential mutations of the HPGD gene. Amplicons containing the mutation spot were generated by nested PCR. The products were then subjected to HRM analysis using the HR-1 instrument. Direct sequencing was carried out in family members and healthy individuals to confirm the result of HRM analysis. A homozygous mutation c.310_311delCT was detected in 2 affected probands, while a heterozygous mutation c.310_311delCT was detected in the third proband. HRM analysis of the fragments encompassing HPGD exon 3 showed 3 curve patterns representing three different genotypes, i.e., the wild type, the c.310_311delCT homozygote, and the c.310_311delCT heterozygote. Result of DNA sequencing was consistent with that of the HRM analysis and phenotype of the subjects. The c.310_311delCT mutation may be the most prevalent mutation among Chinese population. HRM analysis has provided an optimized method for genetic testing of HPGD mutation for its simplicity, rapid turnover and high sensitivity.

Evaluation of a deep learning architecture for MR imaging prediction of ATRX in glioma patients

NASA Astrophysics Data System (ADS)

Korfiatis, Panagiotis; Kline, Timothy L.; Erickson, Bradley J.

2018-02-01

Predicting mutation/loss of alpha-thalassemia/mental retardation syndrome X-linked (ATRX) gene utilizing MR imaging is of high importance since it is a predictor of response and prognosis in brain tumors. In this study, we compare a deep neural network approach based on a residual deep neural network (ResNet) architecture and one based on a classical machine learning approach and evaluate their ability in predicting ATRX mutation status without the need for a distinct tumor segmentation step. We found that the ResNet50 (50 layers) architecture, pre trained on ImageNet data was the best performing model, achieving an accuracy of 0.91 for the test set (classification of a slice as no tumor, ATRX mutated, or mutated) in terms of f1 score in a test set of 35 cases. The SVM classifier achieved 0.63 for differentiating the Flair signal abnormality regions from the test patients based on their mutation status. We report a method that alleviates the need for extensive preprocessing and acts as a proof of concept that deep neural network architectures can be used to predict molecular biomarkers from routine medical images.

Impact of direct-to-consumer advertising for hereditary breast cancer testing on genetic services at a managed care organization: a naturally-occurring experiment.

PubMed

Mouchawar, Judy; Hensley-Alford, Sharon; Laurion, Suzanne; Ellis, Jennifer; Kulchak-Rahm, Alanna; Finucane, Melissa L; Meenan, Richard; Axell, Lisen; Pollack, Rebecca; Ritzwoller, Debra

2005-03-01

To describe the impact of Myriad Genetics, Inc.'s direct-to-consumer advertising (DTC-ad) campaign on cancer genetic services within two Managed Care Organizations, Kaiser Permanente Colorado (KPCO), Denver, Colorado, where the ad campaign occurred, and Henry Ford Health System (HFHS), Detroit, Michigan, where there were no advertisements. The main outcome measures were the changes in number and pretest mutation probability of referrals approved for cancer genetic services at KPCO and HFHS during the campaign versus the year prior, and mutation probability of those undergoing testing. At KPCO, referrals increased 244% during the DTC-ad compared to the same time period a year earlier (P value<0.001). The proportion of referrals at high pretest probability of a mutation (10% or greater) dropped from 69% the previous year to 48% during the campaign (P value<0.001). There was no significant change in pretest mutation probability among women who underwent testing between the two time periods. HFHS reported no significant change between the two time periods for numbers or mutation probability of referrals, or for mutation probability of women tested. The DTC-ad caused significant increase in demand for cancer genetic services. In the face of potential future DTC-ad for inherited cancer risk, providers and payers need to consider the delivery of genetic services and genetic education for persons of all risk levels.

Mitochondrial DNA Mutation Associated with Leber's Hereditary Optic Neuropathy

NASA Astrophysics Data System (ADS)

Wallace, Douglas C.; Singh, Gurparkash; Lott, Marie T.; Hodge, Judy A.; Schurr, Theodore G.; Lezza, Angela M. S.; Elsas, Louis J.; Nikoskelainen, Eeva K.

1988-12-01

Leber's hereditary optic neuropathy is a maternally inherited disease resulting in optic nerve degeneration and cardiac dysrhythmia. A mitochondrial DNA replacement mutation was identified that correlated with this disease in multiple families. This mutation converted a highly conserved arginine to a histidine at codon 340 in the NADH dehydrogenase subunit 4 gene and eliminated an Sfa NI site, thus providing a simple diagnostic test. This finding demonstrated that a nucleotide change in a mitochondrial DNA energy production gene can result in a neurological disease.

Performance of Lynch syndrome predictive models in quantifying the likelihood of germline mutations in patients with abnormal MLH1 immunoexpression.

PubMed

Cabreira, Verónica; Pinto, Carla; Pinheiro, Manuela; Lopes, Paula; Peixoto, Ana; Santos, Catarina; Veiga, Isabel; Rocha, Patrícia; Pinto, Pedro; Henrique, Rui; Teixeira, Manuel R

2017-01-01

Lynch syndrome (LS) accounts for up to 4 % of all colorectal cancers (CRC). Detection of a pathogenic germline mutation in one of the mismatch repair genes is the definitive criterion for LS diagnosis, but it is time-consuming and expensive. Immunohistochemistry is the most sensitive prescreening test and its predictive value is very high for loss of expression of MSH2, MSH6, and (isolated) PMS2, but not for MLH1. We evaluated if LS predictive models have a role to improve the molecular testing algorithm in this specific setting by studying 38 individuals referred for molecular testing and who were subsequently shown to have loss of MLH1 immunoexpression in their tumors. For each proband we calculated a risk score, which represents the probability that the patient with CRC carries a pathogenic MLH1 germline mutation, using the PREMM 1,2,6 and MMRpro predictive models. Of the 38 individuals, 18.4 % had a pathogenic MLH1 germline mutation. MMRpro performed better for the purpose of this study, presenting a AUC of 0.83 (95 % CI 0.67-0.9; P < 0.001) compared with a AUC of 0.68 (95 % CI 0.51-0.82, P = 0.09) for PREMM 1,2,6 . Considering a threshold of 5 %, MMRpro would eliminate unnecessary germline mutation analysis in a significant proportion of cases while keeping very high sensitivity. We conclude that MMRpro is useful to correctly predict who should be screened for a germline MLH1 gene mutation and propose an algorithm to improve the cost-effectiveness of LS diagnosis.

The value of KRAS mutation testing with CEA for the diagnosis of pancreatic mucinous cysts

PubMed Central

Kadayifci, Abdurrahman; Al-Haddad, Mohammad; Atar, Mustafa; Dewitt, John M.; Forcione, David G.; Sherman, Stuart; Casey, Brenna W.; Fernandez-del Castillo, Carlos; Schmidt, C. Max; Pitman, Martha B.; Brugge, William R.

2016-01-01

Background and aims: Pancreatic cyst fluid (PCF) CEA has been shown to be the most accurate preoperative test for detection of cystic mucinous neoplasms (CMNs). This study aimed to assess the added value of PCF KRAS mutational analysis to CEA for diagnosis of CMNs. Patients and methods: This is a retrospective study of prospectively collected endoscopic ultrasonography (EUS) fine-needle aspiration (FNA) data. KRAS mutation was determined by direct sequencing or equivalent methods. Cysts were classified histologically (surgical cohort) or by clinical (EUS or FNA) findings (clinical cohort). Performance characteristics of KRAS, CEA and their combination for detection of a cystic mucinous neoplasm (CMN) and malignancy were calculated. Results: The study cohort consisted of 943 patients: 147 in the surgical cohort and 796 in the clinical cohort. Overall, KRAS and CEA each had high specificity (100 % and 93.2 %), but low sensitivity (48.3 % and 56.3 %) for the diagnosis of a CMN. The positivity of KRAS or CEA increased the diagnostic accuracy (80.8 %) and AUC (0.84) significantly compared to KRAS (65.3 % and 0.74) or CEA (65.8 % and 0.74) alone, but only in the clinical cohort (P < 0.0001 for both). KRAS mutation was significantly more frequent in malignant CMNs compared to histologically confirmed non-malignant CMNs (73 % vs. 37 %, P = 0.001). The negative predictive value of KRAS mutation was 77.6 % in differentiating non-malignant cysts. Conclusions: The detection of a KRAS mutation in PCF is a highly specific test for mucinous cysts. It outperforms CEA for sensitivity in mucinous cyst diagnosis, but the data does not support its routine use. PMID:27092317

High-resolution melting (HRM) assay for the detection of recurrent BRCA1/BRCA2 germline mutations in Tunisian breast/ovarian cancer families.

PubMed

Riahi, Aouatef; Kharrat, Maher; Lariani, Imen; Chaabouni-Bouhamed, Habiba

2014-12-01

Germline deleterious mutations in the BRCA1/BRCA2 genes are associated with an increased risk for the development of breast and ovarian cancer. Given the large size of these genes the detection of such mutations represents a considerable technical challenge. Therefore, the development of cost-effective and rapid methods to identify these mutations became a necessity. High resolution melting analysis (HRM) is a rapid and efficient technique extensively employed as high-throughput mutation scanning method. The purpose of our study was to assess the specificity and sensitivity of HRM for BRCA1 and BRCA2 genes scanning. As a first step we estimate the ability of HRM for detection mutations in a set of 21 heterozygous samples harboring 8 different known BRCA1/BRCA2 variations, all samples had been preliminarily investigated by direct sequencing, and then we performed a blinded analysis by HRM in a set of 68 further sporadic samples of unknown genotype. All tested heterozygous BRCA1/BRCA2 variants were easily identified. However the HRM assay revealed further alteration that we initially had not searched (one unclassified variant). Furthermore, sequencing confirmed all the HRM detected mutations in the set of unknown samples, including homozygous changes, indicating that in this cohort, with the optimized assays, the mutations detections sensitivity and specificity were 100 %. HRM is a simple, rapid and efficient scanning method for known and unknown BRCA1/BRCA2 germline mutations. Consequently the method will allow for the economical screening of recurrent mutations in Tunisian population.

Epidermal Growth Factor Receptor Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung Cancer: An Evidence-Based Analysis.

PubMed

2010-01-01

In February 2010, the Medical Advisory Secretariat (MAS) began work on evidence-based reviews of the literature surrounding three pharmacogenomic tests. This project came about when Cancer Care Ontario (CCO) asked MAS to provide evidence-based analyses on the effectiveness and cost-effectiveness of three oncology pharmacogenomic tests currently in use in Ontario.Evidence-based analyses have been prepared for each of these technologies. These have been completed in conjunction with internal and external stakeholders, including a Provincial Expert Panel on Pharmacogenetics (PEPP). Within the PEPP, subgroup committees were developed for each disease area. For each technology, an economic analysis was also completed by the Toronto Health Economics and Technology Assessment Collaborative (THETA) and is summarized within the reports.THE FOLLOWING REPORTS CAN BE PUBLICLY ACCESSED AT THE MAS WEBSITE AT: http://www.health.gov.on.ca/mas or at www.health.gov.on.ca/english/providers/program/mas/mas_about.htmlGENE EXPRESSION PROFILING FOR GUIDING ADJUVANT CHEMOTHERAPY DECISIONS IN WOMEN WITH EARLY BREAST CANCER: An Evidence-Based AnalysisEpidermal Growth Factor Receptor Mutation (EGFR) Testing for Prediction of Response to EGFR-Targeting Tyrosine Kinase Inhibitor (TKI) Drugs in Patients with Advanced Non-Small-Cell Lung Cancer: an Evidence-Based AnalysisK-RAS testing in Treatment Decisions for Advanced Colorectal Cancer: an Evidence-Based Analysis The Medical Advisory Secretariat undertook a systematic review of the evidence on the clinical effectiveness and cost-effectiveness of epidermal growth factor receptor (EGFR) mutation testing compared with no EGFR mutation testing to predict response to tyrosine kinase inhibitors (TKIs), gefitinib (Iressa(®)) or erlotinib (Tarceva(®)) in patients with advanced non-small cell lung cancer (NSCLC). TARGET POPULATION AND CONDITION With an estimated 7,800 new cases and 7,000 deaths last year, lung cancer is the leading cause of cancer deaths in Ontario. Those with unresectable or advanced disease are commonly treated with concurrent chemoradiation or platinum-based combination chemotherapy. Although response rates to cytotoxic chemotherapy for advanced NSCLC are approximately 30 to 40%, all patients eventually develop resistance and have a median survival of only 8 to 10 months. Treatment for refractory or relapsed disease includes single-agent treatment with docetaxel, pemetrexed or EGFR-targeting TKIs (gefitinib, erlotinib). TKIs disrupt EGFR signaling by competing with adenosine triphosphate (ATP) for the binding sites at the tyrosine kinase (TK) domain, thus inhibiting the phosphorylation and activation of EGFRs and the downstream signaling network. Gefitinib and erlotinib have been shown to be either non-inferior or superior to chemotherapy in the first- or second-line setting (gefitinib), or superior to placebo in the second- or third-line setting (erlotinib). Certain patient characteristics (adenocarcinoma, non-smoking history, Asian ethnicity, female gender) predict for better survival benefit and response to therapy with TKIs. In addition, the current body of evidence shows that somatic mutations in the EGFR gene are the most robust biomarkers for EGFR-targeting therapy selection. Drugs used in this therapy, however, can be costly, up to C$ 2000 to C$ 3000 per month, and they have only approximately a 10% chance of benefiting unselected patients. For these reasons, the predictive value of EGFR mutation testing for TKIs in patients with advanced NSCLC needs to be determined. EGFR MUTATION TESTING The EGFR gene sequencing by polymerase chain reaction (PCR) assays is the most widely used method for EGFR mutation testing. PCR assays can be performed at pathology laboratories across Ontario. According to experts in the province, sequencing is not currently done in Ontario due to lack of adequate measurement sensitivity. A variety of new methods have been introduced to increase the measurement sensitivity of the mutation assay. Some technologies such as single-stranded conformational polymorphism, denaturing high-performance liquid chromatography, and high-resolution melting analysis have the advantage of facilitating rapid mutation screening of large numbers of samples with high measurement sensitivity but require direct sequencing to confirm the identity of the detected mutations. Other techniques have been developed for the simple, but highly sensitive detection of specific EGFR mutations, such as the amplification refractory mutations system (ARMS) and the peptide nucleic acid-locked PCR clamping. Others selectively digest wild-type DNA templates with restriction endonucleases to enrich mutant alleles by PCR. Experts in the province of Ontario have commented that currently PCR fragment analysis for deletion and point mutation conducts in Ontario, with measurement sensitivity of 1% to 5%. In patients with locally-advanced or metastatic NSCLC, what is the clinical effectiveness of EGFR mutation testing for prediction of response to treatment with TKIs (gefitinib, erlotinib) in terms of progression-free survival (PFS), objective response rates (ORR), overall survival (OS), and quality of life (QoL)?What is the impact of EGFR mutation testing on overall clinical decision-making for patients with advanced or metastatic NSCLC?What is the cost-effectiveness of EGFR mutation testing in selecting patients with advanced NSCLC for treatment with gefitinib or erlotinib in the first-line setting?What is the budget impact of EGFR mutation testing in selecting patients with advanced NSCLC for treatment with gefitinib or erlotinib in the second- or third-line setting? A literature search was performed on March 9, 2010 using OVID MEDLINE, MEDLINE In-Process and Other Non-Indexed Citations, OVID EMBASE, Wiley Cochrane, CINAHL, Centre for Reviews and Dissemination/International Agency for Health Technology Assessment for studies published from January 1, 2004 until February 28, 2010 using the following terms: Non-Small-Cell Lung CarcinomaEpidermal Growth Factor ReceptorAn automatic literature update program also extracted all papers published from February 2010 until August 2010. Abstracts were reviewed by a single reviewer and for those studies meeting the eligibility criteria full-text articles were obtained. Reference lists were also examined for any additional relevant studies not identified through the search. Articles with unknown eligibility were reviewed with a second clinical epidemiologist, and then a group of epidemiologists, until consensus was established. The quality of evidence was assessed as high, moderate, low or very low according to GRADE methodology. The inclusion criteria were as follows: patients with locally advanced or metastatic NSCLC (stage IIIB or IV)PROCEDURE: EGFR mutation testing before treatment with gefitinib or erlotinibLANGUAGE: publication in EnglishPublished health technology assessments, guidelines, and peer-reviewed literature (abstracts, full text, conference abstract) progression-free survival (PFS), Objective response rate (ORR), overall survival (OS), quality of life (QoL).The exclusion criteria were as follows: Studies lacking outcomes specific to those of interestStudies focused on erlotinib maintenance therapyStudies focused on gefitinib or erlotinib use in combination with cytotoxic agents or any other drugGrey literature, where relevant, was also reviewed. PFSORR determined by means of the Response Evaluation Criteria in Solid Tumours (RECIST)OSQoL QUALITY OF EVIDENCE: The quality of the Phase II trials and observational studies was based on the method of subject recruitment and sampling, possibility of selection bias, and generalizability to the source population. The overall quality of evidence was assessed as high, moderate, low or very low according to the GRADE Working Group criteria. Since the last published health technology assessment by Blue Cross Blue Shield Association in 2007 there have been a number of phase III trials which provide evidence of predictive value of EGFR mutation testing in patients who were treated with gefitinib compared to chemotherapy in the first- or second-line setting. The Iressa Pan Asian Study (IPASS) trial showed the superiority of gefitinib in terms of PFS in patients with EGFR mutations versus patients with wild-type EGFR (Hazard ratio [HR], 0.48, 95%CI; 0.36-0.64 versus HR, 2.85; 95%CI, 2.05-3.98). Moreover, there was a statistically significant increased ORR in patients who received gefitinib and had EGFR mutations compared to patients with wild-type EGFR (71% versus 1%). The First-SIGNAL trial in patients with similar clinical characteristics as IPASS as well as the NEJ002 and WJTOG3405 trials that included only patients with EGFR mutations, provide confirmation that gefitinib is superior to chemotherapy in terms of improved PFS or higher ORR in patients with EGFR mutations. The INTEREST trial further indicated that patients with EGFR mutations had prolonged PFS and higher ORR when treated with gefitinib compared with docetaxel. In contrast, there is still a paucity of strong evidence regarding the predictive value of EGFR mutation testing for response to erlotinib in the second- or third-line setting. The BR.21 trial randomized 731 patients with NSCLC who were refractory or intolerant to prior first- or second-line chemotherapy to receive erlotinib or placebo. While the HR of 0.61 (95%CI, 0.51-0.74) favored erlotinib in the overall population, this was not a significant in the subsequent retrospective subgroup analysis. A retrospective evaluation of 116 of the BR.21 tumor samples demonstrated that patients with EGFR mutations had significantly higher ORRs when treated with erlotinib compared with placebo (27% versus 7%; P=0.03). (ABSTRACT TRUNCATED)

High risk men's perceptions of pre-implantation genetic diagnosis for hereditary breast and ovarian cancer.

PubMed

Quinn, Gwendolyn P; Vadaparampil, Susan T; Miree, Cheryl A; Lee, Ji-Hyun; Zhao, Xiuhua; Friedman, Susan; Yi, Susan; Mayer, James

2010-10-01

Pre-implantation genetic diagnosis (PGD) is an assisted reproductive technology procedure which provides parents with the option of conducting genetic analyses to determine if a mutation is present in an embryo. Though studies have discussed perceptions of PGD from a general population, couples or high-risk women, no studies to date have specifically examined PGD usage among men. This study sought to explore perceptions and attitudes towards PGD among males who either carry a BRCA mutation or have a partner or first degree relative with a BRCA mutation. A cross-sectional survey was conducted among 228 men visiting the Facing Our Risk of Cancer Empowered or Craigslist website. Eligibility criteria included men who self-reported they had been tested for a BRCA mutation or had a partner or first degree relative tested for a BRCA mutation. A 41-item survey assessed socio-demographic, clinical characteristics, PGD knowledge and attitudinal factors and consideration of the use of PGD. Differences in proportions of subgroups were tested using the Monte Carlo exact test for categorical data. A multiple logistic regression model was then built through a backward elimination procedure. Although 80% of men reported being previously unfamiliar with PGD, after learning the definition of PGD, 34% of the 228 respondents then said they would 'ever consider the use of PGD'. Respondents who thought of PGD only in terms of 'health and safety' were almost three times more likely (OR = 2.82; 95% 1.19-6.71) to 'ever consider the use of PGD' compared with respondents who thought of PGD in terms of both 'health and safety', and 'religion and morality'. As with other anonymous web-based surveys, we cannot verify clinical characteristics that may impact consideration of PGD use. Our findings indicate high-risk men need more information about PGD and may benefit from educational materials to assist them in reproductive decision-making.

Screening for mutations in two exons of FANCG gene in Pakistani population.

PubMed

Aymun, Ujala; Iram, Saima; Aftab, Iram; Khaliq, Saba; Nadir, Ali; Nisar, Ahmed; Mohsin, Shahida

2017-06-01

Fanconi anemia is a rare autosomal recessive disorder of genetic instability. It is both molecularly and clinically, a heterogeneous disorder. Its incidence is 1 in 129,000 births and relatively high in some ethnic groups. Sixteen genes have been identified among them mutations in FANCG gene are most common after FANCA and FANCC gene mutations. To study mutations in exon 3 and 4 of FANCG gene in Pakistani population. Thirty five patients with positive Diepoxybutane test were included in the study. DNA was extracted and amplified for exons 3 and 4. Thereafter Sequencing was done and analyzed for the presence of mutations. No mutation was detected in exon 3 whereas a carrier of known mutation c.307+1 G>T was found in exon 4 of the FANCG gene. Absence of any mutation in exon 3 and only one heterozygous mutation in exon 4 of FANCG gene points to a different spectrum of FA gene pool in Pakistan that needs extensive research in this area.

The first liquid biopsy test approved. Is it a new era of mutation testing for non-small cell lung cancer?

PubMed Central

2017-01-01

Specific mutations in epidermal growth factor receptor (EGFR) gene are predictive for response to the EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer patients (NSCLC). According to international guidelines, the molecular testing in patients with advanced NSCLC of a non-squamous subtype is recommended. However, obtain a tissue sample could be challenging. Liquid biopsy allows to determine patients suitable for EGFR-targeted therapy by analysis of circulating-free tumor DNA (cfDNA) in peripheral blood samples and might replace tissue biopsy. It allows to acquire a material in convenient minimally invasive manner, is easily repeatable, could be used for molecular identification and molecular changes monitoring. Many studies show a high concordance rate between tissue and plasma samples testing. When U.S. Food and Drug Administration (FDA) approved the first liquid biopsy test, analysis of driver gene mutation from cfDNA becomes a reality in clinical practice for patients with NSCLC. PMID:28251125

Frequency of five disease-causing genetic mutations in a large mixed-breed dog population (2011–2012)

PubMed Central

Zierath, Sharon; Hughes, Angela M.; Fretwell, Neale; Dibley, Mark

2017-01-01

Background A large and growing number of inherited genetic disease mutations are now known in the dog. Frequencies of these mutations are typically examined within the breed of discovery, possibly in related breeds, but nearly always in purebred dogs. No report to date has examined the frequencies of specific genetic disease mutations in a large population of mixed-breed dogs. Further, veterinarians and dog owners typically dismiss inherited/genetic diseases as possibilities for health problems in mixed-breed dogs, assuming hybrid vigor will guarantee that single-gene disease mutations are not a cause for concern. Therefore, the objective of this study was to screen a large mixed-breed canine population for the presence of mutant alleles associated with five autosomal recessive disorders: hyperuricosuria and hyperuricemia (HUU), cystinuria (CYST), factor VII deficiency (FVIID), myotonia congenita (MYC) and phosphofructokinase deficiency (PKFD). Genetic testing was performed in conjunction with breed determination via the commercially-available Wisdom PanelTM test. Results From a population of nearly 35,000 dogs, homozygous mutant dogs were identified for HUU (n = 57) and FVIID (n = 65). Homozygotes for HUU and FVIID were identified even among dogs with highly mixed breed ancestry. Carriers were identified for all disorders except MYC. HUU and FVIID were of high enough frequency to merit consideration in any mixed-breed dog, while CYST, MYC, and PKFD are vanishingly rare. Conclusions The assumption that mixed-breed dogs do not suffer from single-gene genetic disorders is shown here to be false. Within the diseases examined, HUU and FVIID should remain on any practitioner’s rule-out list, when clinically appropriate, for all mixed-breed dogs, and judicious genetic testing should be performed for diagnosis or screening. Future testing of large mixed-breed dog populations that include additional known canine genetic mutations will refine our knowledge of which genetic diseases can strike mixed-breed dogs. PMID:29166669

Frequency of five disease-causing genetic mutations in a large mixed-breed dog population (2011-2012).

PubMed

Zierath, Sharon; Hughes, Angela M; Fretwell, Neale; Dibley, Mark; Ekenstedt, Kari J

2017-01-01

A large and growing number of inherited genetic disease mutations are now known in the dog. Frequencies of these mutations are typically examined within the breed of discovery, possibly in related breeds, but nearly always in purebred dogs. No report to date has examined the frequencies of specific genetic disease mutations in a large population of mixed-breed dogs. Further, veterinarians and dog owners typically dismiss inherited/genetic diseases as possibilities for health problems in mixed-breed dogs, assuming hybrid vigor will guarantee that single-gene disease mutations are not a cause for concern. Therefore, the objective of this study was to screen a large mixed-breed canine population for the presence of mutant alleles associated with five autosomal recessive disorders: hyperuricosuria and hyperuricemia (HUU), cystinuria (CYST), factor VII deficiency (FVIID), myotonia congenita (MYC) and phosphofructokinase deficiency (PKFD). Genetic testing was performed in conjunction with breed determination via the commercially-available Wisdom PanelTM test. From a population of nearly 35,000 dogs, homozygous mutant dogs were identified for HUU (n = 57) and FVIID (n = 65). Homozygotes for HUU and FVIID were identified even among dogs with highly mixed breed ancestry. Carriers were identified for all disorders except MYC. HUU and FVIID were of high enough frequency to merit consideration in any mixed-breed dog, while CYST, MYC, and PKFD are vanishingly rare. The assumption that mixed-breed dogs do not suffer from single-gene genetic disorders is shown here to be false. Within the diseases examined, HUU and FVIID should remain on any practitioner's rule-out list, when clinically appropriate, for all mixed-breed dogs, and judicious genetic testing should be performed for diagnosis or screening. Future testing of large mixed-breed dog populations that include additional known canine genetic mutations will refine our knowledge of which genetic diseases can strike mixed-breed dogs.

Time to incorporate germline multigene panel testing into breast and ovarian cancer patient care.

PubMed

Graffeo, Rossella; Livraghi, Luca; Pagani, Olivia; Goldhirsch, Aron; Partridge, Ann H; Garber, Judy E

2016-12-01

Genetic evaluation is increasingly becoming an integral part of the management of women with newly diagnosed breast and ovarian cancer (OC), and of individuals at high risk for these diseases. Genetic counseling and testing have been incorporated into oncological care to help and complete management and treatment strategies. Risk assessment and early detection strategies in individuals with BRCA1/2 mutations and with Lynch syndrome have been quite extensively studied, whereas much less is known about the management of mutation carriers with less common high-penetrance cancer susceptibility genes (PTEN, TP53, STK11, CDH1), and particularly those who carry mutations in moderate-penetrance genes (e.g., PALB2, CHEK2, ATM, NF1, RAD51C, RAD51D, BRIP1). The latter patient groups represent important ongoing research opportunities to enable informed counseling about appropriate clinical management. We summarize the current guidelines for the management of high and moderate-penetrance mutations for breast and OC susceptibility. Continuous updating of guidelines for proper clinical management of these individuals is ongoing because of rapid advances in technology and knowledge in this field. Thus, we exhort the use of multigene panels for the assessment of cancer risk beyond the classic predisposition syndromes as a new standard of care in cancer genetics. We further support an increase of genetic counselors in Europe and use of their expertise to support genetic testing in specialist multidisciplinary teams.

Which BRCA genetic testing programs are ready for implementation in health care? A systematic review of economic evaluations.

PubMed

D'Andrea, Elvira; Marzuillo, Carolina; De Vito, Corrado; Di Marco, Marco; Pitini, Erica; Vacchio, Maria Rosaria; Villari, Paolo

2016-12-01

There is considerable evidence regarding the efficacy and effectiveness of BRCA genetic testing programs, but whether they represent good use of financial resources is not clear. Therefore, we aimed to identify the main health-care programs for BRCA testing and to evaluate their cost-effectiveness. We performed a systematic review of full economic evaluations of health-care programs involving BRCA testing. Nine economic evaluations were included, and four main categories of BRCA testing programs were identified: (i) population-based genetic screening of individuals without cancer, either comprehensive or targeted based on ancestry; (ii) family history (FH)-based genetic screening, i.e., testing individuals without cancer but with FH suggestive of BRCA mutation; (iii) familial mutation (FM)-based genetic screening, i.e., testing individuals without cancer but with known familial BRCA mutation; and (iv) cancer-based genetic screening, i.e., testing individuals with BRCA-related cancers. Currently BRCA1/2 population-based screening represents good value for the money among Ashkenazi Jews only. FH-based screening is potentially very cost-effective, although further studies that include costs of identifying high-risk women are needed. There is no evidence of cost-effectiveness for BRCA screening of all newly diagnosed cases of breast/ovarian cancers followed by cascade testing of relatives, but programs that include tools for identifying affected women at higher risk for inherited forms are promising. Cost-effectiveness is highly sensitive to the cost of BRCA1/2 testing.Genet Med 18 12, 1171-1180.

Genotyping of beta thalassemia trait by high-resolution DNA melting analysis.

PubMed

Saetung, Rattika; Ongchai, Siriwan; Charoenkwan, Pimlak; Sanguansermsri, Torpong

2013-11-01

Beta thalassemia is a common hereditary hemalogogical disease in Thailand, with a prevalence of 5-8%. In this study, we evaluated the high resolution DNA melting (HRM) assay to identify beta thalassemia mutation in samples from 143 carriers of the beta thalassemia traits in at risk couples. The DNA was isolated from venous blood samples and tested for mutation under a series of 5 PCR-HRM (A, B, C, D and E primers) protocols. The A primers were for detection of beta thalassemia mutations in the HBB promoter region, the B primers for mutations in exon I, the C primers for exon II, the D primers for exon III and the E primers for the 3.4 kb deletion mutation. The mutations were diagnosed by comparing the complete melting curve profiles of a wild type control with those for each mutant sample. With the PCR-HRM technique, fourteen types of beta thalassemia mutations were detected. Each mutation had a unique and specific melting profile. The mutations included 36.4% (52 cases) codon 41/42-CTTT, 26.6% (38 cases) codon 17 A-T, 11.2% (16 cases) IVS1-1 G-T, 8.4% (12 cases) codon 71/72 +A, 8.4% (12 cases) of the 3.4 kb deletion and 3.5% (5 cases) -28 A-G. The remainder included one instance each of -87 C-A, -31 A-C, codon 27/28 +C, codon 30 G-A, IVS1-5 G-C, codon 35 C-A, codon 41-C and IVSII -654 C-T. Of the total cases, 85.8% of the mutations could be detected by primers B and C. The PCR-HRM method provides a rapid, simple and highly feasible strategy for mutation screening of beta thalassemia traits.

High Prevalence of Multidrug-Resistant Mycoplasma genitalium in Human Immunodeficiency Virus-Infected Men Who Have Sex With Men in Alabama.

PubMed

Dionne-Odom, Jodie; Geisler, William M; Aaron, Kristal J; Waites, Ken B; Westfall, Andrew O; Van Der Pol, Barbara; Xiao, Li

2018-02-10

We tested for Mycoplasma genitalium in 157 HIV-infected men. Urogenital and rectal prevalence were 10.8% and 6.4%. Macrolide resistance mutations were detected in 70.6% and 80% of urogenital and rectal samples, and fluoroquinolone resistance mutations in 26.7% and 40%, respectively.

A Targeted Q-PCR-Based Method for Point Mutation Testing by Analyzing Circulating DNA for Cancer Management Care.

PubMed

Thierry, Alain R

2016-01-01

Circulating cell-free DNA (cfDNA) is a valuable source of tumor material available with a simple blood sampling enabling a noninvasive quantitative and qualitative analysis of the tumor genome. cfDNA is released by tumor cells and exhibits the genetic and epigenetic alterations of the tumor of origin. Circulating cell-free DNA (cfDNA) analysis constitutes a hopeful approach to provide a noninvasive tumor molecular test for cancer patients. Based upon basic research on the origin and structure of cfDNA, new information on circulating cell-free DNA (cfDNA) structure, and specific determination of cfDNA fragmentation and size, we revisited Q-PCR-based method and recently developed a the allele-specific-Q-PCR-based method with blocker (termed as Intplex) which is the first multiplexed test for cfDNA. This technique, named Intplex(®) and based on a refined Q-PCR method, derived from critical observations made on the specific structure and size of cfDNA. It enables the simultaneous determination of five parameters: the cfDNA total concentration, the presence of a previously known point mutation, the mutant (tumor) cfDNA concentration (ctDNA), the proportion of mutant cfDNA, and the cfDNA fragmentation index. Intplex(®) has enabled the first clinical validation of ctDNA analysis in oncology by detecting KRAS and BRAF point mutations in mCRC patients and has demonstrated that a blood test could replace tumor section analysis for the detection of KRAS and BRAF mutations. The Intplex(®) test can be adapted to all mutations, genes, or cancers and enables rapid, highly sensitive, cost-effective, and repetitive analysis. As regards to the determination of mutations on cfDNA Intplex(®) is limited to the mutational status of known hotspot mutation; it is a "targeted approach." However, it offers the opportunity in detecting quantitatively and dynamically mutation and could constitute a noninvasive attractive tool potentially allowing diagnosis, prognosis, theranostics, therapeutic monitoring, and follow-up of cancer patients expanding the scope of personalized cancer medicine.

High incidence of large deletions in the PMS2 gene in Spanish Lynch syndrome families.

PubMed

Brea-Fernández, A J; Cameselle-Teijeiro, J M; Alenda, C; Fernández-Rozadilla, C; Cubiella, J; Clofent, J; Reñé, J M; Anido, U; Milá, M; Balaguer, F; Castells, A; Castellvi-Bel, S; Jover, R; Carracedo, A; Ruiz-Ponte, C

2014-06-01

Lynch syndrome (LS) is caused by germline mutations in one of the four mismatch repair (MMR) genes. Defects in this pathway lead to microsatellite instability (MSI) in DNA tumors, which constitutes the molecular hallmark of this disease. Selection of patients for genetic testing in LS is usually based on fulfillment of diagnostic clinical criteria (i.e. Amsterdam criteria or the revised Bethesda guidelines). However, following these criteria PMS2 mutations have probably been underestimated as their penetrances appear to be lower than those of the other MMR genes. The use of universal MMR study-based strategies, using MSI testing and immunohistochemical (IHC) staining, is being one proposed alternative. Besides, germline mutation detection in PMS2 is complicated by the presence of highly homologous pseudogenes. Nevertheless, specific amplification of PMS2 by long-range polymerase chain reaction (PCR) and the improvement of the analysis of large deletions/duplications by multiplex ligation-dependent probe amplification (MLPA) overcome this difficulty. By using both approaches, we analyzed 19 PMS2-suspected carriers who have been selected by clinical or universal strategies and found five large deletions and one frameshift mutation in PMS2 in six patients (31%). Owing to the high incidence of large deletions found in our cohort, we recommend MLPA analysis as the first-line method for searching germline mutations in PMS2. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

40 CFR 799.9510 - TSCA bacterial reverse mutation test.

Code of Federal Regulations, 2010 CFR

2010-07-01

... the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Research. 31, 347-364 (1975). (2) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella Mutagenicity Test. Mutation Research. 113... Bridges, B.A. Use of a Simplified Fluctuation Test to Detect Low Levels of Mutagens. Mutation Research. 38...

40 CFR 799.9510 - TSCA bacterial reverse mutation test.

Code of Federal Regulations, 2011 CFR

2011-07-01

... the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Research. 31, 347-364 (1975). (2) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella Mutagenicity Test. Mutation Research. 113... Bridges, B.A. Use of a Simplified Fluctuation Test to Detect Low Levels of Mutagens. Mutation Research. 38...

40 CFR 799.9510 - TSCA bacterial reverse mutation test.

Code of Federal Regulations, 2012 CFR

2012-07-01

... the Salmonella/Mammalian-Microsome Mutagenicity Test. Mutation Research. 31, 347-364 (1975). (2) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella Mutagenicity Test. Mutation Research. 113... Bridges, B.A. Use of a Simplified Fluctuation Test to Detect Low Levels of Mutagens. Mutation Research. 38...

Hereditary diffuse gastric cancer: surgery, surveillance and unanswered questions.

PubMed

Cisco, Robin M; Norton, Jeffrey A

2008-08-01

Hereditary diffuse gastric cancer (HDGC) is an inherited cancer-susceptibility syndrome characterized by autosomal dominance and high penetrance. In 30-50% of cases, a causative germline mutation in CDH1, the E-cadherin gene, may be identified. Female carriers of CDH1 mutations also have an increased (20-40%) risk of lobular breast cancer. Endoscopic surveillance of patients with CDH1 mutations is ineffective because early foci of HDGC are typically small and underlie normal mucosa. CDH1 mutation carriers are therefore offered the option of prophylactic gastrectomy, which commonly reveals early foci of invasive signet-ring cell cancer. We review recommendations for genetic testing, surveillance and prophylactic surgery in HDGC. Areas for future research are discussed, including development of new screening modalities, optimal timing of prophylactic gastrectomy, identification of additional causative mutations in HDGC, management of patients with CDH1 missense mutations and prevention/early detection of lobular breast cancer in CDH1 mutation carriers.

RAS testing in metastatic colorectal cancer: advances in Europe.

PubMed

Van Krieken, J Han J M; Rouleau, Etienne; Ligtenberg, Marjolijn J L; Normanno, Nicola; Patterson, Scott D; Jung, Andreas

2016-04-01

Personalized medicine shows promise for maximizing efficacy and minimizing toxicity of anti-cancer treatment. KRAS exon 2 mutations are predictive of resistance to epidermal growth factor receptor-directed monoclonal antibodies in patients with metastatic colorectal cancer. Recent studies have shown that broader RAS testing (KRAS and NRAS) is needed to select patients for treatment. While Sanger sequencing is still used, approaches based on various methodologies are available. Few CE-approved kits, however, detect the full spectrum of RAS mutations. More recently, "next-generation" sequencing has been developed for research use, including parallel semiconductor sequencing and reversible termination. These techniques have high technical sensitivities for detecting mutations, although the ideal threshold is currently unknown. Finally, liquid biopsy has the potential to become an additional tool to assess tumor-derived DNA. For accurate and timely RAS testing, appropriate sampling and prompt delivery of material is critical. Processes to ensure efficient turnaround from sample request to RAS evaluation must be implemented so that patients receive the most appropriate treatment. Given the variety of methodologies, external quality assurance programs are important to ensure a high standard of RAS testing. Here, we review technical and practical aspects of RAS testing for pathologists working with metastatic colorectal cancer tumor samples. The extension of markers from KRAS to RAS testing is the new paradigm for biomarker testing in colorectal cancer.

Detection of epidermal growth factor receptor gene T790M mutation in cytology samples using the cobas® EGFR mutation test.

PubMed

Satouchi, Miyako; Tanaka, Hiroshi; Yoshioka, Hiroshige; Shimokawaji, Tadasuke; Mizuno, Keiko; Takeda, Koji; Yoshino, Ichiro; Seto, Takashi; Kurata, Takayasu; Tashiro, Naoki; Hagiwara, Koichi

2017-09-01

Detection of epidermal growth factor receptor (EGFR) gene mutations is essential in deciding therapeutic strategy in non-small cell lung cancer (NSCLC) patients at initial diagnosis. Moreover, in EGFR mutation-positive (EGFRm) NSCLC patients, re-biopsy at disease progression to clarify resistance mechanisms is also important. However, collecting histology samples is often difficult because of inaccessibility and invasiveness. In some cases, only cytology samples can be collected, and studies have reported that cytology samples are appropriate for EGFR gene mutation testing. The cobas ® EGFR Mutation Test (Roche Molecular Systems Inc., Branchburg, New Jersey, USA) is approved as a companion diagnostic for osimertinib, a third-generation EGFR-tyrosine kinase inhibitor approved in Japan. However, it is not clear whether the EGFR T790M mutation can be detected in cytology samples using this test. The primary objective of this study was to assess concordance of EGFR T790M gene mutation detection between histology and matched cytology samples using the cobas ® EGFR Mutation Test. We conducted a multicenter, observational study in Japan. Overall, 41 EGFRm NSCLC patients who had both histology and cytology samples collected at the same time at re-biopsy and with the results of EGFR mutation test using histology samples were enrolled. The EGFR mutation status of both sample types was tested using the cobas ® EGFR Mutation Test and the concordance rates were calculated. The EGFR T790M mutation detection rate in histology and cytology samples was 42.5% and 37.5%, respectively. The overall percent agreement between the histology and cytology samples was 91.7%. These data demonstrate that the cobas ® EGFR Mutation Test can detect the EGFR T790M mutation in both cytology and histology samples. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

EGFR Mutation Testing Practices within the Asia Pacific Region

PubMed Central

Kerr, Keith M.; Utomo, Ahmad; Rajadurai, Pathmanathan; Tran, Van Khanh; Du, Xiang; Chou, Teh-Ying; Enriquez, Ma. Luisa D.; Lee, Geon Kook; Iqbal, Jabed; Shuangshoti, Shanop; Chung, Jin-Haeng; Hagiwara, Koichi; Liang, Zhiyong; Normanno, Nicola; Park, Keunchil; Toyooka, Shinichi; Tsai, Chun-Ming; Waring, Paul; Zhang, Li; McCormack, Rose; Ratcliffe, Marianne; Itoh, Yohji; Sugeno, Masatoshi; Mok, Tony

2015-01-01

Introduction: The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in EGFR mutation-positive non–small-cell lung cancer (NSCLC) patients necessitates accurate, timely testing. Although EGFR mutation testing has been adopted by many laboratories in Asia, data are lacking on the proportion of NSCLC patients tested in each country, and the most commonly used testing methods. Methods: A retrospective survey of records from NSCLC patients tested for EGFR mutations during 2011 was conducted in 11 Asian Pacific countries at 40 sites that routinely performed EGFR mutation testing during that period. Patient records were used to complete an online questionnaire at each site. Results: Of the 22,193 NSCLC patient records surveyed, 31.8% (95% confidence interval: 31.2%–32.5%) were tested for EGFR mutations. The rate of EGFR mutation positivity was 39.6% among the 10,687 cases tested. The majority of samples were biopsy and/or cytology samples (71.4%). DNA sequencing was the most commonly used testing method accounting for 40% and 32.5% of tissue and cytology samples, respectively. A pathology report was available only to 60.0% of the sites, and 47.5% were not members of a Quality Assurance Scheme. Conclusions: In 2011, EGFR mutation testing practices varied widely across Asia. These data provide a reference platform from which to improve the molecular diagnosis of NSCLC, and EGFR mutation testing in particular, in Asia. PMID:25376513

EGFR mutation testing practices within the Asia Pacific region: results of a multicenter diagnostic survey.

PubMed

Yatabe, Yasushi; Kerr, Keith M; Utomo, Ahmad; Rajadurai, Pathmanathan; Tran, Van Khanh; Du, Xiang; Chou, Teh-Ying; Enriquez, Ma Luisa D; Lee, Geon Kook; Iqbal, Jabed; Shuangshoti, Shanop; Chung, Jin-Haeng; Hagiwara, Koichi; Liang, Zhiyong; Normanno, Nicola; Park, Keunchil; Toyooka, Shinichi; Tsai, Chun-Ming; Waring, Paul; Zhang, Li; McCormack, Rose; Ratcliffe, Marianne; Itoh, Yohji; Sugeno, Masatoshi; Mok, Tony

2015-03-01

The efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in EGFR mutation-positive non-small-cell lung cancer (NSCLC) patients necessitates accurate, timely testing. Although EGFR mutation testing has been adopted by many laboratories in Asia, data are lacking on the proportion of NSCLC patients tested in each country, and the most commonly used testing methods. A retrospective survey of records from NSCLC patients tested for EGFR mutations during 2011 was conducted in 11 Asian Pacific countries at 40 sites that routinely performed EGFR mutation testing during that period. Patient records were used to complete an online questionnaire at each site. Of the 22,193 NSCLC patient records surveyed, 31.8% (95% confidence interval: 31.2%-32.5%) were tested for EGFR mutations. The rate of EGFR mutation positivity was 39.6% among the 10,687 cases tested. The majority of samples were biopsy and/or cytology samples (71.4%). DNA sequencing was the most commonly used testing method accounting for 40% and 32.5% of tissue and cytology samples, respectively. A pathology report was available only to 60.0% of the sites, and 47.5% were not members of a Quality Assurance Scheme. In 2011, EGFR mutation testing practices varied widely across Asia. These data provide a reference platform from which to improve the molecular diagnosis of NSCLC, and EGFR mutation testing in particular, in Asia.

Adjusting for background mutation frequency biases improves the identification of cancer driver genes.

PubMed

Evans, Perry; Avey, Stefan; Kong, Yong; Krauthammer, Michael

2013-09-01

A common goal of tumor sequencing projects is finding genes whose mutations are selected for during tumor development. This is accomplished by choosing genes that have more non-synonymous mutations than expected from an estimated background mutation frequency. While this background frequency is unknown, it can be estimated using both the observed synonymous mutation frequency and the non-synonymous to synonymous mutation ratio. The synonymous mutation frequency can be determined across all genes or in a gene-specific manner. This choice introduces an interesting trade-off. A gene-specific frequency adjusts for an underlying mutation bias, but is difficult to estimate given missing synonymous mutation counts. Using a genome-wide synonymous frequency is more robust, but is less suited for adjusting biases. Studying four evaluation criteria for identifying genes with high non-synonymous mutation burden (reflecting preferential selection of expressed genes, genes with mutations in conserved bases, genes with many protein interactions, and genes that show loss of heterozygosity), we find that the gene-specific synonymous frequency is superior in the gene expression and protein interaction tests. In conclusion, the use of the gene-specific synonymous mutation frequency is well suited for assessing a gene's non-synonymous mutation burden.

Rare hereditary cause of chronic pancreatitis in a young male: SPINK1 mutation

PubMed Central

Patel, Janaki; Madan, Arina; Gammon, Amanda; Sossenheimer, Michael; Samadder, Niloy Jewel

2017-01-01

Hereditary chronic pancreatitis associated with a mutation in the serine protease inhibitor, Kazal Type-1 (SPINK-1 gene) is extremely rare. The SPINK1 mutation results in trypsinogen activation which predisposes to chronic pancreatitis predominately when combined with CFTR gene mutations. It presents as either chronic or recurrent acute pancreatitis. Symptom control and management of complications is important. Active surveillance with cross-sectional imaging for pancreatic malignancy in individuals with hereditary pancreatitis is advocated due to individuals being high risk. We present an unusual case of a young male who initially presented with renal colic and was incidentally diagnosed with severe chronic pancreatitis on abdominal imaging, with genetic testing confirming a homozygous SPINK1 mutation. PMID:29515728

Rare hereditary cause of chronic pancreatitis in a young male: SPINK1 mutation.

PubMed

Patel, Janaki; Madan, Arina; Gammon, Amanda; Sossenheimer, Michael; Samadder, Niloy Jewel

2017-01-01

Hereditary chronic pancreatitis associated with a mutation in the serine protease inhibitor, Kazal Type-1 (SPINK-1 gene) is extremely rare. The SPINK1 mutation results in trypsinogen activation which predisposes to chronic pancreatitis predominately when combined with CFTR gene mutations. It presents as either chronic or recurrent acute pancreatitis. Symptom control and management of complications is important. Active surveillance with cross-sectional imaging for pancreatic malignancy in individuals with hereditary pancreatitis is advocated due to individuals being high risk. We present an unusual case of a young male who initially presented with renal colic and was incidentally diagnosed with severe chronic pancreatitis on abdominal imaging, with genetic testing confirming a homozygous SPINK1 mutation.

KRAS mutation testing in metastatic colorectal cancer

PubMed Central

Tan, Cong; Du, Xiang

2012-01-01

The KRAS oncogene is mutated in approximately 35%-45% of colorectal cancers, and KRAS mutational status testing has been highlighted in recent years. The most frequent mutations in this gene, point substitutions in codons 12 and 13, were validated as negative predictors of response to anti-epidermal growth factor receptor antibodies. Therefore, determining the KRAS mutational status of tumor samples has become an essential tool for managing patients with colorectal cancers. Currently, a variety of detection methods have been established to analyze the mutation status in the key regions of the KRAS gene; however, several challenges remain related to standardized and uniform testing, including the selection of tumor samples, tumor sample processing and optimal testing methods. Moreover, new testing strategies, in combination with the mutation analysis of BRAF, PIK3CA and loss of PTEN proposed by many researchers and pathologists, should be promoted. In addition, we recommend that microsatellite instability, a prognostic factor, be added to the abovementioned concomitant analysis. This review provides an overview of KRAS biology and the recent advances in KRAS mutation testing. This review also addresses other aspects of status testing for determining the appropriate treatment and offers insight into the potential drawbacks of mutational testing. PMID:23066310

DOE Office of Scientific and Technical Information (OSTI.GOV)

Greenberg, C.R.; Taylor, C.D.; Haworth, J.C.

The authors have discovered a single homoallelic nucleotide substitution as the putative cause of the perinatal (lethal) form of hypophosphatasia in Canadian Mennonites. Previous linkage and haplotype analysis in this population suggested that a single mutational event was responsible for this autosomal recessive form of hypophosphatasia. The mutation is a guanosine-to-adenosine substitution at nucleotide position 1177 in exon 10 of the tissue nonspecific (liver/bone/kidney) alkaline phosphatase gene. This Gly[sup 317] [yields] Asp mutation segregates exclusively with the heterozygote phenotype previously assigned by biochemical testing (maximum combined lod score of 18.24 at [theta] = 0.00). This putative disease-causing mutation has notmore » been described in controls nor in other non-Mennonite probands with both lethal and nonlethal forms of hypophosphatasia studied to date. This Gly[sup 317] [yields] Asp mutation changes a polar glycine to an acidic aspartate at amino acid position 317 within the highly conserved active site region of the 507-amino-acid polypeptide. Carrier screening for this lethal mutation in a high-risk population is now feasible. 15 refs., 2 figs.« less

Detection of somatic mutations by high-resolution DNA melting (HRM) analysis in multiple cancers.

PubMed

Gonzalez-Bosquet, Jesus; Calcei, Jacob; Wei, Jun S; Garcia-Closas, Montserrat; Sherman, Mark E; Hewitt, Stephen; Vockley, Joseph; Lissowska, Jolanta; Yang, Hannah P; Khan, Javed; Chanock, Stephen

2011-01-17

Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples.

Detection of Somatic Mutations by High-Resolution DNA Melting (HRM) Analysis in Multiple Cancers

PubMed Central

Gonzalez-Bosquet, Jesus; Calcei, Jacob; Wei, Jun S.; Garcia-Closas, Montserrat; Sherman, Mark E.; Hewitt, Stephen; Vockley, Joseph; Lissowska, Jolanta; Yang, Hannah P.; Khan, Javed; Chanock, Stephen

2011-01-01

Identification of somatic mutations in cancer is a major goal for understanding and monitoring the events related to cancer initiation and progression. High resolution melting (HRM) curve analysis represents a fast, post-PCR high-throughput method for scanning somatic sequence alterations in target genes. The aim of this study was to assess the sensitivity and specificity of HRM analysis for tumor mutation screening in a range of tumor samples, which included 216 frozen pediatric small rounded blue-cell tumors as well as 180 paraffin-embedded tumors from breast, endometrial and ovarian cancers (60 of each). HRM analysis was performed in exons of the following candidate genes known to harbor established commonly observed mutations: PIK3CA, ERBB2, KRAS, TP53, EGFR, BRAF, GATA3, and FGFR3. Bi-directional sequencing analysis was used to determine the accuracy of the HRM analysis. For the 39 mutations observed in frozen samples, the sensitivity and specificity of HRM analysis were 97% and 87%, respectively. There were 67 mutation/variants in the paraffin-embedded samples, and the sensitivity and specificity for the HRM analysis were 88% and 80%, respectively. Paraffin-embedded samples require higher quantity of purified DNA for high performance. In summary, HRM analysis is a promising moderate-throughput screening test for mutations among known candidate genomic regions. Although the overall accuracy appears to be better in frozen specimens, somatic alterations were detected in DNA extracted from paraffin-embedded samples. PMID:21264207

Rapid detection of rifampin resistance in Mycobacterium tuberculosis isolates from India and Mexico by a molecular beacon assay.

PubMed

Varma-Basil, Mandira; El-Hajj, Hiyam; Colangeli, Roberto; Hazbón, Manzour Hernando; Kumar, Sujeet; Bose, Mridula; Bobadilla-del-Valle, Miriam; García, Lourdes García; Hernández, Araceli; Kramer, Fred Russell; Osornio, Jose Sifuentes; Ponce-de-León, Alfredo; Alland, David

2004-12-01

We assessed the performance of a rapid, single-well, real-time PCR assay for the detection of rifampin-resistant Mycobacterium tuberculosis by using clinical isolates from north India and Mexico, regions with a high incidence of tuberculosis. The assay uses five differently colored molecular beacons to determine if a short region of the M. tuberculosis rpoB gene contains mutations that predict rifampin resistance in most isolates. Until now, the assay had not been sufficiently tested on samples from countries with a high incidence of tuberculosis. In the present study, the assay detected mutations in 16 out of 16 rifampin-resistant isolates from north India (100%) and in 55 of 64 rifampin-resistant isolates from Mexico (86%) compared to results with standard susceptibility testing. The assay did not detect mutations (a finding predictive of rifampin susceptibility) in 37 out of 37 rifampin-susceptible isolates from India (100%) and 125 out of 126 rifampin-susceptible isolates from Mexico (99%). DNA sequencing revealed that none of the nine rifampin-resistant isolates from Mexico, which were misidentified as rifampin susceptible by the molecular beacon assay, contained a mutation in the region targeted by the molecular beacons. The one rifampin-susceptible isolate from Mexico that appeared to be rifampin resistant by the molecular beacon assay contained an S531W mutation, which is usually associated with rifampin resistance. Of the rifampin-resistant isolates that were correctly identified in the molecular beacon assay, one contained a novel L530A mutation and another contained a novel deletion between codons 511 and 514. Overall, the molecular beacon assay appears to have sufficient sensitivity (89%) and specificity (99%) for use in countries with a high prevalence of tuberculosis.

Crystal Genetics, Inc.

PubMed

Kermani, Bahram G

2016-07-01

Crystal Genetics, Inc. is an early-stage genetic test company, focused on achieving the highest possible clinical-grade accuracy and comprehensiveness for detecting germline (e.g., in hereditary cancer) and somatic (e.g., in early cancer detection) mutations. Crystal's mission is to significantly improve the health status of the population, by providing high accuracy, comprehensive, flexible and affordable genetic tests, primarily in cancer. Crystal's philosophy is that when it comes to detecting mutations that are strongly correlated with life-threatening diseases, the detection accuracy of every single mutation counts: a single false-positive error could cause severe anxiety for the patient. And, more importantly, a single false-negative error could potentially cost the patient's life. Crystal's objective is to eliminate both of these error types.

40 CFR 799.9510 - TSCA bacterial reverse mutation test.

Code of Federal Regulations, 2013 CFR

2013-07-01

... Mutagenicity Test. Mutation Research. 31, 347-364 (1975). (2) Maron, D.M. and Ames, B.N. Revised Methods for the Salmonella Mutagenicity Test. Mutation Research. 113, 173-215 (1983). (3) Gatehouse, D., Haworth... Fluctuation Test to Detect Low Levels of Mutagens. Mutation Research. 38, 33-42 (1976). (10) Hubbard, S.A...

EGFR gene copy number alterations are not a useful screening tool for predicting EGFR mutation status in lung adenocarcinoma.

PubMed

Russell, Prudence A; Yu, Yong; Do, Hongdo; Clay, Timothy D; Moore, Melissa M; Wright, Gavin M; Conron, Matthew; Wainer, Zoe; Dobrovic, Alexander; McLachlan, Sue-Anne

2014-01-01

We investigated if gene copy number (GCN) alterations of the epidermal growth factor receptor (EGFR), as detected by silver enhanced in situ hybridisation (SISH), could be used to select patients for EGFR mutation testing. Resected lung adenocarcinoma specimens with adequate tumour were identified. EGFR SISH was performed using the Ventana Benchmark Ultra platform. EGFR GCN was classified according to the Colorado Classification System. EGFR mutations were scanned by high resolution melting and confirmed by Sanger sequencing. Thirty-four of 96 tumours were EGFR SISH positive (35%), and 31 of 96 tumours harboured one or more EGFR mutations (32%). Of 31 EGFR-mutant tumours, 18 were EGFR SISH positive (58%). There was a statistically significant relationship between the presence of an EGFR mutation and EGFR GCN (p = 0.003). Thirteen of 31 EGFR-mutant tumours were EGFR SISH negative (42%), and 16 of 65 EGFR-wild type tumours were EGFR SISH positive (24%). The sensitivity, specificity, positive predictive value and negative predictive value were 58%, 75%, 52.9% and 79%, respectively. Despite a significant relationship between EGFR GCN alterations and EGFR mutations, our results indicate that EGFR GCN as detected by SISH is not a suitable way to select patients for EGFR mutation testing.

The Potential Contribution of BRCA Mutations to Early Onset and Familial Breast Cancer in Uzbekistan.

PubMed

Abdikhakimov, Abdulla; Tukhtaboeva, Mukaddas; Adilov, Bakhtiyar; Turdikulova, Shahlo

2016-01-01

Breast cancer is the most common malignancy in women and affects approximately 1 out of 8 females in the US. Risk of developing breast cancer is strongly influenced by genetic factors. Germ-line mutations in BRCA1 and BRCA2 genes are associated with 5-10% of breast cancer incidence. To reduce the risk of developing cancer and to increase the likelihood of early detection, carriers of BRCA1 or BRCA2 mutations are offered surveillance programs and effective preventive medical interventions. Identification of founder mutations of BRCA1/2 in high risk communities can have a significant impact on the management of hereditary cancer at the level of the national healthcare systems, making genetic testing more affordable and cost-effective. BRCA1 and BRCA2 mutations in breast cancer patients have not been characterized in the Uzbek population. This pilot study aimed to investigate the contribution of BRCA1 and BRCA2 mutation to early onset and familial cases of breast cancer in Uzbekistan. A total of 67 patients with breast cancer and 103 age-matched disease free controls were included in this study. Utilizing SYBR Green based real-time allele-specific PCR, we have analyzed DNA samples of patients with breast cancer and disease free controls to identify the following BRCA1 and BRCA2 mutations: BRCA1 5382insC, BRCA1 4153delA, BRCA1 185delAG, BRCA1 300T>G, BRCA2 6174delT. Three unrelated samples (4.5%) were found to be positive for the heterozygous 5382insCBRCA1 mutation, representing a possible founder mutation in the Uzbek population, supporting the need for larger studies examining the contribution of this mutation to breast cancer incidence in Uzbekistan. We did not find BRCA1 4153delA, BRCA1 185delAG, BRCA1 300T>G, and BRCA2 6174delT mutations. This preliminary evidence suggests a potential contribution of BRCA1 5382insC mutation to breast cancer development in Uzbek population. Taking into account a high disease penetrance in carriers of BRCA1 mutation, it seems reasonable to recommend inclusion of the 5382insC mutation test in future research on the development of screening programs for breast cancer prevention in Uzbekistan.

The relative contribution of target-site mutations in complex acaricide resistant phenotypes as assessed by marker assisted backcrossing in Tetranychus urticae.

PubMed

Riga, Maria; Bajda, Sabina; Themistokleous, Christos; Papadaki, Stavrini; Palzewicz, Maria; Dermauw, Wannes; Vontas, John; Leeuwen, Thomas Van

2017-08-23

The mechanisms underlying insecticide and acaricide resistance in insects and mites are often complex, including additive effects of target-site insensitivity, increased metabolism and transport. The extent to which target-site resistance mutations contribute to the resistance phenotype is, however, not well studied. Here, we used marker-assisted backcrossing to create 30 congenic lines carrying nine mutations (alone, or in combination in a few cases) associated with resistance to avermectins, pyrethroids, mite growth inhibitors and mitochondrial complex III inhibitors (QoI) in a polyphagous arthropod pest, the spider mite Tetranychus urticae. Toxicity tests revealed that mutations in the voltage-gated sodium channel, chitin synthase 1 and cytochrome b confer high levels of resistance and, when fixed in a population, these mutations alone can result in field failure of acaricide treatment. In contrast, although we confirmed the implication of mutations in glutamate-gated chloride channels in abamectin and milbemectin insensitivity, these mutations do not lead to the high resistance levels that are often reported in abamectin resistant strains of T. urticae. Overall, this study functionally validates reported target-site resistance mutations in T. urticae, by uncoupling them from additional mechanisms, allowing to finally investigate the strength of the conferred phenotype in vivo.

The use of high resolution melting analysis to detect Fabry mutations in heterozygous females via dry bloodspots.

PubMed

Tai, Chang-Long; Liu, Mei-Ying; Yu, Hsiao-Chi; Chiang, Chiang-Chuan; Chiang, Hung; Suen, Jeng-Hung; Kao, Shu-Min; Huang, Yu-Hsiu; Wu, Tina Jui-Ting; Yang, Chia-Feng; Tsai, Fang-Chih; Lin, Ching-Yuang; Chang, Jan-Gowth; Chen, Hong-Duo; Niu, Dau-Ming

2012-02-18

As an X-linked genetic disorder, Fabry disease was first thought to affect males only, and females were generally considered to be asymptomatic carriers. However, recent research suggests that female carriers of Fabry disease may still develop vital organ damage causing severe morbidity and mortality. In the previous newborn screening, from 299,007 newborns, we identified a total of 20 different Fabry mutations and 121 newborns with Fabry mutations. However, we found that most female carriers are not detected by enzyme assays. A streamlined method for high resolution melting (HRM) analysis was designed to screen for GLA gene mutations using a same PCR and melting program. Primer sets were designed to cover the 7 exons and the Chinese common intronic mutation, IVS4+919G>A of GLA gene. The HRM analysis was successful in identifying heterozygous and hemizygous patients with the 20 surveyed mutations. We were also successful in using this method to test dry blood spots of newborns afflicted with Fabry mutations without having to determine DNA concentration before PCR amplification. The results of this study show that HRM could be a reliable and sensitive method for use in the rapid screening of females for GLA mutations. Copyright © 2011 Elsevier B.V. All rights reserved.

Constitutive RET tyrosine kinase activation in hereditary medullary thyroid cancer: clinical opportunities.

PubMed

Machens, A; Lorenz, K; Dralle, H

2009-07-01

The ground-breaking discovery of genotype-phenotype relationships in hereditary medullary thyroid cancer has greatly facilitated early prophylactic thyroidectomy. Its timing depends not solely on a positive gene test but, more importantly, on the type of the REarranged during Transfection (RET) mutation and its underlying mode of RET receptor tyrosine kinase activation. In the past decade, the therapeutic corridor opened by molecular information has been defined down to a remarkable level of detail. Based on mutational risk profiles, preemptive thyroidectomy is recommended at 6 months of age for carriers of highest-risk mutations, before the age of 5 years for carriers of high-risk mutations, and before the age of 5 or 10 years for carriers of least-high-risk mutations. Additional lymph node dissection may not be needed in the absence of increased preoperative basal calcitonin levels. Better comprehension of RET function should enable the design of targeted therapies for RET carriers beyond surgical cure in whom the DNA-based 'window of opportunity' has been missed.

Increased frequency of co-existing JAK2 exon-12 or MPL exon-10 mutations in patients with low JAK2(V617F) allelic burden.

PubMed

Nussenzveig, Roberto H; Pham, Ha T; Perkins, Sherrie L; Prchal, Josef T; Agarwal, Archana M; Salama, Mohamed E

2016-01-01

The frequency of co-existing JAK2(V617F)/MPL and JAK2(V617F)/JAK2 exon-12 mutations has not been previously investigated in MPNs. Poor survival was reported in primary myelofibrosis with low JAK2(V617F) allelic burden. However, mutational status of JAK2 exon-12 or MPL were not reported in these patients. This study developed a cost-effective multiplex high resolution melt assay that screens for mutations in JAK2 gene exons-12 and -14 ((V617F)) and MPL gene exon-10. Co-existing mutations with JAK2(V617F) were detected in 2.9% (6/208; two JAK2 exon-12 and four MPL exon-10) patient specimens with known JAK2(V617F) (allelic-burden range: 0.1-96.8%). Co-existing mutations were detected in specimens with < 12% JAK2(V617F) allelic burden. Current WHO guidelines do not recommend further testing once JAK2(V617F) mutation is detected in MPNs. The findings, however, indicate that quantification of JAK2(V617F) allele burden may be clinically relevant in MPNs and in those with low allelic burden additional testing for JAK2 exon-12 and MPL exon-10 mutation should be pursued.

Identification of a founder BRCA1 mutation in the Moroccan population.

PubMed

Quiles, F; Teulé, À; Martinussen Tandstad, N; Feliubadaló, L; Tornero, E; Del Valle, J; Menéndez, M; Salinas, M; Wethe Rognlien, V; Velasco, A; Izquierdo, A; Capellá, G; Brunet, J; Lázaro, C

2016-10-01

Breast cancer (BC) is the most frequent cancer among women in Morocco. However, the role of the most prevalent BC-predisposing genes, BRCA1 and BRCA2, has been largely unexplored. To help define the role of BRCA1 in BC in Morocco, we characterized the first potential BRCA1 founder mutation in this population. Genetic testing of BRCA1 and BRCA2 in BC high-risk families identified mutation BRCA1 c.5309G>T, p.(Gly1770Val) or G1770V in five independent families from Morocco, suggesting a founder effect. To confirm this hypothesis, haplotype construction was performed using seven intragenic and flanking BRCA1 microsatellite markers. Clinical data were also compiled. Clinical data from carriers of mutation G1770V correspond to data from carriers of BRCA1 pathogenic mutations. Microsatellite analysis showed a common haplotype for the five families in a region comprising 1.54 Mb, confirming G1770V as the first specific founder BRCA1 mutation in the Moroccan population. Our findings contribute to a better understanding of BC genetics in the Moroccan population. Nevertheless, comprehensive studies of mutation G1770V in large series of BC patients from Morocco are needed to assess the real prevalence of this mutation and to improve genetic testing and risk assessment in this population. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A novel mutation in the TCOF1 gene found in two Chinese cases of Treacher Collins syndrome.

PubMed

Zhang, Xu; Fan, Yue; Zhang, Ying; Xue, Huadan; Chen, Xiaowei

2013-09-01

To analyze the clinical features, hearing rehabilitation and family related gene mutations in the Chinese cases of Treacher Collins syndrome (TCS). The purpose of this study is to emphasize the genetic research result correlating with the clinical assessment of TCS in Chinese families. Six patients with tentative diagnosis and family members of two patients were analyzed in this study. The analysis included medical histories, clinical analysis, hearing tests and genetic tests. The TCOF1, POLR1C and POLR1D genes were sequenced to identify the pathogenic mutation responsible for the development of TCS. The two TCS cases exhibited high phenotypic variability. One novel heterozygous mutation (c.4420 C>T) in the TCOF1 gene was identified. The mutations were found in the TCS patients but not in any of their unaffected family members or the 200 unrelated control subjects. A novel TCOF1 c.4420 C>T mutation can be a cause of TCS in Chinese. We think that genetic studies to assess patients with mandibulofacial dysostosis may assist in making TCS diagnosis and providing consultant for their families. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Adverse Clinical Outcome Associated With Mutations That Typify African American Colorectal Cancers.

PubMed

Wang, Zhenghe; Li, Li; Guda, Kishore; Chen, Zhengyi; Barnholtz-Sloan, Jill; Park, Young Soo; Markowitz, Sanford D; Willis, Joseph

2016-12-01

African Americans have the highest incidence and mortality from colorectal cancer (CRC) of any US racial group. We recently described a panel of 15 genes that are statistically significantly more likely to be mutated in CRCs from African Americans than in Caucasians (AA-CRC genes). The current study investigated the outcomes associated with these mutations in African American CRCs (AA-CRCs). In a cohort of 66 patients with stage I-III CRCs, eight of 27 CRCs with AA-CRC gene mutations (Mut+) developed metastatic disease vs only four of 39 mutation-negative (Mut-) cases (P = .03, Cox regression model with two-sided Wald test). Moreover, among stage III cases (n = 33), Mut+ cancers were nearly three times more likely to relapse as Mut- cases (7 of 15 Mut+ vs 3 of 18 Mut-; P = .03, Cox regression model with two-sided Wald test). AA-CRC mutations may thus define a high-risk subset of CRCs that contributes to the overall disparity in CRC outcomes observed in African Americans. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

A rare CYP21A2 mutation in a congenital adrenal hyperplasia kindred displaying genotype-phenotype nonconcordance.

PubMed

Khattab, Ahmed; Yuen, Tony; Al-Malki, Sultan; Yau, Mabel; Kazmi, Diya; Sun, Li; Harbison, Madeleine; Haider, Shozeb; Zaidi, Mone; New, Maria I

2016-01-01

Congenital adrenal hyperplasia (CAH) owing to 21-hydroxylase deficiency is caused by the autosomal recessive inheritance of mutations in the gene CYP21A2. CYP21A2 mutations lead to variable impairment of the 21-hydroxylase enzyme, which, in turn, is associated with three clinical phenotypes, namely, salt wasting, simple virilizing, and nonclassical CAH. However, it is known that a given mutation can associate with different clinical phenotypes, resulting in a high rate of genotype-phenotype nonconcordance. We aimed to study the genotype-phenotype nonconcordance in a family with three siblings affected with nonclassical CAH. All had hormonal evidence of nonclassical CAH, but this phenotype could not be explained by the genotype obtained from commercial CYP21A2 genetic testing, which revealed heterozygosity for the maternal 30 kb deletion mutation. We performed Sanger sequencing of the entire CYP21A2 gene in this family to search for a rare mutation that was not covered by commercial testing and found in the three siblings a second, rare c.1097G>A (p.R366H) mutation in exon 8. Computational modeling confirmed that this was a mild mutation consistent with nonclassical CAH. We recommend that sequencing of entire genes for rare mutations should be carried out when genotype-phenotype nonconcordance is observed in patients with autosomal recessive monogenic disorders, including CAH. © 2015 New York Academy of Sciences.

The Norwegian PMS2 founder mutation c.989-1G > T shows high penetrance of microsatellite instable cancers with normal immunohistochemistry.

PubMed

Grindedal, Eli Marie; Aarset, Harald; Bjørnevoll, Inga; Røyset, Elin; Mæhle, Lovise; Stormorken, Astrid; Heramb, Cecilie; Medvik, Heidi; Møller, Pål; Sjursen, Wenche

2014-01-01

Using immunohistochemistry (IHC) to select cases for mismatch repair (MMR) genetic testing, we failed to identify a large kindred with the deleterious PMS2 mutation c.989-1G > T. The purpose of the study was to examine the sensitivity of IHC and microsatellite instability-analysis (MSI) to identify carriers of the mutation, and to estimate its penetrance and expressions. All carriers and obligate carriers of the mutation were identified. All cancer diagnoses were confirmed. IHC and MSI-analysis were performed on available tumours. Penetrances of cancers included in the Amsterdam and the Bethesda Criteria, for MSI-high tumours and MSI-high and low tumours were calculated by the Kaplan-Meier algorithm. Probability for co-segregation of the mutation and cancers by chance was 0.000004. Fifty-six carriers or obligate carriers were identified. There was normal staining for PMS2 in 15/18 (83.3%) of tumours included in the AMS1/AMS2/Bethesda criteria. MSI-analysis showed that 15/21 (71.4%) of tumours were MSI-high and 4/21 (19.0%) were MSI-low. Penetrance at 70 years was 30.6% for AMS1 cancers (colorectal cancers), 42.8% for AMS2 cancers, 47.2% for Bethesda cancers, 55.6% for MSI-high and MSI-low cancers and 52.2% for MSI-high cancers. The mutation met class 5 criteria for pathogenicity. IHC was insensitive in detecting tumours caused by the mutation. Penetrance of cancers that displayed MSI was 56% at 70 years. Besides colorectal cancers, the most frequent expressions were carcinoma of the endometrium and breast in females and stomach and prostate in males.

The Norwegian PMS2 founder mutation c.989-1G > T shows high penetrance of microsatellite instable cancers with normal immunohistochemistry

PubMed Central

2014-01-01

Background Using immunohistochemistry (IHC) to select cases for mismatch repair (MMR) genetic testing, we failed to identify a large kindred with the deleterious PMS2 mutation c.989-1G > T. The purpose of the study was to examine the sensitivity of IHC and microsatellite instability-analysis (MSI) to identify carriers of the mutation, and to estimate its penetrance and expressions. Methods All carriers and obligate carriers of the mutation were identified. All cancer diagnoses were confirmed. IHC and MSI-analysis were performed on available tumours. Penetrances of cancers included in the Amsterdam and the Bethesda Criteria, for MSI-high tumours and MSI-high and low tumours were calculated by the Kaplan-Meier algorithm. Results Probability for co-segregation of the mutation and cancers by chance was 0.000004. Fifty-six carriers or obligate carriers were identified. There was normal staining for PMS2 in 15/18 (83.3%) of tumours included in the AMS1/AMS2/Bethesda criteria. MSI-analysis showed that 15/21 (71.4%) of tumours were MSI-high and 4/21 (19.0%) were MSI-low. Penetrance at 70 years was 30.6% for AMS1 cancers (colorectal cancers), 42.8% for AMS2 cancers, 47.2% for Bethesda cancers, 55.6% for MSI-high and MSI-low cancers and 52.2% for MSI-high cancers. Conclusions The mutation met class 5 criteria for pathogenicity. IHC was insensitive in detecting tumours caused by the mutation. Penetrance of cancers that displayed MSI was 56% at 70 years. Besides colorectal cancers, the most frequent expressions were carcinoma of the endometrium and breast in females and stomach and prostate in males. PMID:24790682

KRAS mutations testing in colorectal carcinoma patients in Italy: from guidelines to external quality assessment.

PubMed

Normanno, Nicola; Pinto, Carmine; Castiglione, Francesca; Bardelli, Alberto; Gambacorta, Marcello; Botti, Gerardo; Nappi, Oscar; Siena, Salvatore; Ciardiello, Fortunato; Taddei, Gianluigi; Marchetti, Antonio

2011-01-01

Monoclonal antibodies directed against the epidermal growth factor receptor (EGFR) have been approved for the treatment of patients with metastatic colorectal carcinoma (mCRC) that do not carry KRAS mutations. Therefore, KRAS testing has become mandatory to chose the most appropriate therapy for these patients. In order to guarantee the possibility for mCRC patients to receive an high quality KRAS testing in every Italian region, the Italian Association of Medical Oncology (AIOM) and the Italian Society of Pathology and Cytopathology -Italian division of the International Academy of Pathology (SIAPEC-IAP) started a program to improve KRAS testing. AIOM and SIAPEC identified a large panel of Italian medical oncologists, pathologists and molecular biologists that outlined guidelines for KRAS testing in mCRC patients. These guidelines include specific information on the target patient population, the biological material for molecular analysis, the extraction of DNA, and the methods for the mutational analysis that are summarized in this paper. Following the publication of the guidelines, the scientific societies started an external quality assessment scheme for KRAS testing. Five CRC specimens with known KRAS mutation status were sent to the 59 centers that participated to the program. The samples were validated by three referral laboratories. The participating laboratories were allowed to use their own preferred method for DNA extraction and mutational analysis and were asked to report the results within 4 weeks. The limit to pass the quality assessment was set at 100% of true responses. In the first round, only two centers did not pass (3%). The two centers were offered to participate to a second round and both centers failed again to pass. The results of this first Italian quality assessment for KRAS testing suggest that KRAS mutational analysis is performed with good quality in the majority of Italian centers. © 2011 Normanno et al.

KRAS Mutations Testing in Colorectal Carcinoma Patients in Italy: From Guidelines to External Quality Assessment

PubMed Central

Normanno, Nicola; Pinto, Carmine; Castiglione, Francesca; Bardelli, Alberto; Gambacorta, Marcello; Botti, Gerardo; Nappi, Oscar; Siena, Salvatore; Ciardiello, Fortunato; Taddei, GianLuigi; Marchetti, Antonio

2011-01-01

Background Monoclonal antibodies directed against the epidermal growth factor receptor (EGFR) have been approved for the treatment of patients with metastatic colorectal carcinoma (mCRC) that do not carry KRAS mutations. Therefore, KRAS testing has become mandatory to chose the most appropriate therapy for these patients. Methodology/Principal Findings In order to guarantee the possibility for mCRC patients to receive an high quality KRAS testing in every Italian region, the Italian Association of Medical Oncology (AIOM) and the Italian Society of Pathology and Cytopathology -Italian division of the International Academy of Pathology (SIAPEC-IAP) started a program to improve KRAS testing. AIOM and SIAPEC identified a large panel of Italian medical oncologists, pathologists and molecular biologists that outlined guidelines for KRAS testing in mCRC patients. These guidelines include specific information on the target patient population, the biological material for molecular analysis, the extraction of DNA, and the methods for the mutational analysis that are summarized in this paper. Following the publication of the guidelines, the scientific societies started an external quality assessment scheme for KRAS testing. Five CRC specimens with known KRAS mutation status were sent to the 59 centers that participated to the program. The samples were validated by three referral laboratories. The participating laboratories were allowed to use their own preferred method for DNA extraction and mutational analysis and were asked to report the results within 4 weeks. The limit to pass the quality assessment was set at 100% of true responses. In the first round, only two centers did not pass (3%). The two centers were offered to participate to a second round and both centers failed again to pass. Conclusions The results of this first Italian quality assessment for KRAS testing suggest that KRAS mutational analysis is performed with good quality in the majority of Italian centers. PMID:22216189

BRCA1 and BRCA2 mutations in ethnic Lebanese Arab women with high hereditary risk breast cancer.

PubMed

El Saghir, Nagi S; Zgheib, Nathalie K; Assi, Hussein A; Khoury, Katia E; Bidet, Yannick; Jaber, Sara M; Charara, Raghid N; Farhat, Rania A; Kreidieh, Firas Y; Decousus, Stephanie; Romero, Pierre; Nemer, Georges M; Salem, Ziad; Shamseddine, Ali; Tfayli, Arafat; Abbas, Jaber; Jamali, Faek; Seoud, Muhieddine; Armstrong, Deborah K; Bignon, Yves-Jean; Uhrhammer, Nancy

2015-04-01

Breast cancer is the most common malignancy among women in Lebanon and in Arab countries, with 50% of cases presenting before the age of 50 years. Between 2009 and 2012, 250 Lebanese women with breast cancer who were considered to be at high risk of carrying BRCA1 or BRCA2 mutations because of presentation at young age and/or positive family history (FH) of breast or ovarian cancer were recruited. Clinical data were analyzed statistically. Coding exons and intron-exon boundaries of BRCA1 and BRCA2 were sequenced from peripheral blood DNA. All patients were tested for BRCA1 rearrangements using multiplex ligation-dependent probe amplification (MLPA). BRCA2 MLPA was done in selected cases. Overall, 14 of 250 patients (5.6%) carried a deleterious BRCA mutation (7 BRCA1, 7 BRCA2) and 31 (12.4%) carried a variant of uncertain significance. Eight of 74 patients (10.8%) aged ≤40 years with positive FH and only 1 of 74 patients (1.4%) aged ≤40 years without FH had a mutated BRCA. Four of 75 patients (5.3%) aged 41-50 years with FH had a deleterious mutation. Only 1 of 27 patients aged >50 years at diagnosis had a BRCA mutation. All seven patients with BRCA1 mutations had grade 3 infiltrating ductal carcinoma and triple-negative breast cancer. Nine BRCA1 and 17 BRCA2 common haplotypes were observed. Prevalence of deleterious BRCA mutations is lower than expected and does not support the hypothesis that BRCA mutations alone cause the observed high percentage of breast cancer in young women of Lebanese and Arab descent. Studies to search for other genetic mutations are recommended. ©AlphaMed Press.

Highly sensitive detection of ESR1 mutations in cell-free DNA from patients with metastatic breast cancer using molecular barcode sequencing.

PubMed

Masunaga, Nanae; Kagara, Naofumi; Motooka, Daisuke; Nakamura, Shota; Miyake, Tomohiro; Tanei, Tomonori; Naoi, Yasuto; Shimoda, Masafumi; Shimazu, Kenzo; Kim, Seung Jin; Noguchi, Shinzaburo

2018-01-01

We aimed to develop a highly sensitive method to detect ESR1 mutations in cell-free DNA (cfDNA) using next-generation sequencing with molecular barcode (MB-NGS) targeting the hotspot segment (c.1600-1713). The sensitivity of MB-NGS was tested using serially diluted ESR1 mutant DNA and then cfDNA samples from 34 patients with metastatic breast cancer were analyzed with MB-NGS. The results of MB-NGS were validated in comparison with conventional NGS and droplet digital PCR (ddPCR). MB-NGS showed a higher sensitivity (0.1%) than NGS without barcode (1%) by reducing background errors. Of the cfDNA samples from 34 patients with metastatic breast cancer, NGS without barcode revealed seven mutations in six patients (17.6%) and MB-NGS revealed six additional mutations including three mutations not reported in the COSMIC database of breast cancer, resulting in total 13 ESR1 mutations in ten patients (29.4%). Regarding the three hotspot mutations, all the patients with mutations detected by MB-NGS had identical mutations detected by droplet digital PCR (ddPCR), and mutant allele frequency correlated very well between both (r = 0.850, p < 0.01). Moreover, all the patients without these mutations by MB-NGS were found to have no mutations by ddPCR. In conclusion, MB-NGS could successfully detect ESR1 mutations in cfDNA with a higher sensitivity of 0.1% than conventional NGS and was considered as clinically useful as ddPCR.

Prevalence of macrolide and fluoroquinolone resistance-mediating mutations in Mycoplasma genitalium in five cities in Russia and Estonia

PubMed Central

Shipitsyna, Elena; Rumyantseva, Tatiana; Golparian, Daniel; Khayrullina, Guzel; Lagos, Amaya C.; Edelstein, Inna; Joers, Kai; Jensen, Jörgen S.; Savicheva, Alevtina; Rudneva, Natalia; Sukhanova, Larisa; Kozlov, Roman; Guschin, Alexander

2017-01-01

Background and objective Resistance in the sexually transmitted bacterium Mycoplasma genitalium to all recommended therapeutic antimicrobials have rapidly emerged. However, to date, internationally reported resistance surveillance data for M. genitalium strains circulating in Eastern Europe are entirely lacking. The aim of this study was to estimate the prevalence of macrolide and fluoroquinolone resistance-associated mutations in M. genitalium in four cities in Russia and one in Estonia, 2013–2016. Materials and methods Consecutive urogenital samples found positive for M. genitalium during diagnostic testing were retrospectively analyzed for resistance-associated mutations in the 23S rRNA and parC genes using pyrosequencing and conventional Sanger sequencing, respectively. Results In total, 867 M. genitalium positive samples from 2013–2016 were analyzed. Macrolide resistance-associated mutations were detected in 4.6% of the samples from Russia (0.7–6.8% in different cities) and in 10% of the samples from Estonia. The mutations A2059G and A2058G were highly predominating in both Russia and Estonia, accounting together for 90.9% of the cases positive for nucleotide substitutions in the 23S rRNA gene. The rates of possible fluoroquinolone resistance-associated mutations were 6.2% in Russia (2.5–7.6% in different cities) and 5% in Estonia. The mutations S83I and S83N were the most frequent ones in Russia (24.4% each), whereas D87N highly predominated in Estonia (83.3% of all fluoroquinolone resistance-associated mutations). Approximately 1% of the samples in both countries harbored both macrolide and possible fluoroquinolone resistance-associated mutations, with A2058G and S83I being the most frequent combination (37.5%). Conclusions The prevalence of macrolide and fluoroquinolone resistance-associated mutations in M. genitalium was 4.6% and 6.2%, respectively, in Russia, and 10% and 5%, respectively, in Estonia. Despite the relatively low rates of macrolide and fluoroquinolone resistance in these countries, antimicrobial resistance surveillance and testing for resistance-associated mutations in M. genitalium positive cases would be valuable. PMID:28407014

Prevalence of macrolide and fluoroquinolone resistance-mediating mutations in Mycoplasma genitalium in five cities in Russia and Estonia.

PubMed

Shipitsyna, Elena; Rumyantseva, Tatiana; Golparian, Daniel; Khayrullina, Guzel; Lagos, Amaya C; Edelstein, Inna; Joers, Kai; Jensen, Jörgen S; Savicheva, Alevtina; Rudneva, Natalia; Sukhanova, Larisa; Kozlov, Roman; Guschin, Alexander; Unemo, Magnus

2017-01-01

Resistance in the sexually transmitted bacterium Mycoplasma genitalium to all recommended therapeutic antimicrobials have rapidly emerged. However, to date, internationally reported resistance surveillance data for M. genitalium strains circulating in Eastern Europe are entirely lacking. The aim of this study was to estimate the prevalence of macrolide and fluoroquinolone resistance-associated mutations in M. genitalium in four cities in Russia and one in Estonia, 2013-2016. Consecutive urogenital samples found positive for M. genitalium during diagnostic testing were retrospectively analyzed for resistance-associated mutations in the 23S rRNA and parC genes using pyrosequencing and conventional Sanger sequencing, respectively. In total, 867 M. genitalium positive samples from 2013-2016 were analyzed. Macrolide resistance-associated mutations were detected in 4.6% of the samples from Russia (0.7-6.8% in different cities) and in 10% of the samples from Estonia. The mutations A2059G and A2058G were highly predominating in both Russia and Estonia, accounting together for 90.9% of the cases positive for nucleotide substitutions in the 23S rRNA gene. The rates of possible fluoroquinolone resistance-associated mutations were 6.2% in Russia (2.5-7.6% in different cities) and 5% in Estonia. The mutations S83I and S83N were the most frequent ones in Russia (24.4% each), whereas D87N highly predominated in Estonia (83.3% of all fluoroquinolone resistance-associated mutations). Approximately 1% of the samples in both countries harbored both macrolide and possible fluoroquinolone resistance-associated mutations, with A2058G and S83I being the most frequent combination (37.5%). The prevalence of macrolide and fluoroquinolone resistance-associated mutations in M. genitalium was 4.6% and 6.2%, respectively, in Russia, and 10% and 5%, respectively, in Estonia. Despite the relatively low rates of macrolide and fluoroquinolone resistance in these countries, antimicrobial resistance surveillance and testing for resistance-associated mutations in M. genitalium positive cases would be valuable.

Low-grade prostate tumors can harbor signs of aggressive cancer | Center for Cancer Research

Cancer.gov

In a new study, Center for Cancer Research investigators found that low-grade and high-grade regions of prostate tumor tissue shared mutations typically linked to aggressive cancer. Testing for mutations to specific genes could help clinicians decide whether a patient with an initial low-grade result should undergo a follow-up biopsy. Learn more...

ColoSeq provides comprehensive lynch and polyposis syndrome mutational analysis using massively parallel sequencing.

PubMed

Pritchard, Colin C; Smith, Christina; Salipante, Stephen J; Lee, Ming K; Thornton, Anne M; Nord, Alex S; Gulden, Cassandra; Kupfer, Sonia S; Swisher, Elizabeth M; Bennett, Robin L; Novetsky, Akiva P; Jarvik, Gail P; Olopade, Olufunmilayo I; Goodfellow, Paul J; King, Mary-Claire; Tait, Jonathan F; Walsh, Tom

2012-07-01

Lynch syndrome (hereditary nonpolyposis colon cancer) and adenomatous polyposis syndromes frequently have overlapping clinical features. Current approaches for molecular genetic testing are often stepwise, taking a best-candidate gene approach with testing of additional genes if initial results are negative. We report a comprehensive assay called ColoSeq that detects all classes of mutations in Lynch and polyposis syndrome genes using targeted capture and massively parallel next-generation sequencing on the Illumina HiSeq2000 instrument. In blinded specimens and colon cancer cell lines with defined mutations, ColoSeq correctly identified 28/28 (100%) pathogenic mutations in MLH1, MSH2, MSH6, PMS2, EPCAM, APC, and MUTYH, including single nucleotide variants (SNVs), small insertions and deletions, and large copy number variants. There was 100% reproducibility of detection mutation between independent runs. The assay correctly identified 222 of 224 heterozygous SNVs (99.4%) in HapMap samples, demonstrating high sensitivity of calling all variants across each captured gene. Average coverage was greater than 320 reads per base pair when the maximum of 96 index samples with barcodes were pooled. In a specificity study of 19 control patients without cancer from different ethnic backgrounds, we did not find any pathogenic mutations but detected two variants of uncertain significance. ColoSeq offers a powerful, cost-effective means of genetic testing for Lynch and polyposis syndromes that eliminates the need for stepwise testing and multiple follow-up clinical visits. Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Germline BRCA1/BRCA2 mutations among high risk breast cancer patients in Jordan.

PubMed

Abdel-Razeq, Hikmat; Al-Omari, Amal; Zahran, Farah; Arun, Banu

2018-02-06

Breast cancer is the most common malignancy and the leading cause of cancer-related deaths among Jordanian women. With a median age of 50 years at diagnosis, a higher prevalence of hereditary breast cancer may be expected. The objective of this pilot study is to evaluate, for the first time, the contribution of germline mutations in BRCA1/2 to breast cancer among Jordanian patients. Jordanian breast cancer women with a selected high risk profile were invited to participate. Peripheral blood samples were obtained for DNA extraction. A detailed 3-generation family history was also collected. BRCA sequencing was performed at a reference laboratory. Mutations were classified as deleterious, suspected deleterious, variant of uncertain significance or favor polymorphisms. Patients' medical records were reviewed for extraction of clinical and tumor pathology data. One hundred patients were enrolled to the study. Median age was 40 (22-75) years. In total, 20 patients had deleterious and 7 suspected deleterious mutations in BRCA1 or BRCA2 genes. Seven variants of uncertain significance were also detected. After excluding patients tested subsequent to the index case in their families, highest mutation rates were observed among triple negatives (9/16, 56.3%) especially among those with positive family history of breast and/or ovarian cancer (9/13, 69.2%), patients with bilateral or second primary breast cancer (10/15, 66.7%) and those with family history of male breast cancer (2/5, 40.0%). BRCA1/2 mutations are not uncommon among selected Jordanian females with breast cancer. The contribution of these findings to much younger age at diagnosis is debatable. Although small, our selected patient cohort shows an important incidence of deleterious and suspected deleterious BRCA1/2 mutations suggesting that genetic testing should be offered to patients with certain high risk features.

Novel Phenotypic and Genotypic Findings in X-Linked Retinoschisis

PubMed Central

Tsang, Stephen H.; Vaclavik, Veronika; Bird, Alan C.; Robson, Anthony G.; Holder, Graham E.

2009-01-01

Objective To describe atypical phenotypes associated with the retinoschisis (X-linked, juvenile) 1 mutation (RS1). Methods Seven patients with multiple fine white dots at the macula and reduced visual acuity were evaluated. Six patients underwent pattern and full-field electroretinography (ERG). On-off ERG, optical coherence tomography, and fundus autofluorescence imaging were performed in some patients. Mutational screening of RS1 was prompted by the ERG findings. Results Fine white dots resembling drusenlike deposits and sometimes associated with retinal pigment epithelial abnormalities were present in the maculae. An electronegative bright-flash ERG configuration was present in all patients tested, and abnormal pattern ERG findings confirmed macular dysfunction. A parafoveal ring of high-density autofluorescence was present in 3 eyes; 1 patient showed high-density foci concordant with the white dots. Optical coherence tomography did not show foveal schisis in 3 of 4 eyes. All patients carried mutations in RS1, including 1 with a novel 206T→C mutation in exon 4. Conclusions Multiple fine white dots at the macula may be the initial fundus feature in RS1 mutation. Electrophysiologic findings suggest dysfunction after phototransduction and enable focused mutational screening. Autofluorescence imaging results suggest early retinal pigment epithelium involvement; a parafoveal ring of high-density autofluorescence has not previously been described in this disorder. PMID:17296904

The influence of mutation gene rpoB of Mycobacterium tuberculosis cluster I (507-534) on the elimination 25-desacetyl rifampicin in urine of tuberculosis subjects

NASA Astrophysics Data System (ADS)

Lily; Sinaga, B. Y. M.; Ardinata, D.; Siregar, Y.

2018-03-01

The study aimed to evaluate the influence of mutation gene rpoB of Mycobacterium (M.) tuberculosis cluster I (507-534) on the elimination of 25-Desacetyl Rifampicin (25-DR) in the urine of tuberculosis (TB) subjects. Early morning sputum took from patient TB before treatment. Urine collected after 2 hours taken Fixed-Dose Combination (FDC) at days 7th treatment. All sputum were sequencing at Macrogen Korea Laboratory. Urine was analyzed by high-Performance Liquid Chromatography (HPLC) using the method of Lily et al. Mean (standard deviation) for mutation and non-mutation of rpoB M. tuberculosis group were 7.6147 (4.4478) and 4.5772 (1.7532) μg/ml, respectively. Shapiro-Wilk test showed normally distributed data, with significance 0.3. Independent t-test performed p-value 0.167 and confidence interval (CI) from -1.648 to 7.723. The mutation gene rpoB of M. tuberculosis cluster, I (507-534) in this study, did not affect elimination 25-DR in theurine of TB subjects statistically and clinically.

JAK2, MPL, and CALR mutations in Chinese Han patients with essential thrombocythemia.

PubMed

Wang, Jing; Zhang, Biao; Chen, Bing; Zhou, Rong-Fu; Zhang, Qi-Guo; Li, Juan; Yang, Yong-Gong; Zhou, Min; Shao, Xiao-Yan; Xu, Yong; Xu, Xi-Hui; Ouyang, Jian; Xu, Jingyan; Ye, Qing

2017-04-01

Mutations in Janus kinase 2 (JAK2), myeloproliferative leukemia (MPL), and CALR are highly relevant to Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms. Assessing the prevalence of molecular mutations in Chinese Han patients with essential thrombocythemia (ET), and correlating their mutational profile with disease characteristics/phenotype. Of the 110 subjects studied, 62 carried the JAK2 V617F mutation, 21 had CALR mutations, one carried an MPL (W515) mutation, and 28 had non-mutated JAK2, CALR, and MPL (so-called triple-negative ET). Mutations in JAK2 exon 12 were not detected in any patient. Two ET patients had both CALR and JAK2 V617F mutations. Comparing the hematological parameters of the patients with JAK2 mutations with those of the patients with CALR mutations showed that the ET patients with CALR mutations were younger (p = 0.045) and had higher platelet counts (p = 0.043). Genotyping for CALR could be a useful diagnostic tool for JAK2/MPL-negative ET, since the data suggest that CALR is much more prevalent than MPL, therefore testing for CALR should be considered in patients who are JAK2 negative as its frequency is almost 20 times that of MPL mutation.

A new sensitive PCR assay for one-step detection of 12 IDH1/2 mutations in glioma.

PubMed

Catteau, Aurélie; Girardi, Hélène; Monville, Florence; Poggionovo, Cécile; Carpentier, Sabrina; Frayssinet, Véronique; Voss, Jesse; Jenkins, Robert; Boisselier, Blandine; Mokhtari, Karima; Sanson, Marc; Peyro-Saint-Paul, Hélène; Giannini, Caterina

2014-06-02

Mutations in isocitrate dehydrogenase genes IDH1 or IDH2 are frequent in glioma, and IDH mutation status is a strong diagnostic and prognostic marker. Current IDH mutation screening is performed with an immunohistochemistry (IHC) assay specific for IDH1 R132H, the most common mutation. Sequencing is recommended as a second-step test for IHC-negative or -equivocal cases. We developed and validated a new real-time quantitative polymerase chain reaction (PCR) assay for single-step detection of IDH1 R132H and 11 rare IDH1/2 mutations in formalin-fixed paraffin-embedded (FFPE) glioma samples. Performance of the IDH1/2 PCR assay was compared to IHC and Sanger sequencing. The IDH1/2 PCR assay combines PCR clamping for detection of 7 IDH1 and 5 IDH2 mutations, and Amplification Refractory Mutation System technology for specific identification of the 3 most common mutations (IDH1 R132H, IDH1 R132C, IDH2 R172K). Analytical sensitivity of the PCR assay for mutation detection was <5% for 11/12 mutations (mean: 3.3%), and sensitivity for mutation identification was very high (0.8% for IDH1 R132H; 1.2% for IDH1 R132C; 0.6% for IDH2 R172K). Assay performance was further validated on 171 clinical glioma FFPE samples; of these, 147 samples met the selection criteria and 146 DNA samples were successfully extracted. IDH1/2 status was successfully obtained in 91% of cases. All but one positive IDH1 R132H-IHC cases were concordantly detected by PCR and 3 were not detected by sequencing. Among the IHC-negative cases (n = 72), PCR detected 12 additional rare mutations (10 IDH1, 2 IDH2). All mutations detected by sequencing (n = 67) were concordantly detected by PCR and 5/66 sequencing-negative cases were PCR-positive (overall concordance: 96%). Analysis of synthetic samples representative of the 11 rare IDH1/2 mutations detected by the assay produced 100% correct results. The new IDH1/2 PCR assay has a high technical success rate and is more sensitive than Sanger sequencing. Positive concordance was 98% with IHC for IDH1 R132H detection and 100% with sequencing. The PCR assay can reliably be performed on FFPE samples and has a faster turnaround time than current IDH mutation detection algorithms. The assay should facilitate implementation of a comprehensive IDH1/2 testing protocol in routine clinical practice.

Effect of genetic background on the stability of sunflower fatty acid composition in different high oleic mutations.

PubMed

Alberio, Constanza; Aguirrezábal, Luis An; Izquierdo, Natalia G; Reid, Roberto; Zuil, Sebastián; Zambelli, Andrés

2018-02-01

The effect of genetic background on the stability of fatty acid composition in sunflower near isogenic lines (NILs) carrying high-oleic Pervenets (P) or high-oleic NM1 mutations was studied. The materials were field-tested in different locations and at different sowing dates to evaluate a wide range of environmental conditions. Relationships were established between the fatty acids and the minimum night temperature (MNT) and the response was characterized. A genetic background effect for the fatty acid composition was found in both groups of NILs. The NM1-NILs showed an oleic level higher than 910 g kg -1 and they were more stable across environments with a zero or low dependence on the genetic background; on the other hand, high oleic materials bearing the P mutation showed lower levels of oleic acid, with a higher variation in fatty acid composition and a highly significant dependence on the genetic background. The NM1 mutation is the best option to develop ultra-high oleic sunflower oil that is stable across environments and genetic backgrounds, making its agronomical production more efficient and predictable. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

Rapid detection of pathological mutations and deletions of the haemoglobin beta gene (HBB) by High Resolution Melting (HRM) analysis and Gene Ratio Analysis Copy Enumeration PCR (GRACE-PCR).

PubMed

Turner, Andrew; Sasse, Jurgen; Varadi, Aniko

2016-10-19

Inherited disorders of haemoglobin are the world's most common genetic diseases, resulting in significant morbidity and mortality. The large number of mutations associated with the haemoglobin beta gene (HBB) makes gene scanning by High Resolution Melting (HRM) PCR an attractive diagnostic approach. However, existing HRM-PCR assays are not able to detect all common point mutations and have only a very limited ability to detect larger gene rearrangements. The aim of the current study was to develop a HBB assay, which can be used as a screening test in highly heterogeneous populations, for detection of both point mutations and larger gene rearrangements. The assay is based on a combination of conventional HRM-PCR and a novel Gene Ratio Analysis Copy Enumeration (GRACE) PCR method. HRM-PCR was extensively optimised, which included the use of an unlabelled probe and incorporation of universal bases into primers to prevent interference from common non-pathological polymorphisms. GRACE-PCR was employed to determine HBB gene copy numbers relative to a reference gene using melt curve analysis to detect rearrangements in the HBB gene. The performance of the assay was evaluated by analysing 410 samples. A total of 44 distinct pathological genotypes were detected. In comparison with reference methods, the assay has a sensitivity of 100 % and a specificity of 98 %. We have developed an assay that detects both point mutations and larger rearrangements of the HBB gene. This assay is quick, sensitive, specific and cost effective making it suitable as an initial screening test that can be used for highly heterogeneous cohorts.

Comparison of Epidermal Growth Factor Receptor Mutations between Metastatic Lymph Node Diagnosed by EBUS-TBNA and Primary Tumor in Non-Small Cell Lung Cancer

PubMed Central

Kang, Hyo Jae; Hwangbo, Bin; Lee, Jin Soo; Kim, Moon Soo; Lee, Jong Mog; Lee, Geon-Kook

2016-01-01

Introduction Although the use of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is increasing for epidermal growth factor receptor (EGFR) testing in lung cancer, the discordance rate in EGFR mutations between lymph node (LN) samples obtained by EBUS-TBNA and primary tumor (PT) is not well known. Thus, we compared the EGFR mutation status of LN samples obtained by EBUS-TBNA and PTs to estimate the efficacy of using EBUS-TBNA specimens for EGFR testing in advanced, non-squamous, non-small cell lung cancer (NSCLC). Materials and Methods Using data of patients from the EBUS-TBNA database (N = 1914) obtained between January 2009 and January 2013, we identified 100 treatment-naïve, advanced, non-squamous NSCLC patients (stage 3 and 4) with matched LN specimens obtained by EBUS-TBNA and PT specimens. Of these, 74 patients with paired specimens were feasible for EGFR mutation analysis, which we performed using a direct sequencing method. Results Of the 74 cases, at least one major [exon 19 deleted (19del) and L858R] or minor (T790M, exon 20 insertion, and other point mutations) EGFR mutation was detected in 31 cases (41.9%), which included PT (n = 31, 41.9%) and LN (n = 28, 37.8%) specimens. Major mutations were detected in 25 PT (33.8%, 19del = 13, L858R = 12) and 22 LN (29.8%, 19del = 11, L858R = 11) specimens. The discordance rate in major mutations between matched PT and LN specimens was 4.1% (3/74). Among minor mutations, T790M was detected in LN specimen only in 2 cases with L858R in PT and LN. The discordance rate major and minor EGFR mutations combined between matched PT and LN specimens was 12% (9/74). Conclusions We observed a high concordance rate of major EGFR mutations between matched LN specimens sampled by EBUS-TBNA and PTs, suggesting that LN samples obtained by EBUS-TBNA from advanced non-squamous NSCLC patients are effective for use in EGFR mutation testing. PMID:27685950

Inhibitor Risk Stratification and Individualized Treatment in Patients With Nonsevere Hemophilia A: A Single-Institution Practice Audit.

PubMed

Sun, Haowei Linda; Chan, Stella; Yenson, Paul; Jackson, Shannon

2018-03-01

Inhibitor risk in nonsevere hemophilia A increases with cumulative factor VIII (FVIII) exposure days and high-risk mutations. A standardized approach to minimize inhibitor risk is warranted. Following establishment of a systematic approach to reduce inhibitor risk in nonsevere hemophilia, we evaluated the uptake of these strategies into clinical practice. All adult males with nonsevere hemophilia A followed by British Columbia Adult Hemophilia Program from 2004 to 2016 were included in this retrospective audit. Quality-of-care indicators on inhibitor prevention were examined. Of 108 patients, 18 patients had high-risk FVIII mutations for inhibitor development. Rates of FVIII genotyping and 1-deamino-8-d-arginine-vasopressin (DDAVP) testing in mild patients without contraindications were both over 90%, although DDAVP was used for surgical prophylaxis in only 70% of procedures. Inhibitor testing and clinic visits occurred at a median interval of 22 months. Over 80% of patients with high-risk mutations had documentation and education on their inhibitor risk. Our practice audit demonstrated a high level of recognition and patient education of individual inhibitor risk. Impact of our standardized approach on the incidence of inhibitor development is yet to be determined.

Detection of katG and inhA mutations to guide isoniazid and ethionamide use for drug-resistant tuberculosis.

PubMed

Bollela, V R; Namburete, E I; Feliciano, C S; Macheque, D; Harrison, L H; Caminero, J A

2016-08-01

Depending on the presence of mutations that determine isoniazid (INH) susceptibility (katG and inhA), Mycobacterium tuberculosis may be susceptible to high doses of INH or ethionamide (ETH). To describe the INH resistance profile and association of katG mutation with previous INH treatment and level of drug resistance based on rapid molecular drug susceptibility testing (DST) in southern Brazil and central Mozambique. Descriptive study of 311 isolates from Ribeirão Preto, São Paulo, Brazil (2011-2014) and 155 isolates from Beira, Mozambique (2014-2015). Drug resistance patterns and specific gene mutations were determined using GenoType(®) MTBDRplus. katG gene mutations were detected in 12/22 (54.5%) Brazilian and 32/38 (84.2%) Mozambican isolates. inhA mutations were observed in 9/22 (40.9%) isolates in Brazil and in 4/38 (10.5%) in Mozambique. Both katG and inhA mutations were detected in respectively 1/22 (5%) and 2/38 (5.2%). The difference in the frequency of katG mutations in Brazil and Mozambique was statistically significant (P = 0.04). katG mutations were present in 68.8% (33/48) of patients previously treated with INH and 31.2% (15/48) of patients without previous INH. This difference was not statistically significant (P = 0.223). INH mutations varied geographically; molecular DST can be used to guide and accelerate decision making in the use of ETH or high doses of INH.

The Use of COLD-PCR and High-Resolution Melting Analysis Improves the Limit of Detection of KRAS and BRAF Mutations in Colorectal Cancer

PubMed Central

Mancini, Irene; Santucci, Claudio; Sestini, Roberta; Simi, Lisa; Pratesi, Nicola; Cianchi, Fabio; Valanzano, Rosa; Pinzani, Pamela; Orlando, Claudio

2010-01-01

Fast and reliable tests to detect mutations in human cancers are required to better define clinical samples and orient targeted therapies. KRAS mutations occur in 30–50% of colorectal cancers (CRCs) and represent a marker of clinical resistance to cetuximab therapy. In addition, the BRAF V600E is mutated in about 10% of CRCs, and the development of a specific inhibitor of mutant BRAF kinase has prompted a growing interest in BRAFV600E detection. Traditional methods, such as PCR and direct sequencing, do not detect low-level mutations in cancer, resulting in false negative diagnoses. In this study, we designed a protocol to detect mutations of KRAS and BRAFV600E in 117 sporadic CRCs based on coamplification at lower denaturation temperature PCR (COLD-PCR) and high-resolution melting (HRM). Using traditional PCR and direct sequencing, we found KRAS mutations in 47 (40%) patients and BRAFV600E in 10 (8.5%). The use of COLD-PCR in apparently wild-type samples allowed us to identify 15 newly mutated CRCs (10 for KRAS and 5 for BRAFV600E), raising the percentage of mutated CRCs to 48.7% for KRAS and to 12.8% for BRAFV600E. Therefore, COLD-PCR combined with HRM permits the correct identification of less represented mutations in CRC and better selection of patients eligible for targeted therapies, without requiring expensive and time-consuming procedures. PMID:20616366

The suitability of small biopsy and cytology specimens for EGFR and other mutation testing in non-small cell lung cancer

PubMed Central

Wang, Shu; Yu, Bing; Ng, Chiu Chin; Mercorella, Belinda; Selinger, Christina I.; O’Toole, Sandra A.

2015-01-01

Background Patients with advanced non-small cell lung cancer (NSCLC) benefit from treatment with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) when their tumor harbors an activating EGFR mutation. As the majority of NSCLC patients present with advanced disease, cytology and small biopsy specimens are frequently the only tissue available for mutation testing, but can pose challenges due to low tumor content. We aim to better define the suitability of these specimens for mutation testing. Methods NSCLC cases referred to our institution for mutation testing over a 15-month period were retrospectively reviewed. Specimens were tested for mutations including EGFR, KRAS, and BRAF, using a multiplex PCR assay (OncoCarta Panel v1.0) and analyzed on the Agena Bioscience MassARRAY platform. Results A total of 146 specimens were tested, comprising 53 (36.3%) resection specimens (including 28 lung resection specimens), 55 (37.7%) small biopsy specimens and 38 (26%) cytology specimens. Of 142 cases with sufficient DNA for mutation testing, EGFR mutations were detected in 31 specimens (21.8%), KRAS mutations in 31 specimens (21.8%) and BRAF mutations in three specimens (2.1%). There was no significant difference in the EGFR mutation rate between lung resection (10 of 28 cases; 35.7%), small biopsy (9 of 53 cases; 17%), and cytology specimens (8 of 36 cases; 22.2%). Conclusions Our results support the utility of small biopsy and cytology specimens for mutation testing. Careful evaluation of the adequacy of small specimens is required to minimize the risk of false negative or positive results. PMID:25870794

Rapid targeted mutational analysis of human tumours: a clinical platform to guide personalized cancer medicine

PubMed Central

Dias-Santagata, Dora; Akhavanfard, Sara; David, Serena S; Vernovsky, Kathy; Kuhlmann, Georgiana; Boisvert, Susan L; Stubbs, Hannah; McDermott, Ultan; Settleman, Jeffrey; Kwak, Eunice L; Clark, Jeffrey W; Isakoff, Steven J; Sequist, Lecia V; Engelman, Jeffrey A; Lynch, Thomas J; Haber, Daniel A; Louis, David N; Ellisen, Leif W; Borger, Darrell R; Iafrate, A John

2010-01-01

Targeted cancer therapy requires the rapid and accurate identification of genetic abnormalities predictive of therapeutic response. We sought to develop a high-throughput genotyping platform that would allow prospective patient selection to the best available therapies, and that could readily and inexpensively be adopted by most clinical laboratories. We developed a highly sensitive multiplexed clinical assay that performs very well with nucleic acid derived from formalin fixation and paraffin embedding (FFPE) tissue, and tests for 120 previously described mutations in 13 cancer genes. Genetic profiling of 250 primary tumours was consistent with the documented oncogene mutational spectrum and identified rare events in some cancer types. The assay is currently being used for clinical testing of tumour samples and contributing to cancer patient management. This work therefore establishes a platform for real-time targeted genotyping that can be widely adopted. We expect that efforts like this one will play an increasingly important role in cancer management. PMID:20432502

Validation of predictive models for germline mutations in DNA mismatch repair genes in colorectal cancer.

PubMed

Monzon, Jose G; Cremin, Carol; Armstrong, Linlea; Nuk, Jennifer; Young, Sean; Horsman, Doug E; Garbutt, Kristy; Bajdik, Chris D; Gill, Sharlene

2010-02-15

Lynch syndrome is defined by the presence of germline mutations in mismatch repair (MMR) genes. Several models have been recently devised that predict mutation carrier status (Myriad Genetics, Wijnen, Barnetson, PREMM and MMRpro models). Families at moderate-high risk for harboring a Lynch-associated mutation, referred to the BC Cancer Agency (BCCA) Hereditary Cancer Program (HCP), underwent mutation analysis, immunohistochemistry and/or microsatellite testing. Seventy-two tested cases were included. Twenty-five patients were mutation positive (34.7%) and 47 were mutation negative (65.3%). Nineteen of 43 patients who were both microsatellite stable and normal on immunohistochemistry for MLH1 and MSH2 were also genotyped for mutations in these genes; all 19 were negative for MMR gene mutations. Model-derived probabilities of harboring a MMR gene mutation in the proband were calculated and compared to observed results. The area under the ROC curves were 0.75 (95%CI; 0.63-0.87), 0.86 (0.7-0.96), 0.89 (0.82-0.97), 0.89 (0.81-0.98) and 0.93 (0.86-0.99) for the Myriad, Barnetson, Wijnen, MMRpro and PREMM models, respectively. The Amsterdam II criteria had a sensitivity and specificity of 0.76 and 0.74, respectively, in this cohort. The PREMM model demonstrated the best performance for predicting carrier status based on the positive likelihood ratios at the >10%, >20% and >30% probability thresholds. In this referred cohort, the PREMM model had the most favorable concordance index and predictive performance for carrier status based on the positive LR. These prediction models (PREMM, MMRPro and Wijnen) may soon replace the Amsterdam II and revised Bethesda criteria as a prescreening tool for Lynch mutations.

EGFR Mutation Analysis for Prospective Patient Selection in Two Phase II Registration Studies of Osimertinib.

PubMed

Jenkins, Suzanne; Chih-Hsin Yang, James; Jänne, Pasi A; Thress, Kenneth S; Yu, Karen; Hodge, Rachel; Weston, Susie; Dearden, Simon; Patel, Sabina; Cantarini, Mireille; Shepherd, Frances A

2017-08-01

Osimertinib is an oral, central nervous system-active, EGFR tyrosine kinase inhibitor (TKI) for the treatment of EGFR T790M-positive advanced NSCLC. Here we have evaluated EGFR mutation frequencies in two phase II studies of osimertinib (AURA extension and AURA2). After progression while receiving their latest line of therapy, patients with EGFR mutation-positive advanced NSCLC provided tumor samples for mandatory central T790M testing for the study selection criteria. Tumor tissue mutation analysis for patient selection was performed with the Roche cobas EGFR Mutation Test (European Conformity-in vitro diagnostic, labeled investigational use only) (Roche Molecular Systems, Pleasanton, CA). Patients should not have been prescreened for T790M mutation status. The cobas test results were compared with those of the MiSeq next-generation sequencing system (Illumina, San Diego, CA), which was used as a reference method. Samples from 324 and 373 patients screened for AURA extension and AURA2, respectively, produced valid cobas test results. The T790M detection rates were similar between AURA extension and AURA2 (64% and 63%, respectively). The pooled T790M rate was 63%, with no difference by ethnicity (63% for Asian and non-Asian patients alike) or immediately prior treatment with an EGFR TKI (afatinib, 69%; erlotinib, 69%; and gefitinib, 63%). A higher proportion of patients had T790M detected against a background of exon 19 deletions versus L858R mutation (73% versus 58% [p = 0.0002]). In both trials the cobas test demonstrated high sensitivity (positive percent agreement) and specificity (negative percent agreement) for T790M detection when compared with the next-generation sequencing reference method: positive percent agreement of 91% versus 89% and negative percent agreement of 97% versus 98%. In both trials, the rate of detection of T790M mutation in patients with advanced NSCLC was approximately 63% and was unaffected by immediately prior treatment with an EGFR TKI or ethnicity. Copyright © 2017 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Impact of fixation artifacts and threshold selection on high resolution melting analysis for KRAS mutation screening.

PubMed

Pérez-Báez, Wendy; García-Latorre, Ethel A; Maldonado-Martínez, Héctor Aquiles; Coronado-Martínez, Iris; Flores-García, Leonardo; Taja-Chayeb, Lucía

2017-10-01

Treatment in metastatic colorectal cancer (mCRC) has expanded with monoclonal antibodies targeting epidermal growth factor receptor, but is restricted to patients with a wild-type (WT) KRAS mutational status. The most sensitive assays for KRAS mutation detection in formalin-fixed paraffin embedded (FFPE) tissues are based on real-time PCR. Among them, high resolution melting analysis (HRMA), is a simple, fast, highly sensitive, specific and cost-effective method, proposed as adjunct for KRAS mutation detection. However the method to categorize WT vs mutant sequences in HRMA is not clearly specified in available studies, besides the impact of FFPE artifacts on HRMA performance hasn't been addressed either. Avowedly adequate samples from 104 consecutive mCRC patients were tested for KRAS mutations by Therascreen™ (FDA Validated test), HRMA, and HRMA with UDG pre-treatment to reverse FFPE fixation artifacts. Comparisons of KRAS status allocation among the three methods were done. Focusing on HRMA as screening test, ROC curve analyses were performed for HRMA and HMRA-UDG against Therascreen™, in order to evaluate their discriminative power and to determine the threshold of profile concordance between WT control and sample for KRAS status determination. Comparing HRMA and HRMA-UDG against Therascreen™ as surrogate gold standard, sensitivity was 1 for both HRMA and HRMA-UDG; and specificity and positive predictive values were respectively 0.838 and 0.939; and 0.777 and 0.913. As evaluated by the McNemar test, HRMA-UDG allocated samples to a WT/mutated genotype in a significatively different way from HRMA (p > 0.001). On the other hand HRMA-UDG did not differ from Therascreen™ (p = 0.125). ROC-curve analysis showed a significant discriminative power for both HRMA and HRMA-UDG against Therascreen™ (respectively, AUC of 0.978, p > 0.0001, CI 95% 0.957-0.999; and AUC of 0.98, p > 0.0001, CI 95% 0.000-1.0). For HRMA as a screening tool, the best threshold (degree of concordance between sample curves and WT control) was attained at 92.14% for HRMA (specificity of 0.887), and at 92.55% for HRMA-UDG (specificity of 0.952). HRMA is a highly sensitive method for KRAS mutation detection, with apparently adequate and statistically significant discriminative power. FFPE sample fixation artifacts have an impact on HRMA results, so for HRMA on FFPE samples pre-treatment with UDG should be strongly suggested. The choice of the threshold for melting curve concordance has also great impact on HRMA performance. A threshold of 93% or greater might be adequate if using HRMA as a screening tool. Further validation of this threshold is required. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of small molecules that improve ATP synthesis defects conferred by Leber’s hereditary optic neuropathy mutations

PubMed Central

Datta, Sandipan; Tomilov, Alexey; Cortopassi, Gino

2016-01-01

Inherited mitochondrial complex I mutations cause blinding Leber's hereditary Optic Neuropathy (LHON), for which no curative therapy exists. A specific biochemical consequence of LHON mutations in the presence of trace rotenone was observed: deficient complex I-dependent ATP synthesis (CIDAS) and mitochondrial O2 consumption, proportional to the clinical severity of the three primary LHON mutations. We optimized a high-throughput assay of CIDAS to screen 1600 drugs to 2, papaverine and zolpidem, which protected CIDAS in LHON cells concentration-dependently. TSPO and cAMP were investigated as protective mechanisms, but a conclusive mechanism remains to be elucidated; next steps include testing in animal models. PMID:27497748

Cost-Effectiveness Analysis of Different Genetic Testing Strategies for Lynch Syndrome in Taiwan.

PubMed

Chen, Ying-Erh; Kao, Sung-Shuo; Chung, Ren-Hua

2016-01-01

Patients with Lynch syndrome (LS) have a significantly increased risk of developing colorectal cancer (CRC) and other cancers. Genetic screening for LS among patients with newly diagnosed CRC aims to identify mutations in the disease-causing genes (i.e., the DNA mismatch repair genes) in the patients, to offer genetic testing for relatives of the patients with the mutations, and then to provide early prevention for the relatives with the mutations. Several genetic tests are available for LS, such as DNA sequencing for MMR genes and tumor testing using microsatellite instability and immunohistochemical analyses. Cost-effectiveness analyses of different genetic testing strategies for LS have been performed in several studies from different countries such as the US and Germany. However, a cost-effectiveness analysis for the testing has not yet been performed in Taiwan. In this study, we evaluated the cost-effectiveness of four genetic testing strategies for LS described in previous studies, while population-specific parameters, such as the mutation rates of the DNA mismatch repair genes and treatment costs for CRC in Taiwan, were used. The incremental cost-effectiveness ratios based on discounted life years gained due to genetic screening were calculated for the strategies relative to no screening and to the previous strategy. Using the World Health Organization standard, which was defined based on Taiwan's Gross Domestic Product per capita, the strategy based on immunohistochemistry as a genetic test followed by BRAF mutation testing was considered to be highly cost-effective relative to no screening. Our probabilistic sensitivity analysis results also suggest that the strategy has a probability of 0.939 of being cost-effective relative to no screening based on the commonly used threshold of $50,000 to determine cost-effectiveness. To the best of our knowledge, this is the first cost-effectiveness analysis for evaluating different genetic testing strategies for LS in Taiwan. The results will be informative for the government when considering offering screening for LS in patients newly diagnosed with CRC.

Mycobacterium tuberculosis drug-resistance testing: challenges, recent developments and perspectives.

PubMed

Schön, T; Miotto, P; Köser, C U; Viveiros, M; Böttger, E; Cambau, E

2017-03-01

Drug-resistance testing, or antimicrobial susceptibility testing (AST), is mandatory for Mycobacterium tuberculosis in cases of failure on standard therapy. We reviewed the different methods and techniques of phenotypic and genotypic approaches. Although multiresistant and extensively drug-resistant (MDR/XDR) tuberculosis is present worldwide, AST for M. tuberculosis (AST-MTB) is still mainly performed according to the resources available rather than the drug-resistance rates. Phenotypic methods, i.e. culture-based AST, are commonly used in high-income countries to confirm susceptibility of new cases of tuberculosis. They are also used to detect resistance in tuberculosis cases with risk factors, in combination with genotypic tests. In low-income countries, genotypic methods screening hot-spot mutations known to confer resistance were found to be easier to perform because they avoid the culture and biosafety constraint. Given that genotypic tests can rapidly detect the prominent mechanisms of resistance, such as the rpoB mutation for rifampicin resistance, we are facing new challenges with the observation of false-resistance (mutations not conferring resistance) and false-susceptibility (mutations different from the common mechanism) results. Phenotypic and genotypic approaches are therefore complementary for obtaining a high sensitivity and specificity for detecting drug resistances and susceptibilities to accurately predict MDR/XDR cure and to gather relevant data for resistance surveillance. Although AST-MTB was established in the 1960s, there is no consensus reference method for MIC determination against which the numerous AST-MTB techniques can be compared. This information is necessary for assessing in vitro activity and setting breakpoints for future anti-tuberculosis agents. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Genetics of pulmonary hypertension in the clinic.

PubMed

Girerd, Barbara; Lau, Edmund; Montani, David; Humbert, Marc

2017-09-01

Heritable pulmonary arterial hypertension (PAH) is an autosomal dominant disease with incomplete penetrance because of mutations in bone morphogenetic protein receptor-II (BMPR2), activin A receptor type II-like kinase 1, endoglin, caveolin-1, potassium channel subfamily K, member 3, and T-box gene 4 genes. Heritable pulmonary veno-occlusive disease and/or pulmonary capillary hemangiomatosis (PVOD/PCH) is an autosomal recessive disease because of biallelic mutations in the eukaryotic translation initiation factor 2 alpha kinase 4 gene. The 2015 european society of cardiology (ESC) and european respiratory society (ERS) pulmonary hypertension guidelines recommend genetic counselling and testing to adults and children with PAH or PVOD/PCH as well as in adult relatives at risk of carrying a predisposing mutation. In France, genetic counseling and testing are offered to all patients displaying sporadic or familial form of PAH or PVOD/PCH and to their relatives at high risk of carrying a predisposing mutation. Patients with a heritable form of PAH are younger at diagnosis with a worse hemodynamic and a dismal prognosis. Patients with a heritable form of PVOD/PCH are younger at diagnosis with a worse response to specific PAH therapies. A program to detect PAH in an early phase was offered to all asymptomatic BMPR2 mutation carriers, according to the 2015 ESC/ERS guidelines. Finally, preimplantation genetic diagnosis has been performed in families with a history of BMPR2 mutations. Genetic counseling and testing has to be implemented in pulmonary hypertension centers.

Underutilization of BRCA1/2 testing to guide breast cancer treatment: black and Hispanic women particularly at risk

PubMed Central

Levy, Douglas E.; Byfield, Stacey D.; Comstock, Catherine B.; Garber, Judy E.; Syngal, Sapna; Crown, William H.; Shields, Alexandra E.

2013-01-01

Purpose Women with early-onset (age ≤40) breast cancer are at high risk of carrying deleterious mutations in the BRCA1/2 genes; genetic assessment is thus recommended. Knowledge of BRCA1/2 mutation status is useful in guiding treatment decisions. To date, there has been no national study of BRCA1/2 testing among newly diagnosed women. Methods We used administrative data (2004–2007) from a national sample of 14.4 million commercially-insured patients to identify newly-diagnosed, early-onset breast cancer cases among women ages 20–40 (n=1,474). Cox models assessed BRCA1/2 testing, adjusting for covariates and differential lengths of follow-up. Results Overall, 30% of women age ≤40 received BRCA1/2 testing. In adjusted analyses, women of Jewish ethnicity were significantly more likely to be tested (HR=2.83, 95% CI 1.52–5.28), while black women (HR=0.34, 95% 0.18–0.64) and Hispanic women (HR=0.52, 95% CI 0.33–0.81) were significantly less likely to be tested than non-Jewish white women. Those enrolled in an HMO (HR=0.73, 95% CI 0.54–0.99) were significantly less likely to receive BRCA1/2 testing than those POS insurance plans. Testing rates rose sharply for women diagnosed in 2007 compared to 2004. Conclusions In this national sample of newly diagnosed breast cancer patients at high risk for BRCA1/2 mutations, genetic assessment was low, with marked racial differences in testing. PMID:21358336

Driver mutations in TP53 are ubiquitous in high grade serous carcinoma of the ovary.

PubMed

Ahmed, Ahmed Ashour; Etemadmoghadam, Dariush; Temple, Jillian; Lynch, Andy G; Riad, Mohamed; Sharma, Raghwa; Stewart, Colin; Fereday, Sian; Caldas, Carlos; Defazio, Anna; Bowtell, David; Brenton, James D

2010-05-01

Numerous studies have tested the association between TP53 mutations in ovarian cancer and prognosis but these have been consistently confounded by limitations in study design, methodology, and/or heterogeneity in the sample cohort. High-grade serous (HGS) carcinoma is the most clinically important histological subtype of ovarian cancer. As these tumours may arise from the ovary, Fallopian tube or peritoneum, they are collectively referred to as high-grade pelvic serous carcinoma (HGPSC). To identify the true prevalence of TP53 mutations in HGPSC, we sequenced exons 2-11 and intron-exon boundaries in tumour DNA from 145 patients. HGPSC cases were defined as having histological grade 2 or 3 and FIGO stage III or IV. Surprisingly, pathogenic TP53 mutations were identified in 96.7% (n = 119/123) of HGPSC cases. Molecular and pathological review of mutation-negative cases showed evidence of p53 dysfunction associated with copy number gain of MDM2 or MDM4, or indicated the exclusion of samples as being low-grade serous tumours or carcinoma of uncertain primary site. Overall, p53 dysfunction rate approached 100% of confirmed HGPSCs. No association between TP53 mutation and progression-free or overall survival was found. From this first comprehensive mapping of TP53 mutation rate in a homogeneous group of HGPSC patients, we conclude that mutant TP53 is a driver mutation in the pathogenesis of HGPSC cancers. Because TP53 mutation is almost invariably present in HGPSC, it is not of substantial prognostic or predictive significance. Copyright (c) 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Clinical validation of a highly sensitive assay to detect EGFR mutations in plasma cell-free DNA from patients with advanced lung adenocarcinoma.

PubMed

Li, Yuping; Xu, Hanyan; Su, Shanshan; Ye, Junru; Chen, Junjie; Jin, Xuru; Lin, Quan; Zhang, Dongqing; Ye, Caier; Chen, Chengshui

2017-01-01

Circulating tumor DNA (ctDNA) is a promising biomarker for noninvasive epidermal growth factor receptor (EGFR) mutations detection in lung cancer patients, but the existing methods have limitations in sensitivity or in availability. In this study, we evaluated the performance of a novel assay called ADx-SuperARMS in detecting EGFR mutations in plasma cell-free DNA from patients with advanced lung adenocarcinoma. A total of 109 patients with metastatic advanced adenocarcinoma were recruited who provided both blood samples and matched tumor tissue samples. EGFR mutation status in plasma samples were tested with ADx-SuperARMS EGFR assay and tumor tissue samples were tested with ADx-ARMS EGFR assay. The clinical sensitivity, specificity, positive prediction value (PPV), and negative prediction value (NPV) of ADx-SuperARMS EGFR assay were calculated by using EGFR mutation status in tumor tissue as standard reference. A receiver operating characteristic (ROC) analysis was implemented and an area under the curve (AUC) was calculated to evaluate sensitivity and specificity of exon 19 deletion (E19Del) and L858R mutation detection. The objective response rate (ORR) were calculated according to the EGFR mutation status determined by ADx-superARMS as well. 0.2% analytical sensitivity and 100% specificity of the ADx-SuperARMS EGFR assays for EGFR E19Del, L858R, and T790M mutants were confirmed by using a series of diluted cell line DNA. In the clinical study, EGFR mutations were detected in 45.9% (50/109) of the plasma samples and in 56.9% (62/109) of the matched tumor tissue samples. The sensitivity, specificity, PPV and NPV of the ADx-SuperARMS EGFR assay for plasma EGFR mutation detection were 82.0% (50/61), 100% (48/48), 100% (50/50), and 81.4% (48/59), respectively. In ROC analysis, ADx-SuperARMS achieved sensitivity and specificity of 88% and 99% in E19Dels as well as sensitivity and specificity of 89% and 100% in L858R, respectively. Among the 35 patients who were plasma EGFR mutation positive and treated with first generation of EGFR-tyrosine kinase inhibitors (TKIs), 23 (65.7%) achieved partial response, 11 (31.4%) sustained disease, and 1 (2.9%) progressive disease. The ORR and disease control rate (DCR) were 65.7% and 97.1%, respectively. ADx-SuperARMS EGFR assay is likely to be a highly sensitive and specific method to noninvasively detect plasma EGFR mutations of patients with advanced lung adenocarcinoma. The EGFR mutations detected by ADx-SuperARMS EGFR assay could predict the efficacy of the treatment with first generation of EGFR-TKIs. Hence, EGFR blood testing with ADx-SuperARMS could address the unmet clinical needs.

Evolution at increased error rate leads to the coexistence of multiple adaptive pathways in an RNA virus.

PubMed

Cabanillas, Laura; Arribas, María; Lázaro, Ester

2013-01-16

When beneficial mutations present in different genomes spread simultaneously in an asexual population, their fixation can be delayed due to competition among them. This interference among mutations is mainly determined by the rate of beneficial mutations, which in turn depends on the population size, the total error rate, and the degree of adaptation of the population. RNA viruses, with their large population sizes and high error rates, are good candidates to present a great extent of interference. To test this hypothesis, in the current study we have investigated whether competition among beneficial mutations was responsible for the prolonged presence of polymorphisms in the mutant spectrum of an RNA virus, the bacteriophage Qβ, evolved during a large number of generations in the presence of the mutagenic nucleoside analogue 5-azacytidine. The analysis of the mutant spectra of bacteriophage Qβ populations evolved at artificially increased error rate shows a large number of polymorphic mutations, some of them with demonstrated selective value. Polymorphisms distributed into several evolutionary lines that can compete among them, making it difficult the emergence of a defined consensus sequence. The presence of accompanying deleterious mutations, the high degree of recurrence of the polymorphic mutations, and the occurrence of epistatic interactions generate a highly complex interference dynamics. Interference among beneficial mutations in bacteriophage Qβ evolved at increased error rate permits the coexistence of multiple adaptive pathways that can provide selective advantages by different molecular mechanisms. In this way, interference can be seen as a positive factor that allows the exploration of the different local maxima that exist in rugged fitness landscapes.

Linking minimum inhibitory concentrations to whole genome sequence-predicted drug resistance in Mycobacterium tuberculosis strains from Romania.

PubMed

Ruesen, Carolien; Riza, Anca Lelia; Florescu, Adriana; Chaidir, Lidya; Editoiu, Cornelia; Aalders, Nicole; Nicolosu, Dragos; Grecu, Victor; Ioana, Mihai; van Crevel, Reinout; van Ingen, Jakko

2018-06-26

Mycobacterium tuberculosis drug resistance poses a major threat to tuberculosis control. Current phenotypic tests for drug susceptibility are time-consuming, technically complex, and expensive. Whole genome sequencing is a promising alternative, though the impact of different drug resistance mutations on the minimum inhibitory concentration (MIC) remains to be investigated. We examined the genomes of 72 phenotypically drug-resistant Mycobacterium tuberculosis isolates from 72 Romanian patients for drug resistance mutations. MICs for first- and second-line drugs were determined using the MycoTB microdilution method. These MICs were compared to macrodilution critical concentration testing by the Mycobacterium Growth Indicator Tube (MGIT) platform and correlated to drug resistance mutations. Sixty-three (87.5%) isolates harboured drug resistance mutations; 48 (66.7%) were genotypically multidrug-resistant. Different drug resistance mutations were associated with different MIC ranges; katG S315T for isoniazid, and rpoB S450L for rifampicin were associated with high MICs. However, several mutations such as in rpoB, rrs and rpsL, or embB were associated with MIC ranges including the critical concentration for rifampicin, aminoglycosides or ethambutol, respectively. Different resistance mutations lead to distinct MICs, some of which may still be overcome by increased dosing. Whole genome sequencing can aid in the timely diagnosis of Mycobacterium tuberculosis drug resistance and guide clinical decision-making.

Quality of Life and Psychological State in Chinese Breast Cancer Patients Who Received BRCA1/2 Genetic Testing

PubMed Central

Qiu, Jiajia; Guan, Jiaqin; Yang, Xiaochen; Wu, Jiong; Liu, Guangyu; Di, Genhong; Chen, Canming; Hou, Yifeng; Han, Qixia; Shen, Zhenzhou; Shao, Zhimin; Hu, Zhen

2016-01-01

Background This study aims to understand the quality of life (QOL) and psychological state (PS) of Chinese breast cancer patients who received BRCA1/2 genetic testing; to examine the psychological changes between BRCA1/2 mutation carriers and non-carriers; and to further explore the psychological experience of BRCA1/2 mutation carriers. Methods This study was combined with quantitative and qualitative designs. First, we performed a quantitative investigation using FACT-B (Chinese version) and Irritability, Depression and Anxiety scale (IDA) to assess the QOL and PS in breast cancer patients who received BRCA1/2 genetic testing. Then semi-structured in-depth qualitative interviews among 13 mutation carriers were conducted in hospital. Results Results from the quantitative study showed QOL scores were relatively high and the IDA scores were relatively low among the patients, and there was no significant difference in the QOL or IDA scores between non-carriers and carriers. Based on the qualitative analysis, four main themes emerged: (1) Finding the reason for having breast cancer; (2) Negative emotions; (3) Behavioral changes; (4) Lack of information. Conclusions The present study showed that QOL and PS are good among the breast cancer patients who received genetic testing. Genetic testing itself does not cause long psychosocial effects. BRCA1/2 mutation carriers may have certain negative emotions at the first stage they knew the testing results and may initiate behavioral and lifestyle changes. The patients with a BRCA1/2 mutation desire knowledge with regard to genetic aspects in mainland China. Professional information and advice can be provided to relieve the patients’ negative emotions when they were informed of gene defect. PMID:27428375

Diphtheria toxin resistance in human lymphocytes and lymphoblasts in the in vivo somatic cell mutation test

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomkins, D.J.; Wei, L.; Laurie, K.E.

1985-01-01

It has been shown that circulating peripheral blood lymphocytes can be used for the enumeration of 6-thioguanine-resistant cells that presumably arise by mutation in vivo. This somatic cell mutation test has been studied in lymphocytes from human populations exposed to known mutagens and/or carcinogens. The sensitivity of the test could be further enhanced by including other gene markers, since there is evidence for locus-specific differences in response to mutagens. Resistance to diphtheria toxin (Dip/sup r/) seemed like a potential marker to incorporate into the test because the mutation acts codominantly, can readily be selected in human diploid fibroblasts and Chinesemore » hamster cells with no evidence for cell density or cross-feeding effects, and can be assayed for in nondividing cells by measuring protein synthesis inhibition. Blood samples were collected from seven individuals, and fresh, cryopreserved, or Epstein-Barr virus (EBV)-transformed lymphocytes were tested for continued DNA synthesis (TH-thymidine, autoradiography) or protein synthesis (TVS-methionine, scintillation counting). Both fresh and cryopreserved lymphocytes, stimulated to divide with phytohemagglutinin (PHA), continued to synthesize DNA in the presence of high doses of diphtheria toxin (DT). Similarly, both dividing (PHA-stimulated) and nondividing fresh lymphocytes carried on significant levels of protein synthesis even 68 hr after exposure to 100 flocculating units (LF)/ml DT. The results suggest that human T and B lymphocytes may not be as sensitive to DT protein synthesis inhibition as human fibroblast and Chinese hamster cells. For this reason, Dip/sup r/ may not be a suitable marker for the somatic cell mutation test.« less

Clinical flow cytometric screening of SAP and XIAP expression accurately identifies patients with SH2D1A and XIAP/BIRC4 mutations.

PubMed

Gifford, Carrie E; Weingartner, Elizabeth; Villanueva, Joyce; Johnson, Judith; Zhang, Kejian; Filipovich, Alexandra H; Bleesing, Jack J; Marsh, Rebecca A

2014-07-01

X-linked lymphoproliferative disease is caused by mutations in two genes, SH2D1A and XIAP/BIRC4. Flow cytometric methods have been developed to detect the gene products, SAP and XIAP. However, there is no literature describing the accuracy of flow cytometric screening performed in a clinical lab setting. We reviewed the clinical flow cytometric testing results for 656 SAP and 586 XIAP samples tested during a 3-year period. Genetic testing was clinically performed as directed by the managing physician in 137 SAP (21%) and 115 XIAP (20%) samples. We included these samples for analyses of flow cytometric test accuracy. SH2D1A mutations were detected in 15/137 samples. SAP expression was low in 13/15 (sensitivity 87%, CI 61-97%). Of the 122 samples with normal sequencing, SAP was normal in 109 (specificity 89%, CI 82-94%). The positive predictive values (PPVs) and the negative predictive values (NPVs) were 50% and 98%, respectively. XIAP/BIRC4 mutations were detected in 19/115 samples. XIAP expression was low in 18/19 (sensitivity 95%, CI 73-100%). Of the 96 samples with normal sequencing, 59 had normal XIAP expression (specificity 61%, CI 51-71%). The PPVs and NPVs were 33% and 98%, respectively. Receiver-operating characteristic analysis was able to improve the specificity to 75%. Clinical flow cytometric screening tests for SAP and XIAP deficiencies offer good sensitivity and specificity for detecting genetic mutations, and are characterized by high NPVs. We recommend these tests for patients suspected of having X-linked lymphoproliferative disease type 1 (XLP1) or XLP2. © 2014 Clinical Cytometry Society.

Diagnostic algorithm for detection of targetable driver mutations in lung adenocarcinomas: Comprehensive analyses of 205 cases with immunohistochemistry, real-time PCR and fluorescence in situ hybridization methods.

PubMed

Kao, Hua-Lin; Yeh, Yi-Chen; Lin, Chin-Hsuan; Hsu, Wei-Fang; Hsieh, Wen-Yu; Ho, Hsiang-Ling; Chou, Teh-Ying

2016-11-01

Analysis of the targetable driver mutations is now recommended in all patients with advanced lung adenocarcinoma. Molecular-based methods are usually adopted, however, along with the implementation of highly sensitive and/or mutation-specific antibodies, immunohistochemistry (IHC) has been considered an alternative method for identifying driver mutations in lung adenocarcinomas. A total of 205 lung adenocarcinomas were examined for EGFR mutations and ALK and ROS1 rearrangements using real-time PCR, fluorescence in situ hybridization (FISH) and IHC in parallel. The performance of different commercially available IHC antibody clones toward targetable driver mutations was evaluated. The association between these driver mutations and clinicopathological characteristics was also analyzed. In 205 cases we studied, 58.5% were found to harbor EGFR mutations, 6.3% ALK rearrangements and 1.0% ROS1 rearrangements. Compared to molecular-based methods, IHC of EGFR mutations showed an excellent specificity but the sensitivity is suboptimal, while IHC of ALK and ROS1 rearrangements demonstrated high sensitivity and specificity. No significant difference regarding the performance of different antibody clones toward these driver mutations was observed, except that clone SP125 showed a higher sensitivity than 43B2 in the detection of p.L858R of EGFR. In circumstances such as poor quality of nucleic acids or low content of tumor cells, IHC of EGFR mutation-specific antibodies could be used as an alternative method. Patients negative for EGFR mutations are subjected to further analysis on ALK and ROS1 rearrangements using IHC methods. Herein, we proposed a lung adenocarcinoma testing algorithm for the application of IHC in therapeutic diagnosis. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Genotype-phenotype associations in French patients with phenylketonuria and importance of genotype for full assessment of tetrahydrobiopterin responsiveness.

PubMed

Jeannesson-Thivisol, Elise; Feillet, François; Chéry, Céline; Perrin, Pascal; Battaglia-Hsu, Shyue-Fang; Herbeth, Bernard; Cano, Aline; Barth, Magalie; Fouilhoux, Alain; Mention, Karine; Labarthe, François; Arnoux, Jean-Baptiste; Maillot, François; Lenaerts, Catherine; Dumesnil, Cécile; Wagner, Kathy; Terral, Daniel; Broué, Pierre; de Parscau, Loïc; Gay, Claire; Kuster, Alice; Bédu, Antoine; Besson, Gérard; Lamireau, Delphine; Odent, Sylvie; Masurel, Alice; Guéant, Jean-Louis; Namour, Fares

2015-12-15

Mutations in Phenylalanine Hydroxylase (PAH) gene cause phenylketonuria. Sapropterin (BH4), the enzyme cofactor, is an important therapeutical strategy in phenylketonuria. However, PAH is a highly polymorphic gene and it is difficult to identify BH4-responsive genotypes. We seek here to improve prediction of BH4-responsiveness through comparison of genotypes, BH4-loading test, predictions of responsiveness according to the literature and types and locations of mutations. A total of 364 French patients among which, 9 % had mild hyperphenylalaninemia, 17.7 % mild phenylketonuria and 73.1 % classical phenylketonuria, benefited from a 24-hour BH4-loading test and had the PAH gene sequenced and analyzed by Multiplex Ligation Probe Amplification. Overall, 31.6 % of patients were BH4-responsive. The number of different mutations found was 127, including 26 new mutations. The mutations c.434A > T, c.500A > T, c.529G > C, c.1045 T > G and c.1196 T > C were newly classified as being BH4-responsive. We identified 261 genotypes, among which 46 were newly recognized as being BH4-responsive. Even though patients carry 2 responsive alleles, BH4-responsiveness cannot be predicted with certainty unless they present mild hyperphenylalaninemia. BH4-responsiveness cannot be predicted in patients carrying one responsive mutation only. In general, the milder the phenotype is, the stronger the BH4-response is. Almost exclusively missense mutations, particularly in exons 12, 11 and 8, are associated with BH4-responsiveness and any other type of mutation predicts a negative response. This study is the first of its kind, in a French population, to identify the phenotype associated with several combinations of PAH mutations. As others, it highlights the necessity of performing simultaneously BH4 loading test and molecular analysis in monitoring phenylketonuria patients.

High resolution melting: improvements in the genetic diagnosis of hypertrophic cardiomyopathy in a Portuguese cohort

PubMed Central

2012-01-01

Background Hypertrophic Cardiomyopathy (HCM) is a complex myocardial disorder with a recognized genetic heterogeneity. The elevated number of genes and mutations involved in HCM limits a gene-based diagnosis that should be considered of most importance for basic research and clinical medicine. Methodology In this report, we evaluated High Resolution Melting (HRM) robustness, regarding HCM genetic testing, by means of analyzing 28 HCM-associated genes, including the most frequent 4 HCM-associated sarcomere genes, as well as 24 genes with lower reported HCM-phenotype association. We analyzed 80 Portuguese individuals with clinical phenotype of HCM allowing simultaneously a better characterization of this disease in the Portuguese population. Results HRM technology allowed us to identify 60 mutated alleles in 72 HCM patients: 49 missense mutations, 3 nonsense mutations, one 1-bp deletion, one 5-bp deletion, one in frame 3-bp deletion, one insertion/deletion, 3 splice mutations, one 5'UTR mutation in MYH7, MYBPC3, TNNT2, TNNI3, CSRP3, MYH6 and MYL2 genes. Significantly 22 are novel gene mutations. Conclusions HRM was proven to be a technique with high sensitivity and a low false positive ratio allowing a rapid, innovative and low cost genotyping of HCM. In a short return, HRM as a gene scanning technique could be a cost-effective gene-based diagnosis for an accurate HCM genetic diagnosis and hopefully providing new insights into genotype/phenotype correlations. PMID:22429680

Significant clinical impact of recurrent BRCA1 and BRCA2 mutations in Mexico

PubMed Central

Villarreal-Garza, Cynthia; Alvarez-Gómez, Rosa María; Pérez-Plasencia, Carlos; Herrera, Luis A.; Herzog, Josef; Castillo, Danielle; Mohar, Alejandro; Castro, Clementina; Gallardo, Lenny N.; Gallardo, Dolores; Santibáñez, Miguel; Blazer, Kathleen R.; Weitzel, Jeffrey N.

2014-01-01

Background Frequent recurrent BRCA1 and BRCA2 gene (BRCA) mutations among Hispanics, including a large rearrangement Mexican founder mutation (BRCA1 ex9-12del), suggest that an ancestry-informed BRCA-testing strategy could reduce disparities and promote cancer prevention by enabling economical screening for hereditary breast and ovarian cancer in Mexico. Methods In a multistage approach, 188 cancer cases unselected for family cancer history (92 ovarian cancer and 96 breast cancer) were screened for BRCA mutations using a Hispanic mutation panel (HISPANEL®) of 115 recurrent mutations in a multiplex assay (114 on a mass spectroscopy platform, and a PCR assay for the BRCA1 ex9-12del mutation), followed by sequencing of all BRCA exons and adjacent intronic regions, and BRCA1 multiplex ligation-dependent probe amplification assay (MLPA) for HISPANEL negative cases. BRCA mutation prevalence was calculated and correlated with histology and tumor receptor status, and HISPANEL sensitivity was estimated. Results BRCA mutations were detected in 28% (26/92) of ovarian cancer cases and 15% (14/96) of breast cancer cases overall and 27% (9/33) of triple negative breast cancer. Most breast cancer cases were diagnosed with locally advanced disease. The Mexican founder mutation (BRCA1 ex9-12del) accounted for 35% of the BRCA-associated ovarian cancer cases and 29% of the BRCA-associated breast cancer cases. At 2% of the sequencing and MLPA cost, the HISPANEL detected 68% of all BRCA mutations. Conclusion In this study, we found a remarkably high prevalence of BRCA mutations among ovarian and breast cases not selected for family history, and BRCA1 ex9-12del explained one third of the total. The remarkable frequency of BRCA1 ex9-12del in Mexico City supports a nearby origin of this Mexican founder mutation and may constitute a regional public health problem. The HISPANEL presents a translational opportunity for cost-effective genetic testing to enable breast and ovarian cancer prevention. PMID:25236687

Genomic Characterization of Upper Tract Urothelial Carcinoma.

PubMed

Sfakianos, John P; Cha, Eugene K; Iyer, Gopa; Scott, Sasinya N; Zabor, Emily C; Shah, Ronak H; Ren, Qinghu; Bagrodia, Aditya; Kim, Philip H; Hakimi, A Ari; Ostrovnaya, Irina; Ramirez, Ricardo; Hanrahan, Aphrothiti J; Desai, Neil B; Sun, Arony; Pinciroli, Patrizia; Rosenberg, Jonathan E; Dalbagni, Guido; Schultz, Nikolaus; Bajorin, Dean F; Reuter, Victor E; Berger, Michael F; Bochner, Bernard H; Al-Ahmadie, Hikmat A; Solit, David B; Coleman, Jonathan A

2015-12-01

Despite a similar histologic appearance, upper tract urothelial carcinoma (UTUC) and urothelial carcinoma of the bladder (UCB) tumors have distinct epidemiologic and clinicopathologic differences. To investigate whether the differences between UTUC and UCB result from intrinsic biological diversity. Tumor and germline DNA from patients with UTUC (n=83) and UCB (n=102) were analyzed using a custom next-generation sequencing assay to identify somatic mutations and copy number alterations in 300 cancer-associated genes. We described co-mutation patterns and copy number alterations in UTUC. We also compared mutation frequencies in high-grade UTUC (n=59) and high-grade UCB (n=102). Comparison of high-grade UTUC and UCB revealed significant differences in the prevalence of somatic alterations. Genes altered more commonly in high-grade UTUC included FGFR3 (35.6% vs 21.6%; p=0.065), HRAS (13.6% vs 1.0%; p=0.001), and CDKN2B (15.3% vs 3.9%; p=0.016). Genes less frequently mutated in high-grade UTUC included TP53 (25.4% vs 57.8%; p<0.001), RB1 (0.0% vs 18.6%; p<0.001), and ARID1A (13.6% vs 27.5%; p=0.050). Because our assay was restricted to genomic alterations in a targeted panel, rare mutations and epigenetic changes were not analyzed. High-grade UTUC tumors display a spectrum of genetic alterations similar to high-grade UCB. However, there were significant differences in the prevalence of several recurrently mutated genes including HRAS, TP53, and RB1. As relevant targeted inhibitors are being developed and tested, these results may have important implications for the site-specific management of patients with urothelial carcinoma. Comparison of next-generation sequencing of upper tract urothelial carcinoma (UTUC) with urothelial bladder cancer identified that similar mutations were present in both cancer types but at different frequencies, indicating a potential need for unique management strategies. UTUC tumors were found to have a high rate of mutations that could be targeted with novel therapies. Copyright © 2015 European Association of Urology. Published by Elsevier B.V. All rights reserved.

Hereditary diffuse gastric cancer: updated clinical guidelines with an emphasis on germline CDH1 mutation carriers.

PubMed

van der Post, Rachel S; Vogelaar, Ingrid P; Carneiro, Fátima; Guilford, Parry; Huntsman, David; Hoogerbrugge, Nicoline; Caldas, Carlos; Schreiber, Karen E Chelcun; Hardwick, Richard H; Ausems, Margreet G E M; Bardram, Linda; Benusiglio, Patrick R; Bisseling, Tanya M; Blair, Vanessa; Bleiker, Eveline; Boussioutas, Alex; Cats, Annemieke; Coit, Daniel; DeGregorio, Lynn; Figueiredo, Joana; Ford, James M; Heijkoop, Esther; Hermens, Rosella; Humar, Bostjan; Kaurah, Pardeep; Keller, Gisella; Lai, Jennifer; Ligtenberg, Marjolijn J L; O'Donovan, Maria; Oliveira, Carla; Pinheiro, Hugo; Ragunath, Krish; Rasenberg, Esther; Richardson, Susan; Roviello, Franco; Schackert, Hans; Seruca, Raquel; Taylor, Amy; Ter Huurne, Anouk; Tischkowitz, Marc; Joe, Sheena Tjon A; van Dijck, Benjamin; van Grieken, Nicole C T; van Hillegersberg, Richard; van Sandick, Johanna W; Vehof, Rianne; van Krieken, J Han; Fitzgerald, Rebecca C

2015-06-01

Germline CDH1 mutations confer a high lifetime risk of developing diffuse gastric (DGC) and lobular breast cancer (LBC). A multidisciplinary workshop was organised to discuss genetic testing, surgery, surveillance strategies, pathology reporting and the patient's perspective on multiple aspects, including diet post gastrectomy. The updated guidelines include revised CDH1 testing criteria (taking into account first-degree and second-degree relatives): (1) families with two or more patients with gastric cancer at any age, one confirmed DGC; (2) individuals with DGC before the age of 40 and (3) families with diagnoses of both DGC and LBC (one diagnosis before the age of 50). Additionally, CDH1 testing could be considered in patients with bilateral or familial LBC before the age of 50, patients with DGC and cleft lip/palate, and those with precursor lesions for signet ring cell carcinoma. Given the high mortality associated with invasive disease, prophylactic total gastrectomy at a centre of expertise is advised for individuals with pathogenic CDH1 mutations. Breast cancer surveillance with annual breast MRI starting at age 30 for women with a CDH1 mutation is recommended. Standardised endoscopic surveillance in experienced centres is recommended for those opting not to have gastrectomy at the current time, those with CDH1 variants of uncertain significance and those that fulfil hereditary DGC criteria without germline CDH1 mutations. Expert histopathological confirmation of (early) signet ring cell carcinoma is recommended. The impact of gastrectomy and mastectomy should not be underestimated; these can have severe consequences on a psychological, physiological and metabolic level. Nutritional problems should be carefully monitored. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Hereditary diffuse gastric cancer: updated clinical guidelines with an emphasis on germline CDH1 mutation carriers

PubMed Central

van der Post, Rachel S; Vogelaar, Ingrid P; Carneiro, Fátima; Guilford, Parry; Huntsman, David; Hoogerbrugge, Nicoline; Caldas, Carlos; Schreiber, Karen E Chelcun; Hardwick, Richard H; Ausems, Margreet G E M; Bardram, Linda; Benusiglio, Patrick R; Bisseling, Tanya M; Blair, Vanessa; Bleiker, Eveline; Boussioutas, Alex; Cats, Annemieke; Coit, Daniel; DeGregorio, Lynn; Figueiredo, Joana; Ford, James M; Heijkoop, Esther; Hermens, Rosella; Humar, Bostjan; Kaurah, Pardeep; Keller, Gisella; Lai, Jennifer; Ligtenberg, Marjolijn J L; O'Donovan, Maria; Oliveira, Carla; Ragunath, Krish; Rasenberg, Esther; Richardson, Susan; Roviello, Franco; Schackert, Hans; Seruca, Raquel; Taylor, Amy; ter Huurne, Anouk; Tischkowitz, Marc; Joe, Sheena Tjon A; van Dijck, Benjamin; van Grieken, Nicole C T; van Hillegersberg, Richard; van Sandick, Johanna W; Vehof, Rianne; van Krieken, J Han; Fitzgerald, Rebecca C

2015-01-01

Germline CDH1 mutations confer a high lifetime risk of developing diffuse gastric (DGC) and lobular breast cancer (LBC). A multidisciplinary workshop was organised to discuss genetic testing, surgery, surveillance strategies, pathology reporting and the patient's perspective on multiple aspects, including diet post gastrectomy. The updated guidelines include revised CDH1 testing criteria (taking into account first-degree and second-degree relatives): (1) families with two or more patients with gastric cancer at any age, one confirmed DGC; (2) individuals with DGC before the age of 40 and (3) families with diagnoses of both DGC and LBC (one diagnosis before the age of 50). Additionally, CDH1 testing could be considered in patients with bilateral or familial LBC before the age of 50, patients with DGC and cleft lip/palate, and those with precursor lesions for signet ring cell carcinoma. Given the high mortality associated with invasive disease, prophylactic total gastrectomy at a centre of expertise is advised for individuals with pathogenic CDH1 mutations. Breast cancer surveillance with annual breast MRI starting at age 30 for women with a CDH1 mutation is recommended. Standardised endoscopic surveillance in experienced centres is recommended for those opting not to have gastrectomy at the current time, those with CDH1 variants of uncertain significance and those that fulfil hereditary DGC criteria without germline CDH1 mutations. Expert histopathological confirmation of (early) signet ring cell carcinoma is recommended. The impact of gastrectomy and mastectomy should not be underestimated; these can have severe consequences on a psychological, physiological and metabolic level. Nutritional problems should be carefully monitored. PMID:25979631

Founder mutations characterise the mutation panorama in 200 Swedish index cases referred for Long QT syndrome genetic testing.

PubMed

Stattin, Eva-Lena; Boström, Ida Maria; Winbo, Annika; Cederquist, Kristina; Jonasson, Jenni; Jonsson, Björn-Anders; Diamant, Ulla-Britt; Jensen, Steen M; Rydberg, Annika; Norberg, Anna

2012-10-25

Long QT syndrome (LQTS) is an inherited arrhythmic disorder characterised by prolongation of the QT interval on ECG, presence of syncope and sudden death. The symptoms in LQTS patients are highly variable, and genotype influences the clinical course. This study aims to report the spectrum of LQTS mutations in a Swedish cohort. Between March 2006 and October 2009, two hundred, unrelated index cases were referred to the Department of Clinical Genetics, Umeå University Hospital, Sweden, for LQTS genetic testing. We scanned five of the LQTS-susceptibility genes (KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2) for mutations by DHPLC and/or sequencing. We applied MLPA to detect large deletions or duplications in the KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2 genes. Furthermore, the gene RYR2 was screened in 36 selected LQTS genotype-negative patients to detect cases with the clinically overlapping disease catecholaminergic polymorphic ventricular tachycardia (CPVT). In total, a disease-causing mutation was identified in 103 of the 200 (52%) index cases. Of these, altered exon copy numbers in the KCNH2 gene accounted for 2% of the mutations, whereas a RYR2 mutation accounted for 3% of the mutations. The genotype-positive cases stemmed from 64 distinct mutations, of which 28% were novel to this cohort. The majority of the distinct mutations were found in a single case (80%), whereas 20% of the mutations were observed more than once. Two founder mutations, KCNQ1 p.Y111C and KCNQ1 p.R518*, accounted for 25% of the genotype-positive index cases. Genetic cascade screening of 481 relatives to the 103 index cases with an identified mutation revealed 41% mutation carriers who were at risk of cardiac events such as syncope or sudden unexpected death. In this cohort of Swedish index cases with suspected LQTS, a disease-causing mutation was identified in 52% of the referred patients. Copy number variations explained 2% of the mutations and 3 of 36 selected cases (8%) harboured a mutation in the RYR2 gene. The mutation panorama is characterised by founder mutations (25%), even so, this cohort increases the amount of known LQTS-associated mutations, as approximately one-third (28%) of the detected mutations were unique.

β-Thalassemia mutations in Western India: outcome of prenatal diagnosis in a hemoglobinopathies project.

PubMed

Patel, Ashwin P; Patel, Rupesh B; Patel, Saumyaa A; Vaniawala, Salil N; Patel, Dipika S; Shrivastava, Naina S; Sharma, Narmadeshwar P; Zala, Jayendrasinh V; Parmar, Prakash H; Naik, Madhuben R

2014-01-01

Prenatal diagnosis (PND) is one of the most cost effective preventive methods, but it is available only in the large cities of India. Therefore, we initiated a program that offers PND and allows us to determine the prevalence of various mutations. Pregnant females (n = 111,426) were screened for hemoglobinopathies using complete blood count (CBC) and high performance liquid chromatography (HPLC). If the female had a hemoglobinopathy, her husband was then tested. If hemoglobinopathies were seen in both partners, a genetic mutation study was performed on the couple. Fetal samples were obtained by either chorionic villus sampling (CVS) in 70.6% or amniocentesis in 29.4%. The study included 282 couples. IVS-I-5 (G > C) was the most common mutation in all castes except in the Sindhis and Lohanas, where the 619 bp deletion was the most common. Prenatal testing was informative in 97.9% of the couples. A significant number of couples (41.0%) underwent PND during their first pregnancy. Seven patients with β-thalassemia (β-thal) trait had normal Hb A2 levels. The Hb A2 and Hb F values varied significantly (p < 0.0001 and 0.0082, respectively) among mutations associated with β-thal. The IVS-I-5, 619 bp deletion, codons 41/42 (-CTTT), codons 8/9 (+G) and IVS-I-1 (G > T or G > A), were present in 81.0% of the couples tested. β-Thalassemia mutation frequency varied among the different castes, underlining the need for evolving a testing strategy that considers the caste system. Targeting antenatal clinics could also prove to be a most cost effective way of preventing hemoglobinopathies.

Epitope-positive truncating MLH1 mutation and loss of PMS2: implications for IHC-directed genetic testing for Lynch syndrome.

PubMed

Zighelboim, Israel; Powell, Matthew A; Babb, Sheri A; Whelan, Alison J; Schmidt, Amy P; Clendenning, Mark; Senter, Leigha; Thibodeau, Stephen N; de la Chapelle, Albert; Goodfellow, Paul J

2009-01-01

We assessed mismatch repair by immunohistochemistry (IHC) and microsatellite instability (MSI) analysis in an early onset endometrial cancer and a sister's colon cancer. We demonstrated high-level MSI and normal expression for MLH1, MSH2 and MSH6. PMS2 failed to stain in both tumors, strongly implicating a PMS2 defect. This family did not meet clinical criteria for Lynch syndrome. However, early onset endometrial cancers in the proband and her sister, a metachronous colorectal cancer in the sister as well as MSI in endometrial and colonic tumors suggested a heritable mismatch repair defect. PCR-based direct exonic sequencing and multiplex ligation-dependent probe amplification (MLPA) were undertaken to search for PMS2 mutations in the germline DNA from the proband and her sister. No mutation was identified in the PMS2 gene. However, PMS2 exons 3, 4, 13, 14, 15 were not evaluated by MLPA and as such, rearrangements involving those exons cannot be excluded. Clinical testing for MLH1 and MSH2 mutation revealed a germline deletion of MLH1 exons 14 and 15. This MLH1 germline deletion leads to an immunodetectable stable C-terminal truncated MLH1 protein which based on the IHC staining must abrogate PMS2 stabilization. To the best of our knowledge, loss of PMS2 in MLH1 truncating mutation carriers that express MLH1 in their tumors has not been previously reported. This family points to a potential limitation of IHC-directed gene testing for suspected Lynch syndrome and the need to consider comprehensive MLH1 testing for individuals whose tumors lack PMS2 but for whom PMS2 mutations are not identified.

Rapid genotyping of common MeCP2 mutations with an electronic DNA microchip using serial differential hybridization.

PubMed

Thistlethwaite, William A; Moses, Linda M; Hoffbuhr, Kristen C; Devaney, Joseph M; Hoffman, Eric P

2003-05-01

Rett syndrome is a neurodevelopmental disorder that affects females almost exclusively, and in which eight common point mutations on the X-linked MeCP2 gene are knows to cause over 70% of mutation-positive cases. We explored the use of a novel platform to detect the eight common mutations in Rett syndrome patients to expedite and simplify the process of identification of known genotypes. The Nanogen workstation consists of a two-color assay based on electric hybridization and thermal discrimination, all performed on an electronically active NanoChip. This genotyping platform was tested on 362 samples of a pre-determined genotype, which had been previously identified by a combination of DHPLC (denaturing high performance liquid chromatography) and direct sequencing. This genotyping technique proved to be rapid, facile, and displayed a specificity of 100% with 3% ambiguity. In addition, we present consecutive testing of seven mutations on a single pad of the NanoChip. This was accomplished by tagging down two amplimers together and serially hybridizing for seven different loci, allowing us to genotype samples for seven of the eight common Rett mutations on a single pad. This novel method displayed the same level of specificity and accuracy as the single amplimer reactions, and proved to be faster and more economical.

The mitochondrial DNA G13513A MELAS mutation in the NADH dehydrogenase 5 gene is a frequent cause of Leigh-like syndrome with isolated complex I deficiency.

PubMed

Chol, M; Lebon, S; Bénit, P; Chretien, D; de Lonlay, P; Goldenberg, A; Odent, S; Hertz-Pannier, L; Vincent-Delorme, C; Cormier-Daire, V; Rustin, P; Rötig, A; Munnich, A

2003-03-01

Leigh syndrome is a subacute necrotising encephalomyopathy frequently ascribed to mitochondrial respiratory chain deficiency. This condition is genetically heterogeneous, as mutations in both mitochondrial (mt) and nuclear genes have been reported. Here, we report the G13513A transition in the ND5 mtDNA gene in three unrelated children with complex I deficiency and a peculiar MRI aspect distinct from typical Leigh syndrome. Brain MRI consistently showed a specific involvement of the substantia nigra and medulla oblongata sparing the basal ganglia. Variable degrees of heteroplasmy were found in all tissues tested and a high percentage of mutant mtDNA was observed in muscle. The asymptomatic mothers presented low levels of mutant mtDNA in blood leucocytes. This mutation, which affects an evolutionary conserved amino acid (D393N), has been previously reported in adult patients with MELAS or LHON/MELAS syndromes, emphasising the clinical heterogeneity of mitochondrial DNA mutations. Since the G13513A mutation was found in 21% of our patients with Leigh syndrome and complex I deficiency (3/14), it appears that this mutation represents a frequent cause of Leigh-like syndrome, which should be systematically tested for molecular diagnosis in affected children and for genetic counselling in their maternal relatives.

MPL mutation profile in JAK2 mutation-negative patients with myeloproliferative disorders.

PubMed

Ma, Wanlong; Zhang, Xi; Wang, Xiuqiang; Zhang, Zhong; Yeh, Chen-Hsiung; Uyeji, Jennifer; Albitar, Maher

2011-03-01

Mutations in the thrombopoietin receptor gene (myeloproliferative leukemia, MPL) have been reported in patients with JAK2 V617F-negative chronic myeloproliferative disorders (MPDs). We evaluated the prevalence of MPL mutations relative to JAK2 mutations in patients with suspected MPDs. A total of 2790 patient samples submitted for JAK2 mutation analysis were tested using real-time polymerase chain reaction and bidirectional sequencing of plasma RNA. JAK2 V617F-negative samples were tested for JAK2 exons 12 to 14 mutations, and those with negative results were then tested for mutations in MPL exons 10 and 11. Of the 2790 patients, 529 (18.96%) had V617F, 12 (0.43%) had small insertions or deletions in exon 12, and 7 (0.25%) had other JAK2 mutations in exons 12 to 14. Of the 2242 JAK2 mutation-negative patients, 68 (3.03%) had MPL mutations. W515L was the predominant MPL mutation (n=46; 68%), and 10 (15%) patients had other W515 variants. The remaining MPL mutations (n=12, 17%) were detected at other locations in exons 10 and 11 and included 3 insertion/deletion mutations. The S505N mutation, associated with familial MPD, was detected in 3 patients. Overall, for every 100 V617F mutations in patients with suspected MPDs, there were 12.9 MPL mutations, 2.3 JAK2 exon 12 mutations, and 1.3 JAK2 exons 13 to 14 mutations. These findings suggest that MPL mutation screening should be performed before JAK2 exons 12 to 14 testing in JAK2 V617F-negative patients with suspected MPDs.

Assessment of real-time PCR method for detection of EGFR mutation using both supernatant and cell pellet of malignant pleural effusion samples from non-small-cell lung cancer patients.

PubMed

Shin, Saeam; Kim, Juwon; Kim, Yoonjung; Cho, Sun-Mi; Lee, Kyung-A

2017-10-26

EGFR mutation is an emerging biomarker for treatment selection in non-small-cell lung cancer (NSCLC) patients. However, optimal mutation detection is hindered by complications associated with the biopsy procedure, tumor heterogeneity and limited sensitivity of test methodology. In this study, we evaluated the diagnostic utility of real-time PCR using malignant pleural effusion samples. A total of 77 pleural fluid samples from 77 NSCLC patients were tested using the cobas EGFR mutation test (Roche Molecular Systems). Pleural fluid was centrifuged, and separated cell pellets and supernatants were tested in parallel. Results were compared with Sanger sequencing and/or peptide nucleic acid (PNA)-mediated PCR clamping of matched tumor tissue or pleural fluid samples. All samples showed valid real-time PCR results in one or more DNA samples extracted from cell pellets and supernatants. Compared with other molecular methods, the sensitivity of real-time PCR method was 100%. Concordance rate of real-time PCR and Sanger sequencing plus PNA-mediated PCR clamping was 98.7%. We have confirmed that real-time PCR using pleural fluid had a high concordance rate compared to conventional methods, with no failed samples. Our data demonstrated that the parallel real-time PCR testing using supernatant and cell pellet could offer reliable and robust surrogate strategy when tissue is not available.

Detection of MPL mutations by a novel allele-specific PCR-based strategy.

PubMed

Furtado, Larissa V; Weigelin, Helmut C; Elenitoba-Johnson, Kojo S J; Betz, Bryan L

2013-11-01

MPL mutation testing is recommended in patients with suspected primary myelofibrosis or essential thrombocythemia who lack the JAK2 V617F mutation. MPL mutations can occur at allelic levels below 15%, which may escape detection by commonly used mutation screening methods such as Sanger sequencing. We developed a novel multiplexed allele-specific PCR assay capable of detecting most recurrent MPL exon 10 mutations associated with primary myelofibrosis and essential thrombocythemia (W515L, W515K, W515A, and S505N) down to a sensitivity of 2.5% mutant allele. Test results were reviewed from 15 reference cases and 1380 consecutive specimens referred to our laboratory for testing. Assay performance was compared to Sanger sequencing across a series of 58 specimens with MPL mutations. Positive cases consisted of 45 with W515L, 6 with S505N, 5 with W515K, 1 with W515A, and 1 with both W515L and S505N. Seven cases had mutations below 5% that were undetected by Sanger sequencing. Ten additional cases had mutation levels between 5% and 15% that were not consistently detected by sequencing. All results were easily interpreted in the allele-specific test. This assay offers a sensitive and reliable solution for MPL mutation testing. Sanger sequencing appears insufficiently sensitive for robust MPL mutation detection. Our data also suggest the relative frequency of S505N mutations may be underestimated, highlighting the necessity for inclusion of this mutation in MPL test platforms. Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Next-Generation Genotyping by Digital PCR to Detect and Quantify the BRAF V600E Mutation in Melanoma Biopsies.

PubMed

Lamy, Pierre-Jean; Castan, Florence; Lozano, Nicolas; Montélion, Cécile; Audran, Patricia; Bibeau, Frédéric; Roques, Sylvie; Montels, Frédéric; Laberenne, Anne-Claire

2015-07-01

The detection of the BRAF V600E mutation in melanoma samples is used to select patients who should respond to BRAF inhibitors. Different techniques are routinely used to determine BRAF status in clinical samples. However, low tumor cellularity and tumor heterogeneity can affect the sensitivity of somatic mutation detection. Digital PCR (dPCR) is a next-generation genotyping method that clonally amplifies nucleic acids and allows the detection and quantification of rare mutations. Our aim was to evaluate the clinical routine performance of a new dPCR-based test to detect and quantify BRAF mutation load in 47 paraffin-embedded cutaneous melanoma biopsies. We compared the results obtained by dPCR with high-resolution melting curve analysis and pyrosequencing or with one of the allele-specific PCR methods available on the market. dPCR showed the lowest limit of detection. dPCR and allele-specific amplification detected the highest number of mutated samples. For the BRAF mutation load quantification both dPCR and pyrosequencing gave similar results with strong disparities in allele frequencies in the 47 tumor samples under study (from 0.7% to 79% of BRAF V600E mutations/sample). In conclusion, the four methods showed a high degree of concordance. dPCR was the more-sensitive method to reliably and easily detect mutations. Both pyrosequencing and dPCR could quantify the mutation load in heterogeneous tumor samples. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Detection of katG and inhA mutations to guide isoniazid and ethionamide use for drug-resistant tuberculosis

PubMed Central

Bollela, V. R.; Namburete, E. I.; Feliciano, C. S.; Macheque, D.; Harrison, L. H.; Caminero, J. A.

2017-01-01

SUMMARY BACKGROUND Depending on the presence of mutations that determine isoniazid (INH) susceptibility (katG and inhA), Mycobacterium tuberculosis may be susceptible to high doses of INH or ethionamide (ETH). OBJECTIVE To describe the INH resistance profile and association of katG mutation with previous INH treatment and level of drug resistance based on rapid molecular drug susceptibility testing (DST) in southern Brazil and central Mozambique. DESIGN Descriptive study of 311 isolates from Ribeirão Preto, São Paulo, Brazil (2011–2014) and 155 isolates from Beira, Mozambique (2014–2015). Drug resistance patterns and specific gene mutations were determined using GenoType® MTBDRplus. RESULTS katG gene mutations were detected in 12/22 (54.5%) Brazilian and 32/38 (84.2%) Mozambican isolates. inhA mutations were observed in 9/22 (40.9%) isolates in Brazil and in 4/38 (10.5%) in Mozambique. Both katG and inhA mutations were detected in respectively 1/22 (5%) and 2/38 (5.2%). The difference in the frequency of katG mutations in Brazil and Mozambique was statistically significant (P = 0.04). katG mutations were present in 68.8% (33/48) of patients previously treated with INH and 31.2% (15/48) of patients without previous INH. This difference was not statistically significant (P = 0.223). CONCLUSION INH mutations varied geographically; molecular DST can be used to guide and accelerate decision making in the use of ETH or high doses of INH. PMID:27393546

Extensive scanning of the calpain-3 gene broadens the spectrum of LGMD2A phenotypes.

PubMed

Piluso, G; Politano, L; Aurino, S; Fanin, M; Ricci, E; Ventriglia, V M; Belsito, A; Totaro, A; Saccone, V; Topaloglu, H; Nascimbeni, A C; Fulizio, L; Broccolini, A; Canki-Klain, N; Comi, L I; Nigro, G; Angelini, C; Nigro, V

2005-09-01

The limb girdle muscular dystrophies (LGMD) are a heterogeneous group of Mendelian disorders highlighted by weakness of the pelvic and shoulder girdle muscles. Seventeen autosomal loci have been so far identified and genetic tests are mandatory to distinguish among the forms. Mutations at the calpain 3 locus (CAPN3) cause LGMD type 2A. To obtain unbiased information on the consequences of CAPN3 mutations. 530 subjects with different grades of symptoms and 300 controls. High throughput denaturing HPLC analysis of DNA pools. 141 LGMD2A cases were identified, carrying 82 different CAPN3 mutations (45 novel), along with 18 novel polymorphisms/variants. Females had a more favourable course than males. In 94% of the more severely affected patient group, the defect was also discovered in the second allele. This proves the sensitivity of the approach. CAPN3 mutations were found in 35.1% of classical LGMD phenotypes. Mutations were also found in 18.4% of atypical patients and in 12.6% of subjects with high serum creatine kinase levels. A non-invasive and cost-effective strategy, based on the high throughput denaturing HPLC analysis of DNA pools, was used to obtain unbiased information on the consequences of CAPN3 mutations in the largest genetic study ever undertaken. This broadens the spectrum of LGMD2A phenotypes and sets the carrier frequency at 1:103.

Mutations in Splicing Factor Genes Are a Major Cause of Autosomal Dominant Retinitis Pigmentosa in Belgian Families

PubMed Central

Coppieters, Frauke; Roels, Dimitri; De Jaegere, Sarah; Flipts, Helena; De Zaeytijd, Julie; Walraedt, Sophie; Claes, Charlotte; Fransen, Erik; Van Camp, Guy; Depasse, Fanny; Casteels, Ingele; de Ravel, Thomy

2017-01-01

Purpose Autosomal dominant retinitis pigmentosa (adRP) is characterized by an extensive genetic heterogeneity, implicating 27 genes, which account for 50 to 70% of cases. Here 86 Belgian probands with possible adRP underwent genetic testing to unravel the molecular basis and to assess the contribution of the genes underlying their condition. Methods Mutation detection methods evolved over the past ten years, including mutation specific methods (APEX chip analysis), linkage analysis, gene panel analysis (Sanger sequencing, targeted next-generation sequencing or whole exome sequencing), high-resolution copy number screening (customized microarray-based comparative genomic hybridization). Identified variants were classified following American College of Medical Genetics and Genomics (ACMG) recommendations. Results Molecular genetic screening revealed mutations in 48/86 cases (56%). In total, 17 novel pathogenic mutations were identified: four missense mutations in RHO, five frameshift mutations in RP1, six mutations in genes encoding spliceosome components (SNRNP200, PRPF8, and PRPF31), one frameshift mutation in PRPH2, and one frameshift mutation in TOPORS. The proportion of RHO mutations in our cohort (14%) is higher than reported in a French adRP population (10.3%), but lower than reported elsewhere (16.5–30%). The prevalence of RP1 mutations (10.5%) is comparable to other populations (3.5%-10%). The mutation frequency in genes encoding splicing factors is unexpectedly high (altogether 19.8%), with PRPF31 the second most prevalent mutated gene (10.5%). PRPH2 mutations were found in 4.7% of the Belgian cohort. Two families (2.3%) have the recurrent NR2E3 mutation p.(Gly56Arg). The prevalence of the recurrent PROM1 mutation p.(Arg373Cys) was higher than anticipated (3.5%). Conclusions Overall, we identified mutations in 48 of 86 Belgian adRP cases (56%), with the highest prevalence in RHO (14%), RP1 (10.5%) and PRPF31 (10.5%). Finally, we expanded the molecular spectrum of PRPH2, PRPF8, RHO, RP1, SNRNP200, and TOPORS-associated adRP by the identification of 17 novel mutations. PMID:28076437

Quadruple-first line drug resistance in Mycobacterium tuberculosis in Vietnam: What can we learn from genes?

PubMed

Nguyen, Huy Quang; Nguyen, Nhung Viet; Contamin, Lucie; Tran, Thanh Hoa Thi; Vu, Thuong Thi; Nguyen, Hung Van; Nguyen, Ngoc Lan Thi; Nguyen, Son Thai; Dang, Anh Duc; Bañuls, Anne-Laure; Nguyen, Van Anh Thi

2017-06-01

In Vietnam, a country with high tuberculosis (137/100.000 population) and multidrug-resistant (MDR)-TB burdens (7.8/100.000 population), little is known about the molecular signatures of drug resistance in general and more particularly of second line drug (SLD) resistance. This study is specifically focused on Mycobacterium tuberculosis isolates resistant to four first-line drugs (FLDs) that make TB much more difficult to treat. The aim is to determine the proportion of SLD resistance in these quadruple drug resistant isolates and the genetic determinants linked to drug resistance to better understand the genetic processes leading to quadruple and extremely drug resistance (XDR). 91 quadruple (rifampicin, isoniazid, ethambutol and streptomycin) FLD resistant and 55 susceptible isolates were included. Spoligotyping and 24-locus MIRU-VNTR techniques were performed and 9 genes and promoters linked to FLD and SLD resistance were sequenced. SLD susceptibility testing was carried out on a subsample of isolates. High proportion of quadruple-FLD resistant isolates was resistant to fluoroquinolones (27%) and second-line injectable drugs (30.2%) by drug susceptibility testing. The sequencing revealed high mutation diversity with prevailing mutations at positions katG315, inhA-15, rpoB531, embB306, rrs1401, rpsL43 and gyrA94. The sensitivity and specificity were high for most drug resistances (>86%), but the sensitivity was lower for injectable drug resistances (<69%). The mutation patterns revealed 23.1% of pre-XDR and 7.7% of XDR isolates, mostly belonging to Beijing family. The genotypic diversity and the variety of mutations reflect the existence of various evolutionary paths leading to FLD and SLD resistance. Nevertheless, particular mutation patterns linked to high-level resistance and low fitness costs seem to be favored. Copyright © 2017 Elsevier B.V. All rights reserved.

Optimized p53 immunohistochemistry is an accurate predictor of TP53 mutation in ovarian carcinoma.

PubMed

Köbel, Martin; Piskorz, Anna M; Lee, Sandra; Lui, Shuhong; LePage, Cecile; Marass, Francesco; Rosenfeld, Nitzan; Mes Masson, Anne-Marie; Brenton, James D

2016-10-01

TP53 mutations are ubiquitous in high-grade serous ovarian carcinomas (HGSOC), and the presence of TP53 mutation discriminates between high and low-grade serous carcinomas and is now an important biomarker for clinical trials targeting mutant p53. p53 immunohistochemistry (IHC) is widely used as a surrogate for TP53 mutation but its accuracy has not been established. The objective of this study was to test whether improved methods for p53 IHC could reliably predict TP53 mutations independently identified by next generation sequencing (NGS). Four clinical p53 IHC assays and tagged-amplicon NGS for TP53 were performed on 171 HGSOC and 80 endometrioid carcinomas (EC). p53 expression was scored as overexpression (OE), complete absence (CA), cytoplasmic (CY) or wild type (WT). p53 IHC was evaluated as a binary classifier where any abnormal staining predicted deleterious TP53 mutation and as a ternary classifier where OE, CA or WT staining predicted gain-of-function (GOF or nonsynonymous), loss-of-function (LOF including stopgain, indel, splicing) or no detectable TP53 mutations (NDM), respectively. Deleterious TP53 mutations were detected in 169/171 (99%) HGSOC and 7/80 (8.8%) EC. The overall accuracy for the best performing IHC assay for binary and ternary prediction was 0.94 and 0.91 respectively, which improved to 0.97 (sensitivity 0.96, specificity 1.00) and 0.95 after secondary analysis of discordant cases. The sensitivity for predicting LOF mutations was lower at 0.76 because p53 IHC detected mutant p53 protein in 13 HGSOC with LOF mutations. CY staining associated with LOF was seen in 4 (2.3%) of HGSOC. Optimized p53 IHC can approach 100% specificity for the presence of TP53 mutation and its high negative predictive value is clinically useful as it can exclude the possibility of a low-grade serous tumour. 4.1% of HGSOC cases have detectable WT staining while harboring a TP53 LOF mutation, which limits sensitivity for binary prediction of mutation to 96%.

Changes in screening behaviors and attitudes toward screening from pre-test genetic counseling to post-disclosure in Lynch syndrome families

PubMed Central

Burton-Chase, Allison M.; Hovick, Shelly R.; Peterson, Susan K.; Marani, Salma K.; Vernon, Sally W.; Amos, Christopher I.; Frazier, Marsha L.; Lynch, Patrick M.; Gritz, Ellen R.

2013-01-01

Purpose This study examined colonoscopy adherence and attitudes towards colorectal cancer (CRC) screening in individuals who underwent Lynch syndrome genetic counseling and testing. Methods We evaluated changes in colonoscopy adherence and CRC screening attitudes in 78 cancer-unaffected relatives of Lynch syndrome mutation carriers before pre-test genetic counseling (baseline) and at 6 and 12 months post-disclosure of test results (52 mutation-negative, 26 mutation-positive). Results While both groups were similar at baseline, at 12 months post-disclosure, a greater number of mutation-positive individuals had had a colonoscopy compared with mutation-negative individuals. From baseline to 12 months post-disclosure, the mutation-positive group demonstrated an increase in mean scores on measures of colonoscopy commitment, self-efficacy, and perceived benefits of CRC screening, and a decrease in mean scores for perceived barriers to CRC screening. Mean scores on colonoscopy commitment decreased from baseline to 6 months in the mutation-negative group. Conclusion Adherence to risk-appropriate guidelines for CRC surveillance improved after genetic counseling and testing for Lynch syndrome. Mutation-positive individuals reported increasingly positive attitudes toward CRC screening after receiving genetic test results, potentially reinforcing longer term colonoscopy adherence. PMID:23414081

A new mutation in MT-ND1 m.3928G>C p.V208L causes Leigh disease with infantile spasms.

PubMed

Wray, Carter D; Friederich, Marisa W; du Sart, Desiree; Pantaleo, Sarah; Smet, Joél; Kucera, Cathlin; Fenton, Laura; Scharer, Gunter; Van Coster, Rudy; Van Hove, Johan L K

2013-11-01

New mutations in mitochondrial DNA encoded genes of complex I are rarely reported. An infant developed Leigh disease with infantile spasms. Complex I enzyme activity was deficient and response to increasing coenzyme Q concentrations was reduced. Complex I assembly was intact. A new mutation in MT-ND1 m.3928G>C p.V208L, affecting a conserved amino acid in a critical domain, part of the coenzyme Q binding pocket, was present at high heteroplasmy. The unaffected mother did not carry measurable mutant mitochondrial DNA, but concern remained for gonadal mosaicism. Prenatal testing was possible for a subsequent sibling. The ND1 p.V208L mutation causes Leigh disease. © 2013.

The Evolving Landscape of HIV Drug Resistance Diagnostics for Expanding Testing in Resource-Limited Settings.

PubMed

Inzaule, Seth C; Hamers, Ralph L; Paredes, Roger; Yang, Chunfu; Schuurman, Rob; Rinke de Wit, Tobias F

2017-01-01

Global scale-up of antiretroviral treatment has dramatically changed the prospects of HIV/AIDS disease, rendering life-long chronic care and treatment a reality for millions of HIV-infected patients. Affordable technologies to monitor antiretroviral treatment are needed to ensure long-term durability of limited available drug regimens. HIV drug resistance tests can complement existing strategies in optimizing clinical decision-making for patients with treatment failure, in addition to facilitating population-based surveillance of HIV drug resistance. This review assesses the current landscape of HIV drug resistance technologies and discusses the strengths and limitations of existing assays available for expanding testing in resource-limited settings. These include sequencing-based assays (Sanger sequencing assays and nextgeneration sequencing), point mutation assays, and genotype-free data-based prediction systems. Sanger assays are currently considered the gold standard genotyping technology, though only available at a limited number of resource-limited setting reference and regional laboratories, but high capital and test costs have limited their wide expansion. Point mutation assays present opportunities for simplified laboratory assays, but HIV genetic variability, extensive codon redundancy at or near the mutation target sites with limited multiplexing capability have restricted their utility. Next-generation sequencing, despite high costs, may have potential to reduce the testing cost significantly through multiplexing in high-throughput facilities, although the level of bioinformatics expertise required for data analysis is currently still complex and expensive and lacks standardization. Web-based genotype-free prediction systems may provide enhanced antiretroviral treatment decision-making without the need for laboratory testing, but require further clinical field evaluation and implementation scientific research in resource-limited settings.

Development and Validation of an Ultradeep Next-Generation Sequencing Assay for Testing of Plasma Cell-Free DNA from Patients with Advanced Cancer.

PubMed

Janku, Filip; Zhang, Shile; Waters, Jill; Liu, Li; Huang, Helen J; Subbiah, Vivek; Hong, David S; Karp, Daniel D; Fu, Siqing; Cai, Xuyu; Ramzanali, Nishma M; Madwani, Kiran; Cabrilo, Goran; Andrews, Debra L; Zhao, Yue; Javle, Milind; Kopetz, E Scott; Luthra, Rajyalakshmi; Kim, Hyunsung J; Gnerre, Sante; Satya, Ravi Vijaya; Chuang, Han-Yu; Kruglyak, Kristina M; Toung, Jonathan; Zhao, Chen; Shen, Richard; Heymach, John V; Meric-Bernstam, Funda; Mills, Gordon B; Fan, Jian-Bing; Salathia, Neeraj S

2017-09-15

Purpose: Tumor-derived cell-free DNA (cfDNA) in plasma can be used for molecular testing and provide an attractive alternative to tumor tissue. Commonly used PCR-based technologies can test for limited number of alterations at the time. Therefore, novel ultrasensitive technologies capable of testing for a broad spectrum of molecular alterations are needed to further personalized cancer therapy. Experimental Design: We developed a highly sensitive ultradeep next-generation sequencing (NGS) assay using reagents from TruSeqNano library preparation and NexteraRapid Capture target enrichment kits to generate plasma cfDNA sequencing libraries for mutational analysis in 61 cancer-related genes using common bioinformatics tools. The results were retrospectively compared with molecular testing of archival primary or metastatic tumor tissue obtained at different points of clinical care. Results: In a study of 55 patients with advanced cancer, the ultradeep NGS assay detected 82% (complete detection) to 87% (complete and partial detection) of the aberrations identified in discordantly collected corresponding archival tumor tissue. Patients with a low variant allele frequency (VAF) of mutant cfDNA survived longer than those with a high VAF did ( P = 0.018). In patients undergoing systemic therapy, radiological response was positively associated with changes in cfDNA VAF ( P = 0.02), and compared with unchanged/increased mutant cfDNA VAF, decreased cfDNA VAF was associated with longer time to treatment failure (TTF; P = 0.03). Conclusions: Ultradeep NGS assay has good sensitivity compared with conventional clinical mutation testing of archival specimens. A high VAF in mutant cfDNA corresponded with shorter survival. Changes in VAF of mutated cfDNA were associated with TTF. Clin Cancer Res; 23(18); 5648-56. ©2017 AACR . ©2017 American Association for Cancer Research.

[Detection of gene mutation in glucose-6-phosphate dehydrogenase deficiency by RT-PCR sequencing].

PubMed

Lyu, Rong-Yu; Chen, Xiao-Wen; Zhang, Min; Chen, Yun-Sheng; Yu, Jie; Wen, Fei-Qiu

2016-07-01

Since glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common hereditary hemolytic erythrocyte enzyme deficiency, most cases have single nucleotide mutations in the coding region, and current test methods for gene mutation have some missed detections, this study aimed to investigate the feasibility of RT-PCR sequencing in the detection of gene mutation in G6PD deficiency. According to the G6PD/6GPD ratio, 195 children with anemia of unknown cause or who underwent physical examination between August 2013 and July 2014 were classified into G6PD-deficiency group with 130 children (G6PD/6GPD ratio <1.00) and control group with 65 children (G6PD/6GPD ratio≥1.00). The primer design and PCR amplification conditions were optimized, and RT-PCR sequencing was used to analyze the complete coding sequence and verify the genomic DNA sequence in the two groups. In the G6PD-deficiency group, the detection rate of gene mutation was 100% and 13 missense mutations were detected, including one new mutation. In the control group, no missense mutation was detected in 28 boys; 13 heterozygous missense mutations, 1 homozygous same-sense mutation (C1191T) which had not been reported in China and abroad, and 14 single nucleotide polymorphisms of C1311T were detected in 37 girls. The control group showed a high rate of missed detection of G6PD deficiency (carriers) in the specimens from girls (35%, 13/37). RT-PCR sequencing has a high detection rate of G6PD gene mutation and a certain value in clinical diagnosis of G6PD deficiency.

Preconceptional identification of cystic fibrosis carriers in the Sardinian population: A pilot screening program.

PubMed

Coiana, Alessandra; Faa', Valeria; Carta, Daniela; Puddu, Rosalba; Cao, Antonio; Rosatelli, Maria Cristina

2011-05-01

In Sardinia the mutational spectrum of CFTR gene is well defined. A mutation detection rate of 94% can be achieved by screening for 15 CFTR mutations with a frequency higher than 0.5%. The efficiency of this molecular test suggests that Sardinians may represent a suitable population for a preconceptional screening. Five hundred couples of Sardinia descent were screened for 38 mutations using a semi-automated reverse-dot blot and PCR-gel electrophoresis assays. This mutation panel included the 15 most frequent CF alleles in Sardinia. We identified 38 CF carriers, revealing an overall frequency of 1/25 (4%). The most common CF allele was the p.Thr338Ile (T338I) (65%), followed by the p.Phe508del (F508del) (22.5%). We also identified one couple at risk and an asymptomatic female homozygote for the p.Thr338Ile allele. In spite of the low number of the couples tested, the results herein reported demonstrate the efficacy and efficiency of the preconceptional screening program and the high participation rate of the Sardinian population (99%). Copyright © 2010 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Diagnostic genetic testing for patients with bilateral optic neuropathy and comparison of clinical features according to OPA1 mutation status.

PubMed

Gaier, Eric D; Boudreault, Katherine; Nakata, Isao; Janessian, Maria; Skidd, Philip; DelBono, Elizabeth; Allen, Keri F; Pasquale, Louis R; Place, Emily; Cestari, Dean M; Stacy, Rebecca C; Rizzo, Joseph F; Wiggs, Janey L

2017-01-01

Inherited optic neuropathy is genetically heterogeneous, and genetic testing has an important role in risk assessment and counseling. The purpose of this study is to determine the prevalence and spectrum of mutations in a group of patients referred for genetic testing to a tertiary center in the United States. In addition, we compared the clinical features of patients with and without mutations in OPA1 , the gene most commonly involved in dominantly inherited optic atrophy. Clinical data and genetic testing results were reviewed for 74 unrelated, consecutive patients referred with a history of insidious, relatively symmetric, bilateral visual loss secondary to an optic neuropathy. Patients were evaluated for disease-causing variants in OPA1 , OPA3 , WFS1 , and the entire mitochondrial genome with DNA sequencing and copy number variation (CNV) testing. Pathogenic DNA variants were found in 25 cases, with the majority (24 patients) located in OPA1 . Demographics, clinical history, and clinical features for the group of patients with mutations in OPA1 were compared to those without disease-causing variants. Compared to the patients without mutations, cases with mutations in OPA1 were more likely to have a family history of optic nerve disease (p = 0.027); however, 30.4% of patients without a family history of disease also had mutations in OPA1 . OPA1 mutation carriers had less severe mean deviation and pattern standard deviation on automated visual field testing than patients with optic atrophy without mutations in OPA1 (p<0.005). Other demographic and ocular features were not statistically significantly different between the two groups, including the fraction of patients with central scotomas (42.9% of OPA1 mutation positive and 66.0% of OPA1 mutation negative). Genetic testing identified disease-causing mutations in 34% of referred cases, with the majority of these in OPA1. Patients with mutations in OPA1 were more likely to have a family history of disease; however, 30.4% of patients without a family history were also found to have an OPA1 mutation. This observation, as well as similar frequencies of central scotomas in the groups with and without mutations in OPA1 , underscores the need for genetic testing to establish an OPA1 genetic diagnosis.

Identification of a Comprehensive Spectrum of Genetic Factors for Hereditary Breast Cancer in a Chinese Population by Next-Generation Sequencing

PubMed Central

Yang, Xiaochen; Wu, Jiong; Lu, Jingsong; Liu, Guangyu; Di, Genhong; Chen, Canming; Hou, Yifeng; Sun, Menghong; Yang, Wentao; Xu, Xiaojing; Zhao, Ying; Hu, Xin; Li, Daqiang; Cao, Zhigang; Zhou, Xiaoyan; Huang, Xiaoyan; Liu, Zhebin; Chen, Huan; Gu, Yanzi; Chi, Yayun; Yan, Xia; Han, Qixia; Shen, Zhenzhou; Shao, Zhimin; Hu, Zhen

2015-01-01

The genetic etiology of hereditary breast cancer has not been fully elucidated. Although germline mutations of high-penetrance genes such as BRCA1/2 are implicated in development of hereditary breast cancers, at least half of all breast cancer families are not linked to these genes. To identify a comprehensive spectrum of genetic factors for hereditary breast cancer in a Chinese population, we performed an analysis of germline mutations in 2,165 coding exons of 152 genes associated with hereditary cancer using next-generation sequencing (NGS) in 99 breast cancer patients from families of cancer patients regardless of cancer types. Forty-two deleterious germline mutations were identified in 21 genes of 34 patients, including 18 (18.2%) BRCA1 or BRCA2 mutations, 3 (3%) TP53 mutations, 5 (5.1%) DNA mismatch repair gene mutations, 1 (1%) CDH1 mutation, 6 (6.1%) Fanconi anemia pathway gene mutations, and 9 (9.1%) mutations in other genes. Of seven patients who carried mutations in more than one gene, 4 were BRCA1/2 mutation carriers, and their average onset age was much younger than patients with only BRCA1/2 mutations. Almost all identified high-penetrance gene mutations in those families fulfill the typical phenotypes of hereditary cancer syndromes listed in the National Comprehensive Cancer Network (NCCN) guidelines, except two TP53 and three mismatch repair gene mutations. Furthermore, functional studies of MSH3 germline mutations confirmed the association between MSH3 mutation and tumorigenesis, and segregation analysis suggested antagonism between BRCA1 and MSH3. We also identified a lot of low-penetrance gene mutations. Although the clinical significance of those newly identified low-penetrance gene mutations has not been fully appreciated yet, these new findings do provide valuable epidemiological information for the future studies. Together, these findings highlight the importance of genetic testing based on NCCN guidelines and a multi-gene analysis using NGS may be a supplement to traditional genetic counseling. PMID:25927356

Interplay between DMD Point Mutations and Splicing Signals in Dystrophinopathy Phenotypes

PubMed Central

Juan-Mateu, Jonàs; González-Quereda, Lidia; Rodríguez, Maria José; Verdura, Edgard; Lázaro, Kira; Jou, Cristina; Nascimento, Andrés; Jiménez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jesús; Baiget, Montserrat; Gallano, Pia

2013-01-01

DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements. PMID:23536893

Analytical validation of BRAF mutation testing from circulating free DNA using the amplification refractory mutation testing system.

PubMed

Aung, Kyaw L; Donald, Emma; Ellison, Gillian; Bujac, Sarah; Fletcher, Lynn; Cantarini, Mireille; Brady, Ged; Orr, Maria; Clack, Glen; Ranson, Malcolm; Dive, Caroline; Hughes, Andrew

2014-05-01

BRAF mutation testing from circulating free DNA (cfDNA) using the amplification refractory mutation testing system (ARMS) holds potential as a surrogate for tumor mutation testing. Robust assay validation is needed to establish the optimal clinical matrix for measurement and cfDNA-specific mutation calling criteria. Plasma- and serum-derived cfDNA samples from 221 advanced melanoma patients were analyzed for BRAF c.1799T>A (p.V600E) mutation using ARMS in two stages in a blinded fashion. cfDNA-specific mutation calling criteria were defined in stage 1 and validated in stage 2. cfDNA concentrations in serum and plasma, and the sensitivities and specificities of BRAF mutation detection in these two clinical matrices were compared. Sensitivity of BRAF c.1799T>A (p.V600E) mutation detection in cfDNA was increased by using mutation calling criteria optimized for cfDNA (these criteria were adjusted from those used for archival tumor biopsies) without compromising specificity. Sensitivity of BRAF mutation detection in serum was 44% (95% CI, 35% to 53%) and in plasma 52% (95% CI, 43% to 61%). Specificity was 96% (95% CI, 90% to 99%) in both matrices. Serum contains significantly higher total cfDNA than plasma, whereas the proportion of tumor-derived mutant DNA was significantly higher in plasma. Using mutation calling criteria optimized for cfDNA improves sensitivity of BRAF c.1799T>A (p.V600E) mutation detection. The proportion of tumor-derived cfDNA in plasma was significantly higher than in serum. Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Accurate clinical genetic testing for autoinflammatory diseases using the next-generation sequencing platform MiSeq.

PubMed

Nakayama, Manabu; Oda, Hirotsugu; Nakagawa, Kenji; Yasumi, Takahiro; Kawai, Tomoki; Izawa, Kazushi; Nishikomori, Ryuta; Heike, Toshio; Ohara, Osamu

2017-03-01

Autoinflammatory diseases occupy one of a group of primary immunodeficiency diseases that are generally thought to be caused by mutation of genes responsible for innate immunity, rather than by acquired immunity. Mutations related to autoinflammatory diseases occur in 12 genes. For example, low-level somatic mosaic NLRP3 mutations underlie chronic infantile neurologic, cutaneous, articular syndrome (CINCA), also known as neonatal-onset multisystem inflammatory disease (NOMID). In current clinical practice, clinical genetic testing plays an important role in providing patients with quick, definite diagnoses. To increase the availability of such testing, low-cost high-throughput gene-analysis systems are required, ones that not only have the sensitivity to detect even low-level somatic mosaic mutations, but also can operate simply in a clinical setting. To this end, we developed a simple method that employs two-step tailed PCR and an NGS system, MiSeq platform, to detect mutations in all coding exons of the 12 genes responsible for autoinflammatory diseases. Using this amplicon sequencing system, we amplified a total of 234 amplicons derived from the 12 genes with multiplex PCR. This was done simultaneously and in one test tube. Each sample was distinguished by an index sequence of second PCR primers following PCR amplification. With our procedure and tips for reducing PCR amplification bias, we were able to analyze 12 genes from 25 clinical samples in one MiSeq run. Moreover, with the certified primers designed by our short program-which detects and avoids common SNPs in gene-specific PCR primers-we used this system for routine genetic testing. Our optimized procedure uses a simple protocol, which can easily be followed by virtually any office medical staff. Because of the small PCR amplification bias, we can analyze simultaneously several clinical DNA samples with low cost and can obtain sufficient read numbers to detect a low level of somatic mosaic mutations.

CMV

MedlinePlus

... Mutation Mycophenolic Acid Mycoplasma Myoglobin Nicotine and Cotinine Non-High Density Lipoprotein Cholesterol Opioid Testing Osmolality Ova ... and abdominal pain), lungs (causing pneumonia with a non-productive cough and shortness of breath), and brain ( ...

Alternative Computational Protocols for Supercharging Protein Surfaces for Reversible Unfolding and Retention of Stability

PubMed Central

Der, Bryan S.; Kluwe, Christien; Miklos, Aleksandr E.; Jacak, Ron; Lyskov, Sergey; Gray, Jeffrey J.; Georgiou, George; Ellington, Andrew D.; Kuhlman, Brian

2013-01-01

Reengineering protein surfaces to exhibit high net charge, referred to as “supercharging”, can improve reversibility of unfolding by preventing aggregation of partially unfolded states. Incorporation of charged side chains should be optimized while considering structural and energetic consequences, as numerous mutations and accumulation of like-charges can also destabilize the native state. A previously demonstrated approach deterministically mutates flexible polar residues (amino acids DERKNQ) with the fewest average neighboring atoms per side chain atom (AvNAPSA). Our approach uses Rosetta-based energy calculations to choose the surface mutations. Both protocols are available for use through the ROSIE web server. The automated Rosetta and AvNAPSA approaches for supercharging choose dissimilar mutations, raising an interesting division in surface charging strategy. Rosetta-supercharged variants of GFP (RscG) ranging from −11 to −61 and +7 to +58 were experimentally tested, and for comparison, we re-tested the previously developed AvNAPSA-supercharged variants of GFP (AscG) with +36 and −30 net charge. Mid-charge variants demonstrated ∼3-fold improvement in refolding with retention of stability. However, as we pushed to higher net charges, expression and soluble yield decreased, indicating that net charge or mutational load may be limiting factors. Interestingly, the two different approaches resulted in GFP variants with similar refolding properties. Our results show that there are multiple sets of residues that can be mutated to successfully supercharge a protein, and combining alternative supercharge protocols with experimental testing can be an effective approach for charge-based improvement to refolding. PMID:23741319

A New Microarray Substrate for Ultra-Sensitive Genotyping of KRAS and BRAF Gene Variants in Colorectal Cancer

PubMed Central

Pinzani, Pamela; Mancini, Irene; Vinci, Serena; Chiari, Marcella; Orlando, Claudio; Cremonesi, Laura; Ferrari, Maurizio

2013-01-01

Molecular diagnostics of human cancers may increase accuracy in prognosis, facilitate the selection of the optimal therapeutic regimen, improve patient outcome, reduce costs of treatment and favour development of personalized approaches to patient care. Moreover sensitivity and specificity are fundamental characteristics of any diagnostic method. We developed a highly sensitive microarray for the detection of common KRAS and BRAF oncogenic mutations. In colorectal cancer, KRAS and BRAF mutations have been shown to identify a cluster of patients that does not respond to anti-EGFR therapies; the identification of these mutations is therefore clinically extremely important. To verify the technical characteristics of the microarray system for the correct identification of the KRAS mutational status at the two hotspot codons 12 and 13 and of the BRAFV600E mutation in colorectal tumor, we selected 75 samples previously characterized by conventional and CO-amplification at Lower Denaturation temperature-PCR (COLD-PCR) followed by High Resolution Melting analysis and direct sequencing. Among these samples, 60 were collected during surgery and immediately steeped in RNAlater while the 15 remainders were formalin-fixed and paraffin-embedded (FFPE) tissues. The detection limit of the proposed method was different for the 7 KRAS mutations tested and for the V600E BRAF mutation. In particular, the microarray system has been able to detect a minimum of about 0.01% of mutated alleles in a background of wild-type DNA. A blind validation displayed complete concordance of results. The excellent agreement of the results showed that the new microarray substrate is highly specific in assigning the correct genotype without any enrichment strategy. PMID:23536897

Surveillance of Helicobacter pylori Antibiotic Susceptibility in Indonesia: Different Resistance Types among Regions and with Novel Genetic Mutations

PubMed Central

Miftahussurur, Muhammad; Syam, Ari Fahrial; Nusi, Iswan Abbas; Makmun, Dadang; Waskito, Langgeng Agung; Zein, Lukman Hakim; Akil, Fardah; Uwan, Willy Brodus; Simanjuntak, David; Wibawa, I Dewa Nyoman; Waleleng, Jimmy Bradley; Saudale, Alexander Michael Joseph; Yusuf, Fauzi; Mustika, Syifa; Adi, Pangestu; Maimunah, Ummi; Maulahela, Hasan; Rezkitha, Yudith Annisa Ayu; Subsomwong, Phawinee; Nasronudin; Rahardjo, Dadik; Suzuki, Rumiko; Akada, Junko; Yamaoka, Yoshio

2016-01-01

Information regarding Helicobacter pylori antibiotic resistance in Indonesia was previously inadequate. We assessed antibiotic susceptibility for H. pylori in Indonesia, and determined the association between virulence genes or genetic mutations and antibiotic resistance. We recruited 849 dyspeptic patients who underwent endoscopy in 11 cities in Indonesia. E-test was used to determine the minimum inhibitory concentration of five antibiotics. PCR-based sequencing assessed mutations in 23S rRNA, rdxA, gyrA, gyrB, and virulence genes. Next generation sequencing was used to obtain full-length sequences of 23S rRNA, infB, and rpl22. We cultured 77 strains and identified 9.1% with clarithromycin resistance. Low prevalence was also found for amoxicillin and tetracycline resistance (5.2% and 2.6%, respectively). In contrast, high resistance rates to metronidazole (46.7%) and levofloxacin (31.2%) were demonstrated. Strains isolated from Sumatera Island had significantly higher metronidazole resistance than those from other locations. Metronidazole resistant strains had highly distributed rdxA amino acid substitutions and the 23S rRNA A2143G mutation was associated with clarithromycin resistance (42.9%). However, one strain with the highest MIC value had a novel mutation in rpl22 without an A2143G mutation. Mutation at Asn-87 and/or Asp-91 of gyrA was associated with levofloxacin-resistance and was related to gyrB mutations. In conclusions, although this is a pilot study for a larger survey, our current data show that Indonesian strains had the high prevalence of metronidazole and levofloxacin resistance with low prevalence of clarithromycin, amoxicillin, and tetracycline resistance. Nevertheless, clarithromycin- or metronidazole-based triple therapy should be administered with caution in some regions of Indonesia. PMID:27906990

Surveillance of Helicobacter pylori Antibiotic Susceptibility in Indonesia: Different Resistance Types among Regions and with Novel Genetic Mutations.

PubMed

Miftahussurur, Muhammad; Syam, Ari Fahrial; Nusi, Iswan Abbas; Makmun, Dadang; Waskito, Langgeng Agung; Zein, Lukman Hakim; Akil, Fardah; Uwan, Willy Brodus; Simanjuntak, David; Wibawa, I Dewa Nyoman; Waleleng, Jimmy Bradley; Saudale, Alexander Michael Joseph; Yusuf, Fauzi; Mustika, Syifa; Adi, Pangestu; Maimunah, Ummi; Maulahela, Hasan; Rezkitha, Yudith Annisa Ayu; Subsomwong, Phawinee; Nasronudin; Rahardjo, Dadik; Suzuki, Rumiko; Akada, Junko; Yamaoka, Yoshio

2016-01-01

Information regarding Helicobacter pylori antibiotic resistance in Indonesia was previously inadequate. We assessed antibiotic susceptibility for H. pylori in Indonesia, and determined the association between virulence genes or genetic mutations and antibiotic resistance. We recruited 849 dyspeptic patients who underwent endoscopy in 11 cities in Indonesia. E-test was used to determine the minimum inhibitory concentration of five antibiotics. PCR-based sequencing assessed mutations in 23S rRNA, rdxA, gyrA, gyrB, and virulence genes. Next generation sequencing was used to obtain full-length sequences of 23S rRNA, infB, and rpl22. We cultured 77 strains and identified 9.1% with clarithromycin resistance. Low prevalence was also found for amoxicillin and tetracycline resistance (5.2% and 2.6%, respectively). In contrast, high resistance rates to metronidazole (46.7%) and levofloxacin (31.2%) were demonstrated. Strains isolated from Sumatera Island had significantly higher metronidazole resistance than those from other locations. Metronidazole resistant strains had highly distributed rdxA amino acid substitutions and the 23S rRNA A2143G mutation was associated with clarithromycin resistance (42.9%). However, one strain with the highest MIC value had a novel mutation in rpl22 without an A2143G mutation. Mutation at Asn-87 and/or Asp-91 of gyrA was associated with levofloxacin-resistance and was related to gyrB mutations. In conclusions, although this is a pilot study for a larger survey, our current data show that Indonesian strains had the high prevalence of metronidazole and levofloxacin resistance with low prevalence of clarithromycin, amoxicillin, and tetracycline resistance. Nevertheless, clarithromycin- or metronidazole-based triple therapy should be administered with caution in some regions of Indonesia.

KRAS Mutation Test in Korean Patients with Colorectal Carcinomas: A Methodological Comparison between Sanger Sequencing and a Real-Time PCR-Based Assay.

PubMed

Lee, Sung Hak; Chung, Arthur Minwoo; Lee, Ahwon; Oh, Woo Jin; Choi, Yeong Jin; Lee, Youn-Soo; Jung, Eun Sun

2017-01-01

Mutations in the KRAS gene have been identified in approximately 50% of colorectal cancers (CRCs). KRAS mutations are well established biomarkers in anti-epidermal growth factor receptor therapy. Therefore, assessment of KRAS mutations is needed in CRC patients to ensure appropriate treatment. We compared the analytical performance of the cobas test to Sanger sequencing in 264 CRC cases. In addition, discordant specimens were evaluated by 454 pyrosequencing. KRAS mutations for codons 12/13 were detected in 43.2% of cases (114/264) by Sanger sequencing. Of 257 evaluable specimens for comparison, KRAS mutations were detected in 112 cases (43.6%) by Sanger sequencing and 118 cases (45.9%) by the cobas test. Concordance between the cobas test and Sanger sequencing for each lot was 93.8% positive percent agreement (PPA) and 91.0% negative percent agreement (NPA) for codons 12/13. Results from the cobas test and Sanger sequencing were discordant for 20 cases (7.8%). Twenty discrepant cases were subsequently subjected to 454 pyrosequencing. After comprehensive analysis of the results from combined Sanger sequencing-454 pyrosequencing and the cobas test, PPA was 97.5% and NPA was 100%. The cobas test is an accurate and sensitive test for detecting KRAS -activating mutations and has analytical power equivalent to Sanger sequencing. Prescreening using the cobas test with subsequent application of Sanger sequencing is the best strategy for routine detection of KRAS mutations in CRC.

Impact of 226C>T MSH2 gene mutation on cancer phenotypes in two HNPCC-associated highly-consanguineous families from Kuwait: emphasis on premarital genetic testing.

PubMed

Marafie, Makia J; Al-Awadi, Sadiqa; Al-Mosawi, Fatemah; Elshafey, Alaa; Al-Ali, Waleed; Al-Mulla, Fahd

2009-01-01

Lynch syndrome or hereditary nonpolyposis colorectal cancer (HNPCC) is one of the commonest cancer susceptibility syndromes. It is characterized by early onset colon cancer and a variety of extracolonic tumours. Germline mutations in the DNA mismatch repair genes (MLH1, MSH2, MSH6, PMS1, and PMS2) are responsible for this disorder. Identifying an affected individual depends on the tumour histopathology, family history that fulfils the Amsterdam and/or Bethesda criteria, tumour immunohistochemistry, microsatellite instability, and finally molecular analysis of an affected member. It is a laborious, time consuming and expensive procedure, which needs the effort of a multi-disciplinary team. However, once the diagnosis is established and germline defect is identified, other high risk pre-symptomatic carriers could be offered intensive surveillance and management as a preventive measure against cancer development. Here, we present two large highly consanguineous HNPCC-families from Kuwait in whom a founder MSH2 mutation was identified. The relationship between this mutation and cancer expressivity in two large consanguineous families harbouring other genetic defects is discussed. Moreover, we shed light on the challenges pertaining to diagnosis, screening, premarital counselling of couples and prenatal diagnosis of offspring with biallelic MSH2 gene mutation.

Development of a Molecular-Beacon Assay To Detect the G1896A Precore Mutation in Hepatitis B Virus-Infected Individuals

PubMed Central

Waltz, Therese L.; Marras, Salvatore; Rochford, Gemma; Nolan, John; Lee, Eugenia; Melegari, Margherita; Pollack, Henry

2005-01-01

The 1896 precore (PC) mutation is the most frequent cause of hepatitis B virus e-antigen (HBeAg)-negative chronic hepatitis B virus (HBV) infection. Detection of the 1896 PC mutation has application in studies monitoring antiviral therapy and the natural history of the disease. Identification of this mutation is usually performed by direct sequencing, which is both costly and laborious. The aim of this study was to develop a rapid, high-throughput assay to detect the 1896 PC mutation using real-time PCR and molecular-beacon technology. The assay was initially standardized on oligonucleotide targets and plasmids containing the wild-type (WT) and PC mutation and then tested on plasma samples from children with HBV DNA of >106 copies/ml. Nine individuals were HBeAg negative and suspected to harbor HBeAg mutations, while 12 children were HBeAg positive and selected as controls. Ninety percent (19 of 21) of plasma samples tested with molecular beacons were in complete agreement with sequencing results. The remaining 10% (2 of 21) of samples were identified as heterogeneous mixtures of WT and mutant virus by molecular beacons, though sequencing found only a homogeneous mutant in both cases. Overall, the 1896 PC mutation was detected by this assay in 55.5% of the children with HBeAg-negative infection. In summary, this assay is a rapid, sensitive, and specific technique that effectively discriminates WT from 1896 PC mutant HBV and may be useful in clinical and epidemiological studies. PMID:15634980

A prognostic mutation panel for predicting cancer recurrence in stages II and III colorectal cancer.

PubMed

Sho, Shonan; Court, Colin M; Winograd, Paul; Russell, Marcia M; Tomlinson, James S

2017-12-01

Approximately 20-40% of stage II/III colorectal cancer (CRC) patients develop relapse. Clinicopathological factors alone are limited in detecting these patients, resulting in potential under/over-treatment. We sought to identify a prognostic tumor mutational profile that could predict CRC recurrence. Whole-exome sequencing data were obtained for 207 patients with stage II/III CRC from The Cancer Genome Atlas. Mutational landscape in relapse-free versus relapsed cohort was compared using Fisher's exact test, followed by multivariate Cox regression to identify genes associated with cancer recurrence. Bootstrap-validation was used to examine internal/external validity. We identified five prognostic genes (APAF1, DIAPH2, NTNG1, USP7, and VAV2), which were combined to form a prognostic mutation panel. Patients with ≥1 mutation(s) within this five-gene panel had worse prognosis (3-yr relapse-free survival [RFS]: 53.0%), compared to patients with no mutation (3-yr RFS: 84.3%). In multivariate analysis, the five-gene panel remained prognostic for cancer recurrence independent of stage and high-risk features (hazard ratio 3.63, 95%CI [1.93-6.83], P < 0.0001). Furthermore, its prognostic accuracy was superior to the American Joint Commission on Cancer classification (concordance-index: 0.70 vs 0.54). Our proposed mutation panel identifies CRC patients at high-risk for recurrence, which may help guide adjuvant therapy and post-operative surveillance protocols. © 2017 Wiley Periodicals, Inc.

Metastatic site location influences the diagnostic accuracy of ctDNA EGFR- mutation testing in NSCLC patients: a pooled analysis.

PubMed

Passiglia, Francesco; Rizzo, Sergio; Rolfo, Christian; Galvano, Antonio; Bronte, Enrico; Incorvaia, Lorena; Listi, Angela; Barraco, Nadia; Castiglia, Marta; Calo, Valentina; Bazan, Viviana; Russo, Antonio

2018-03-08

Recent studies evaluated the diagnostic accuracy of circulating tumor DNA (ctDNA) in the detection of epidermal growth factor receptor (EGFR) mutations from plasma of NSCLC patients, overall showing a high concordance as compared to standard tissue genotyping. However it is less clear if the location of metastatic site may influence the ability to identify EGFR mutations in plasma. This pooled analysis aims to evaluate the association between the metastatic site location and the sensitivity of ctDNA analysis in detecting EGFR mutations in NSCLC patients. Data from all published studies, evaluating the sensitivity of plasma-based EGFR-mutation testing, stratified by metastatic site location (extrathoracic (M1b) vs intrathoracic (M1a)) were collected by searching in PubMed, Cochrane Library, American Society of Clinical Oncology, and World Conference of Lung Cancer, meeting proceedings. Pooled Odds ratio (OR) and 95% confidence intervals (95% CIs) were calculated for the ctDNA analysis sensitivity, according to metastatic site location. A total of ten studies, with 1425 patients, were eligible. Pooled analysis showed that the sensitivity of ctDNA-based EGFR-mutation testing is significantly higher in patients with M1b vs M1a disease (OR: 5.09; 95% CIs: 2.93 - 8.84). A significant association was observed for both EGFR-activating (OR: 4.30, 95% CI: 2.35-7.88) and resistant T790M mutations (OR: 11.89, 95% CI: 1.45-97.22), regardless of the use of digital-PCR (OR: 5.85, 95% CI: 3.56-9.60) or non-digital PCR technologies (OR: 2.96, 95% CI: 2.24-3.91). These data suggest that the location of metastatic sites significantly influences the diagnostic accuracy of ctDNA analysis in detecting EGFR mutations in NSCLC patients. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Haplotype diversity of the myostatin gene among beef cattle breeds

PubMed Central

Dunner, Susana; Miranda, M Eugenia; Amigues, Yves; Cañón, Javier; Georges, Michel; Hanset, Roger; Williams, John; Ménissier, François

2003-01-01

A total of 678 individuals from 28 European bovine breeds were both phenotyped and analysed at the myostatin locus by the Single Strand Conformation Polymorphism (SSCP) method. Seven new mutations were identified which contribute to the high polymorphism (1 SNP every 100 bp) present in this small gene; twenty haplotypes were described and a genotyping method was set up using the Oligonucleotide Ligation Assay (OLA) method. Some haplotypes appeared to be exclusive to a particular breed; this was the case for 5 in the Charolaise (involving mutation Q204X) and 7 in the Maine-Anjou (involving mutation E226X). The relationships between the different haplotypes were studied, thus allowing to test the earlier hypothesis on the origin of muscular hypertrophy in Europe: muscular hypertrophy (namely nt821(del11)) was mainly spread in different waves from northern Europe milk purpose populations in most breeds; however, other mutations (mostly disruptive) arose in a single breed, were highly selected and have since scarcely evolved to other populations. PMID:12605853

Analysis of mutational spectra by denaturant capillary electrophoresis

PubMed Central

Ekstrøm, Per O.; Khrapko, Konstantin; Li-Sucholeiki, Xiao-Cheng; Hunter, Ian W.; Thilly, William G.

2009-01-01

Numbers and kinds of point mutant within DNA from cells, tissues and human population may be discovered for nearly any 75–250bp DNA sequence. High fidelity DNA amplification incorporating a thermally stable DNA “clamp” is followed by separation by denaturing capillary electrophoresis (DCE). DCE allows for peak collection and verification sequencing. DCE in a mode of cycling temperature, e.g.+/− 5°C, CyDCE, permits high resolution of mutant sequences using computer defined analytes without preliminary optimization experiments. DNA sequencers have been modified to permit higher throughput CyDCE and a massively parallel,~25,000 capillary system, has been designed for pangenomic scans in large human populations. DCE has been used to define quantitative point mutational spectra for study a wide variety of genetic phenomena: errors of DNA polymerases, mutations induced in human cells by chemicals and irradiation, testing of human gene-common disease associations and the discovery of origins of point mutations in human development and carcinogenesis. PMID:18600220

Autosomal dominant polycystic kidney disease caused by somatic and germline mosaicism.

PubMed

Tan, A Y; Blumenfeld, J; Michaeel, A; Donahue, S; Bobb, W; Parker, T; Levine, D; Rennert, H

2015-04-01

Autosomal dominant polycystic kidney disease (ADPKD) is a heterogeneous genetic disorder caused by loss of function mutations of PKD1 or PKD2 genes. Although PKD1 is highly polymorphic and the new mutation rate is relatively high, the role of mosaicism is incompletely defined. Herein, we describe the molecular analysis of ADPKD in a 19-year-old female proband and her father. The proband had a PKD1 truncation mutation c.10745dupC (p.Val3584ArgfsX43), which was absent in paternal peripheral blood lymphocytes (PBL). However, very low quantities of this mutation were detected in the father's sperm DNA, but not in DNA from his buccal cells or urine sediment. Next generation sequencing (NGS) analysis determined the level of this mutation in the father's PBL, buccal cells and sperm to be ∼3%, 4.5% and 10%, respectively, consistent with somatic and germline mosaicism. The PKD1 mutation in ∼10% of her father's sperm indicates that it probably occurred early in embryogenesis. In ADPKD cases where a de novo mutation is suspected because of negative PKD gene testing of PBL, additional evaluation with more sensitive methods (e.g. NGS) of the proband PBL and paternal sperm can enhance detection of mosaicism and facilitate genetic counseling. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Development of a human live attenuated West Nile infectious DNA vaccine: Suitability of attenuating mutations found in SA14-14-2 for WN vaccine design

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamshchikov, Vladimir, E-mail: yaximik@gmail.com; Manuvakhova, Marina; Rodriguez, Efrain

Direct attenuation of West Nile (WN) virus strain NY99 for the purpose of vaccine development is not feasible due to its high virulence and pathogenicity. Instead, we created highly attenuated chimeric virus W1806 with the serological identity of NY99. To further attenuate W1806, we investigated effects of mutations found in Japanese encephalitis virus vaccine SA14-14-2. WN viruses carrying all attenuating mutations lost infectivity in mammalian, but not in mosquito cells. No single reversion restored infectivity in mammalian cells, although increased infectivity in mosquito cells was observed. To identify a subset of mutations suitable for further attenuation of W1806, we analyzedmore » effects of E{sub 138}K and K{sub 279}M changes on virulence, growth properties, and immunogenicity of derivatized W956, from which chimeric W1806 inherited its biological properties and attenuation profile. Despite strong dominant attenuating effect, introduction of only two mutations was not sufficient for attenuating W1806 to the safety level acceptable for human use. - Highlights: • Further attenuation of a WN vaccine precursor is outlined. • Effect of SA14-14-2 attenuating mutations is tested. • Mechanism of attenuation is proposed and illustrated. • The need for additional attenuating mutations is justified.« less

Low load for disruptive mutations in autism genes and their biased transmission

PubMed Central

Iossifov, Ivan; Levy, Dan; Allen, Jeremy; Ye, Kenny; Ronemus, Michael; Lee, Yoon-ha; Yamrom, Boris; Wigler, Michael

2015-01-01

We previously computed that genes with de novo (DN) likely gene-disruptive (LGD) mutations in children with autism spectrum disorders (ASD) have high vulnerability: disruptive mutations in many of these genes, the vulnerable autism genes, will have a high likelihood of resulting in ASD. Because individuals with ASD have lower fecundity, such mutations in autism genes would be under strong negative selection pressure. An immediate prediction is that these genes will have a lower LGD load than typical genes in the human gene pool. We confirm this hypothesis in an explicit test by measuring the load of disruptive mutations in whole-exome sequence databases from two cohorts. We use information about mutational load to show that lower and higher intelligence quotients (IQ) affected individuals can be distinguished by the mutational load in their respective gene targets, as well as to help prioritize gene targets by their likelihood of being autism genes. Moreover, we demonstrate that transmission of rare disruptions in genes with a lower LGD load occurs more often to affected offspring; we show transmission originates most often from the mother, and transmission of such variants is seen more often in offspring with lower IQ. A surprising proportion of transmission of these rare events comes from genes expressed in the embryonic brain that show sharply reduced expression shortly after birth. PMID:26401017

Next-generation sequencing in familial breast cancer patients from Lebanon.

PubMed

Jalkh, Nadine; Chouery, Eliane; Haidar, Zahraa; Khater, Christina; Atallah, David; Ali, Hamad; Marafie, Makia J; Al-Mulla, Mohamed R; Al-Mulla, Fahd; Megarbane, Andre

2017-02-15

Familial breast cancer (BC) represents 5 to 10% of all BC cases. Mutations in two high susceptibility BRCA1 and BRCA2 genes explain 16-40% of familial BC, while other high, moderate and low susceptibility genes explain up to 20% more of BC families. The Lebanese reported prevalence of BRCA1 and BRCA2 deleterious mutations (5.6% and 12.5%) were lower than those reported in the literature. In the presented study, 45 Lebanese patients with a reported family history of BC were tested using Whole Exome Sequencing (WES) technique followed by Sanger sequencing validation. Nineteen pathogenic mutations were identified in this study. These 19 mutations were found in 13 different genes such as: ABCC12, APC, ATM, BRCA1, BRCA2, CDH1, ERCC6, MSH2, POLH, PRF1, SLX4, STK11 and TP53. In this first application of WES on BC in Lebanon, we detected six BRCA1 and BRCA2 deleterious mutations in seven patients, with a total prevalence of 15.5%, a figure that is lower than those reported in the Western literature. The p.C44F mutation in the BRCA1 gene appeared twice in this study, suggesting a founder effect. Importantly, the overall mutation prevalence was equal to 40%, justifying the urgent need to deploy WES for the identification of genetic variants responsible for familial BC in the Lebanese population.

Novel mutations in the SOX10 gene in the first two Chinese cases of type IV Waardenburg syndrome.

PubMed

Jiang, Lu; Chen, Hongsheng; Jiang, Wen; Hu, Zhengmao; Mei, Lingyun; Xue, Jingjie; He, Chufeng; Liu, Yalan; Xia, Kun; Feng, Yong

2011-05-20

We analyzed the clinical features and family-related gene mutations for the first two Chinese cases of type IV Waardenburg syndrome (WS4). Two families were analyzed in this study. The analysis included a medical history, clinical analysis, a hearing test and a physical examination. In addition, the EDNRB, EDN3 and SOX10 genes were sequenced in order to identify the pathogenic mutation responsible for the WS4 observed in these patients. The two WS4 cases presented with high phenotypic variability. Two novel heterozygous mutations (c.254G>A and c.698-2A>T) in the SOX10 gene were detected. The mutations identified in the patients were not found in unaffected family members or in 200 unrelated control subjects. This is the first report of WS4 in Chinese patients. In addition, two novel mutations in SOX10 gene have been identified. Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.

DNA mutations of the cat: the good, the bad and the ugly.

PubMed

Lyons, Leslie A

2015-03-01

The health of the cat is a complex interaction between its environment (nurture) and its genetics (nature). Over 70 genetic mutations (variants) have been defined in the cat, many involving diseases, structural abnormalities and clinically relevant health concerns. As more of the cat's genome is deciphered, less commonly will the term 'idiopathic' be used regarding the diagnosis of diseases and unique health conditions. State-of-the-art health care will include DNA profiling of the individual cat, and perhaps its tumor, to establish the best treatment approaches. Genetic testing and eventually whole genome sequencing should become routine diagnostics for feline health care. Cat breeds have disseminated around the world. Thus, practitioners should be aware of the breeds common to their region and the mutations found in those regional populations. Specific random-bred populations can also have defined genetic characteristics and mutations. This review of 'the good, the bad and the ugly' DNA variants provides the current state of knowledge for genetic testing and genetic health management for cats. It is aimed at feline and general practitioners wanting to update and review the basics of genetics, what tests are available for cats and sources for genetic testing. The tables are intended to be used as references in the clinic. Practitioners with a high proportion of cat breeder clientele will especially benefit from the review. The data presented is extracted from peer-reviewed publications pertaining to mutation identification, and relevant articles concerning the heritable trait and/or disease. The author also draws upon personal experience and expertise in feline genetics. © ISFM and AAFP 2015.

Interest of proviral HIV-1 DNA genotypic resistance testing in virologically suppressed patients candidate for maintenance therapy.

PubMed

Allavena, C; Rodallec, A; Leplat, A; Hall, N; Luco, C; Le Guen, L; Bernaud, C; Bouchez, S; André-Garnier, E; Boutoille, D; Ferré, V; Raffi, F

2018-01-01

Switch of antiretroviral therapy in virologically suppressed HIV-infected patients is frequent, to prevent toxicities, for simplification or convenience reasons. Pretherapeutic genotypic resistance testing on RNA can be lacking in some patients, which could enhance the risk of virologic failure, if resistance-associated mutations of the new regimen are not taken into account. Proviral DNA resistance testing in 69 virologically suppressed patients on antiretroviral treatment with no history of virological failure were pair-wised compared with pre-ART plasma RNA resistance testing. The median time between plasma (RNA testing) and whole blood (proviral DNA testing) was 47 months (IQR 29-63). A stop codon was evidenced in 23% (16/69) of proviral DNA sequences; these strains were considered as defective, non-replicative, and not taken into consideration. Within the non defective strains, concordance rate between plasma RNA and non-defective proviral DNA was high both on protease (194/220 concordant resistance-associated mutations=88%) and reverse transcriptase (28/37 concordant resistance-associated mutations=76%) genes. This study supports that proviral DNA testing might be an informative tool before switching antiretrovirals in virologically suppressed patients with no history of virological failure, but the interpretation should be restricted to non-defective viruses. Copyright © 2017 Elsevier B.V. All rights reserved.

Increased T-cell receptor mutation frequency in radiation-exposed residents living near the Semipalatinsk nuclear test site.

PubMed

Taooka, Yasuyuki; Takeichi, Nobuo; Noso, Yoshihiro; Kawano, Noriyuki; Apsalikov, Kazbek N; Hoshi, Masaharu

2006-02-01

From 1949 to 1989, 488 nuclear explosions were carried out in Semipalatinsk, and the cancer risk is increased in this region. Measuring somatic-cell mutation frequencies may be a useful tool for evaluating cancer risk within radiation-exposed populations. Here, we report the first evidence of increased T-cell receptor (TCR) mutations in peripheral blood from radiation-exposed residents of Semipalatinsk. The TCR mutation frequency in the highly exposed residents (Dolon and Sarzhal) was significantly higher than in the control group (Kokpekti). There was no statistically significant difference between the control group and the weakly exposed group (Kaynar and Semipalatinsk-city). The TCR mutation assay appeared to be a useful biological dosimeter even after a period of 40 years since radiation exposure. This may be the result of specific conditions, such as the presence of internal exposure.

Mutation Analysis of SLC26A4 for Pendred Syndrome and Nonsyndromic Hearing Loss by High-Resolution Melting

PubMed Central

Chen, Neng; Tranebjærg, Lisbeth; Rendtorff, Nanna Dahl; Schrijver, Iris

2011-01-01

Pendred syndrome and DFNB4 (autosomal recessive nonsyndromic congenital deafness, locus 4) are associated with autosomal recessive congenital sensorineural hearing loss and mutations in the SLC26A4 gene. Extensive allelic heterogeneity, however, necessitates analysis of all exons and splice sites to identify mutations for individual patients. Although Sanger sequencing is the gold standard for mutation detection, screening methods supplemented with targeted sequencing can provide a cost-effective alternative. One such method, denaturing high-performance liquid chromatography, was developed for clinical mutation detection in SLC26A4. However, this method inherently cannot distinguish homozygous changes from wild-type sequences. High-resolution melting (HRM), on the other hand, can detect heterozygous and homozygous changes cost-effectively, without any post-PCR modifications. We developed a closed-tube HRM mutation detection method specific for SLC26A4 that can be used in the clinical diagnostic setting. Twenty-eight primer pairs were designed to cover all 21 SLC26A4 exons and splice junction sequences. Using the resulting amplicons, initial HRM analysis detected all 45 variants previously identified by sequencing. Subsequently, a 384-well plate format was designed for up to three patient samples per run. Blinded HRM testing on these plates of patient samples collected over 1 year in a clinical diagnostic laboratory accurately detected all variants identified by sequencing. In conclusion, HRM with targeted sequencing is a reliable, simple, and cost-effective method for SLC26A4 mutation screening and detection. PMID:21704276

Dissecting protein function: an efficient protocol for identifying separation-of-function mutations that encode structurally stable proteins.

PubMed

Lubin, Johnathan W; Rao, Timsi; Mandell, Edward K; Wuttke, Deborah S; Lundblad, Victoria

2013-03-01

Mutations that confer the loss of a single biochemical property (separation-of-function mutations) can often uncover a previously unknown role for a protein in a particular biological process. However, most mutations are identified based on loss-of-function phenotypes, which cannot differentiate between separation-of-function alleles vs. mutations that encode unstable/unfolded proteins. An alternative approach is to use overexpression dominant-negative (ODN) phenotypes to identify mutant proteins that disrupt function in an otherwise wild-type strain when overexpressed. This is based on the assumption that such mutant proteins retain an overall structure that is comparable to that of the wild-type protein and are able to compete with the endogenous protein (Herskowitz 1987). To test this, the in vivo phenotypes of mutations in the Est3 telomerase subunit from Saccharomyces cerevisiae were compared with the in vitro secondary structure of these mutant proteins as analyzed by circular-dichroism spectroscopy, which demonstrates that ODN is a more sensitive assessment of protein stability than the commonly used method of monitoring protein levels from extracts. Reverse mutagenesis of EST3, which targeted different categories of amino acids, also showed that mutating highly conserved charged residues to the oppositely charged amino acid had an increased likelihood of generating a severely defective est3(-) mutation, which nevertheless encoded a structurally stable protein. These results suggest that charge-swap mutagenesis directed at a limited subset of highly conserved charged residues, combined with ODN screening to eliminate partially unfolded proteins, may provide a widely applicable and efficient strategy for generating separation-of-function mutations.

Small-scale high-throughput sequencing-based identification of new therapeutic tools in cystic fibrosis.

PubMed

Bonini, Jennifer; Varilh, Jessica; Raynal, Caroline; Thèze, Corinne; Beyne, Emmanuelle; Audrezet, Marie-Pierre; Ferec, Claude; Bienvenu, Thierry; Girodon, Emmanuelle; Tuffery-Giraud, Sylvie; Des Georges, Marie; Claustres, Mireille; Taulan-Cadars, Magali

2015-10-01

Although 97-99% of CFTR mutations have been identified, great efforts must be made to detect yet-unidentified mutations. We developed a small-scale next-generation sequencing approach for reliably and quickly scanning the entire gene, including noncoding regions, to identify new mutations. We applied this approach to 18 samples from patients suffering from cystic fibrosis (CF) in whom only one mutation had hitherto been identified. Using an in-house bioinformatics pipeline, we could rapidly identify a second disease-causing CFTR mutation for 16 of 18 samples. Of them, c.1680-883A>G was found in three unrelated CF patients. Analysis of minigenes and patients' transcripts showed that this mutation results in aberrantly spliced transcripts because of the inclusion of a pseudoexon. It is located only three base pairs from the c.1680-886A>G mutation (1811+1.6kbA>G), the fourth most frequent mutation in southwestern Europe. We next tested the effect of antisense oligonucleotides targeting splice sites on these two mutations on pseudoexon skipping. Oligonucleotide transfection resulted in the restoration of the full-length, in-frame CFTR transcript, demonstrating the effect of antisense oligonucleotide-induced pseudoexon skipping in CF. Our data confirm the importance of analyzing noncoding regions to find unidentified mutations, which is essential to designing targeted therapeutic approaches.

Factors predicting the occurrence of germline mutations in candidate genes among patients with cutaneous malignant melanoma from South Italy.

PubMed

Casula, Milena; Colombino, Maria; Satta, Maria P; Cossu, Antonio; Lissia, Amelia; Budroni, Mario; Simeone, Ester; Calemma, Rosa; Loddo, Cinzia; Caracò, Corrado; Mozzillo, Nicola; Daponte, Antonio; Comella, Giuseppe; Canzanella, Sergio; Guida, Michele; Castello, Giuseppe; Ascierto, Paolo A; Palmieri, Giuseppe

2007-01-01

Clinical predictors for germline mutations of candidate genes in large clinic based population of patients with cutaneous malignant melanoma (CMM) are widely awaited. Using denaturing high-performance liquid chromatography (DHPLC) analysis and DNA sequencing, 557 consecutively-collected CMM patients originating from South Italy were screened for CDKN2A germline mutations; subsets of them were screened for mutations in the BRAF and BRCA2 genes. Seven CDKN2A mutations were detected in 14 (2.5%) CMM patients. Relative risk of carrying a CDKN2A mutation for CMM patients was demonstrated to significantly increase with the presence of familial recurrence of melanoma (risk ratio (RR)=6.31; p=0.0009), multiple primary melanomas (RR=3.43; p=0.0014), and early onset age (RR=4.56; p=0.0026). All CDKN2A mutations were observed in non-Sardinian patients (14/441; 3.2%), whereas BRAF and BRCA2 genes were found mutated in Sardinian patients (3/116; 2.6%). Such indicators of the presence of CDKN2A mutations will be useful in counselling patients about undergoing genetic testing. Our findings strongly suggest that mutation rates of candidate cancer genes may deeply vary among CMM patients from different geographical areas.

Clinical evaluation and mutational analysis of GALK and GALE genes in patients with galactosemia in Greece: one novel mutation and two rare cases.

PubMed

Schulpis, Kleopatra H; Thodi, Georgia; Iakovou, Konstantinos; Chatzidaki, Maria; Dotsikas, Yannis; Molou, Elina; Triantafylli, Olga; Loukas, Yannis L

2017-07-26

Deficiencies of galactokinase (GALK) and UDP-epimerase (GALE) are implicated with galactose metabolic disorders. The aim of the study was the identification of mutations in GALK and GALE genes and clinical evaluation of patients. Five patients with GALK and five with GALE deficiency were picked up via the Neonatal Screening Program. Additionally, two females, 4 years old, were referred with late diagnosed galactosemia, as rare cases. Mutational analysis was conducted via Sanger sequencing, while in silico analysis tools were utilized for the novel mutation. Psychomotor and speech development tests were performed, as well. The mutation p.Pro28Thr was identified in both alleles in GALK-deficient patients of Roma (gypsy) origin, whereas the novel p.Asn39Ser was detected in two non-Roma patients. In GALE-deficient patients benign and/or likely benign mutations were found. Psychomotor and speech delay were determined in the Roma GALK patients. In each of the late diagnosed females, four mutations were identified in all galactosemia-related genes. The mutational spectrums of GALE- and GALK-deficient patients in Greece are presented for the first time along with a clinical evaluation. Mutational analysis in all galactosemia-related genes of symptomatic patients is highly recommended for future cases.

Changes in screening behaviors and attitudes toward screening from pre-test genetic counseling to post-disclosure in Lynch syndrome families.

PubMed

Burton-Chase, A M; Hovick, S R; Peterson, S K; Marani, S K; Vernon, S W; Amos, C I; Frazier, M L; Lynch, P M; Gritz, E R

2013-03-01

The purpose of this study was to examine colonoscopy adherence and attitudes toward colorectal cancer (CRC) screening in individuals who underwent Lynch syndrome genetic counseling and testing. We evaluated changes in colonoscopy adherence and CRC screening attitudes in 78 cancer-unaffected relatives of Lynch syndrome mutation carriers before pre-test genetic counseling (baseline) and at 6 and 12 months post-disclosure of test results (52 mutation negative and 26 mutation positive). While both groups were similar at baseline, at 12 months post-disclosure, a greater number of mutation-positive individuals had had a colonoscopy compared with mutation-negative individuals. From baseline to 12 months post-disclosure, the mutation-positive group demonstrated an increase in mean scores on measures of colonoscopy commitment, self-efficacy, and perceived benefits of CRC screening, and a decrease in mean scores for perceived barriers to CRC screening. Mean scores on colonoscopy commitment decreased from baseline to 6 months in the mutation-negative group. To conclude, adherence to risk-appropriate guidelines for CRC surveillance improved after genetic counseling and testing for Lynch syndrome. Mutation-positive individuals reported increasingly positive attitudes toward CRC screening after receiving genetic test results, potentially reinforcing longer term colonoscopy adherence. © 2013 John Wiley & Sons A/S.

Online self-test identifies women at high familial breast cancer risk in population-based breast cancer screening without inducing anxiety or distress.

PubMed

van Erkelens, A; Sie, A S; Manders, P; Visser, A; Duijm, L E; Mann, R M; Ten Voorde, M; Kroeze, H; Prins, J B; Hoogerbrugge, N

2017-06-01

Identifying high familial breast cancer (FBC) risk improves detection of yet unknown BRCA1/2-mutation carriers, for whom BC risk is both highly likely and potentially preventable. We assessed whether a new online self-test could identify women at high FBC risk in population-based BC screening without inducing anxiety or distress. After their visit for screening mammography, women were invited by email to take an online self-test for identifying highly increased FBC risk-based on Dutch guidelines. Exclusion criteria were previously diagnosed as increased FBC risk or a personal history of BC. Anxiety (State-Trait Anxiety Inventory Dutch Version), distress (Hospital Anxiety Depression Scale) and BC risk perception were assessed using questionnaires, which were completed immediately before and after taking the online self-test and 2 weeks later. Of the 562 women invited by email, 406 (72%) completed the online self-test while 304 also completed questionnaires (response rate 54%). After exclusion criteria, 287 (51%) were included for data analysis. Median age was 56 years (range 50-74). A high or moderate FBC risk was identified in 12 (4%) and three (1%) women, respectively. After completion of the online self-test, anxiety and BC risk perception were decreased while distress scores remained unchanged. Levels were below clinical relevance. Most women (85%) would recommend the self-test; few (3%) would not. The online self-test identified previously unknown women at high FBC risk (4%), who may carry a BRCA1/2-mutation, without inducing anxiety or distress. We therefore recommend offering this self-test to women who attend population-based screening mammography for the first time. Copyright © 2017 Elsevier Ltd. All rights reserved.

Relationship between p53 dysfunction, CD38 expression, and IgV(H) mutation in chronic lymphocytic leukemia.

PubMed

Lin, Ke; Sherrington, Paul D; Dennis, Michael; Matrai, Zoltan; Cawley, John C; Pettitt, Andrew R

2002-08-15

Established adverse prognostic factors in chronic lymphocytic leukemia (CLL) include CD38 expression, relative lack of IgV(H) mutation, and defects of the TP53 gene. However, disruption of the p53 pathway can occur through mechanisms other than TP53 mutation, and we have recently developed a simple screening test that detects p53 dysfunction due to mutation of the genes encoding either p53 or ATM, a kinase that regulates p53. The present study was conducted to examine the predictive value of this test and to establish the relationship between p53 dysfunction, CD38 expression, and IgV(H) mutation. CLL cells from 71 patients were examined for IgV(H) mutation, CD38 expression, and p53 dysfunction (detected as an impaired p53/p21 response to ionizing radiation). Survival data obtained from 69 patients were analyzed according to each of these parameters. Relative lack of IgV(H) mutation (less than 5%; n = 45), CD38 positivity (antigen expressed on more than 20% of malignant cells; n = 19), and p53 dysfunction (n = 19) were independently confirmed as adverse prognostic factors. Intriguingly, all p53-dysfunctional patients and all but one of the CD38(+) patients had less [corrected] than 5% IgV(H) mutation. Moreover, patients with p53 dysfunction and/or CD38 positivity (n = 31) accounted for the short survival of the less mutated group. These findings indicate that the poor outcome associated with having less than 5% IgV(H) mutation may be due to the overrepresentation of high-risk patients with p53 dysfunction and/or CD38 positivity within this group, and that CD38(-) patients with functionally intact p53 may have a prolonged survival regardless of the extent of IgV(H) mutation.

Immuno-Oncology Biomarkers for Gastric and Gastroesophageal Junction Adenocarcinoma: Why PD-L1 Testing May Not Be Enough.

PubMed

Weinberg, Benjamin A; Xiu, Joanne; Hwang, Jimmy J; Shields, Anthony F; Salem, Mohamed E; Marshall, John L

2018-04-27

The treatment of patients with advanced gastric and gastroesophageal junction (G/GEJ) adenocarcinomas has been transformed by the U.S. Food and Drug Administration approval of pembrolizumab. Tumor and adjacent tissue must stain positively for the programmed cell death ligand 1 (PD-L1) protein by companion diagnostic testing. However, some patients with PD-L1-negative tumors also benefit from pembrolizumab. High microsatellite instability (MSI) and tumor mutational load (TML) are positive predictive biomarkers for immune checkpoint inhibition (ICI) in other tumors. We sought to identify more patients who could benefit from ICI using alternative PD-L1 thresholds, MSI, and TML. Tumor specimens underwent next-generation sequencing (NGS) and PD-L1 testing using immunohistochemistry. NGS was used to determine TML and MSI. We profiled 581 G/GEJ adenocarcinoma specimens. PD-L1 staining was scored for intensity (0, none; 1+, weak; 2+, moderate; 3+, strong). Using 2+ staining at a 5% threshold, 9.3% of tumors were PD-L1 positive, and using 1+ staining at 1%, 16.2% were PD-L1 positive. 6.9% of tumors had high MSI. High TML (≥17 mutations per megabase) was seen in 6.9%, and medium TML (≥7) was seen in 56.5% of tumors. Thirty (5.2%) PD-L1-negative tumors at the 1+, 1% threshold had high TML or high MSI. Primary tumors had higher rates of high TML (8.8% vs. 3.9%; p  = .0377) and high MSI (8.5% vs. 3.9%; p  = .0471) than metastases. PD-L1 testing alone fails to detect patients who may benefit from ICI. Lower PD-L1 thresholds and TML testing should be considered in future clinical trials. Pembrolizumab is approved by the U.S. Food and Drug Administration for patients with refractory gastric and gastroesophageal cancers if the tumor and adjacent tissue stain positively for the programmed cell death ligand 1 (PD-L1) protein by companion diagnostic testing. Tumor mutational load, microsatellite instability (MSI), and alternative PD-L1 testing thresholds may serve as predictive biomarkers for response to immune checkpoint inhibition, and standard PD-L1 testing will not identify all patients who may benefit from this therapy. © AlphaMed Press 2018.

A mutation in the 14 alpha-demethylase gene of Uncinula necator that correlates with resistance to a sterol biosynthesis inhibitor.

PubMed Central

Délye, C; Laigret, F; Corio-Costet, M F

1997-01-01

We investigated the molecular basis of resistance of the obligate biotrophic grape powdery mildew fungus Uncinula necator to sterol demethylation-inhibiting fungicides (DMIs). The sensitivity of 91 single-spore field isolates of U. necator to triadimenol was assessed by using a leaf disc assay. Resistance factors (RF) ranged from 1.8 to 26.0. The gene encoding the target of DMIs (eburicol 14 alpha-demethylase) from five sensitive and seven resistant isolates was cloned and sequenced. A single mutation, leading to the substitution of a phenylalanine residue for a tyrosine residue at position 136, was found in all isolates exhibiting an RF higher than 5. No mutation was found in sensitive or weakly resistant (RF, < 5) isolates. An allele-specific PCR assay was developed to detect the mutation. Among the 91 isolates tested, only isolates with RF higher than 5 carried the mutation. Three of the 19 resistant isolates and all sensitive and weakly resistant isolates did not possess the mutation. The mutation at codon 136 is thus clearly associated with high levels of resistance to triadimenol. PMID:9251183

Erythrocytic Pyruvate Kinase Mutations Causing Hemolytic Anemia, Osteosclerosis, and Secondary Hemochromatosis in Dogs

PubMed Central

Gultekin, G. Inal; Raj, K.; Foureman, P.; Lehman, S.; Manhart, K.; Abdulmalik, O.; Giger, U.

2013-01-01

Background Erythrocytic pyruvate kinase (PK) deficiency, first documented in Basenjis, is the most common inherited erythroenzymopathy in dogs. Objectives To report 3 new breed-specific PK-LR gene mutations and a retrospective survey of PK mutations in a small and selected group of Beagles and West Highland White Terriers (WHWT). Animals Labrador Retrievers (2 siblings, 5 unrelated), Pugs (2 siblings, 1 unrelated), Beagles (39 anemic, 29 other), WHWTs (22 anemic, 226 nonanemic), Cairn Terrier (n = 1). Methods Exons of the PK-LR gene were sequenced from genomic DNA of young dogs (<2 years) with persistent highly regenerative hemolytic anemia. Results A nonsense mutation (c.799C>T) resulting in a premature stop codon was identified in anemic Labrador Retriever siblings that had osteosclerosis, high serum ferritin concentrations, and severe hepatic secondary hemochromatosis. Anemic Pug and Beagle revealed 2 different missense mutations (c.848T>C, c.994G>A, respectively) resulting in intolerable amino acid changes to protein structure and enzyme function. Breed-specific mutation tests were developed. Among the biased group of 248 WHWTs, 9% and 35% were homozygous (affected) and heterozygous, respectively, for the previously described mutation (mutant allele frequency 0.26). A PK-deficient Cairn Terrier had the same insertion mutation as the affected WHWTs. Of the selected group of 68 Beagles, 35% were PK-deficient and 3% were carriers (0.37). Conclusions and Clinical Importance Erythrocytic PK deficiency is caused by different mutations in different dog breeds and causes chronic severe hemolytic anemia, hemosiderosis, and secondary hemochromatosis because of chronic hemolysis and, an as yet unexplained osteosclerosis. The newly developed breed-specific mutation assays simplify the diagnosis of PK deficiency. PMID:22805166

Screening for germline BRCA1, BRCA2, TP53 and CHEK2 mutations in families at-risk for hereditary breast cancer identified in a population-based study from Southern Brazil.

PubMed

Palmero, Edenir Inêz; Alemar, Bárbara; Schüler-Faccini, Lavínia; Hainaut, Pierre; Moreira-Filho, Carlos Alberto; Ewald, Ingrid Petroni; Santos, Patricia Koehler Dos; Ribeiro, Patricia Lisbôa Izetti; Oliveira, Cristina Brinkmann de Netto; Calvez-Kelm, Florence Le; Tavtigian, Sean; Cossio, Silvia Liliana; Giugliani, Roberto; Caleffi, Maira; Ashton-Prolla, Patricia

2016-05-24

In Brazil, breast cancer is a public health care problem due to its high incidence and mortality rates. In this study, we investigated the prevalence of hereditary breast cancer syndromes (HBCS) in a population-based cohort in Brazils southernmost capital, Porto Alegre. All participants answered a questionnaire about family history (FH) of breast, ovarian and colorectal cancer and those with a positive FH were invited for genetic cancer risk assessment (GCRA). If pedigree analysis was suggestive of HBCS, genetic testing of the BRCA1, BRCA2, TP53, and CHEK2 genes was offered. Of 902 women submitted to GCRA, 214 had pedigrees suggestive of HBCS. Fifty of them underwent genetic testing: 18 and 40 for BRCA1/BRCA2 and TP53 mutation screening, respectively, and 7 for CHEK2 1100delC testing. A deleterious BRCA2 mutation was identified in one of the HBOC probands and the CHEK2 1100delC mutation occurred in one of the HBCC families. No deleterious germline alterations were identified in BRCA1 or TP53. Although strict inclusion criteria and a comprehensive testing approach were used, the suspected genetic risk in these families remains unexplained. Further studies in a larger cohort are necessary to better understand the genetic component of hereditary breast cancer in Southern Brazil.

A novel method for objective vision testing in canine models of inherited retinal disease.

PubMed

Gearhart, Patricia M; Gearhart, Chris C; Petersen-Jones, Simon M

2008-08-01

The use of canine models of retinal disease in the development of therapeutic strategies for inherited retinal disorders is a growing area of research. To evaluate accurately the success of potential vision-enhancing treatments, reliable methods for objectively assessing visual function in canine models is necessary. A simple vision-testing device was constructed that consisted of a junction box with four exit tunnels. Dogs were placed in the junction box and given one vision-based choice for exit. The first-choice tunnel and time to exit were recorded and analyzed. Two canine models of retinal disease with distinct molecular defects, a null mutation in the gene encoding the alpha subunit of rod cyclic GMP phosphodiesterase (PDE6A), and a null mutation in the gene encoding a retinal pigment epithelium-specific protein (RPE65) were tested and compared to those in unaffected dogs. With the use of bright light versus dim red light, the test differentiated between unaffected dogs and dogs affected with either mutation with a high degree of certainty. The white-light intensity series showed a significantly different performance between the unaffected and affected dogs. A significant difference in performance was detected between the dogs with each mutation. The results indicate that this novel canine vision-testing method is an accurate and sensitive means of distinguishing between unaffected dogs and dogs affected with two different forms of inherited retinal disease and should be useful as a means of assessing response to therapy in future studies.

Liquid Biopsy Analysis of FGFR3 and PIK3CA Hotspot Mutations for Disease Surveillance in Bladder Cancer.

PubMed

Christensen, Emil; Birkenkamp-Demtröder, Karin; Nordentoft, Iver; Høyer, Søren; van der Keur, Kirstin; van Kessel, Kim; Zwarthoff, Ellen; Agerbæk, Mads; Ørntoft, Torben Falck; Jensen, Jørgen Bjerggaard; Dyrskjøt, Lars

2017-06-01

Disease surveillance in patients with bladder cancer is important for early diagnosis of progression and metastasis and for optimised treatment. To develop urine and plasma assays for disease surveillance for patients with FGFR3 and PIK3CA tumour mutations. Droplet digital polymerase chain reaction (ddPCR) assays were developed and tumour DNA from two patient cohorts was screened for FGFR3 and PIK3CA hotspot mutations. One cohort included 363 patients with non-muscle-invasive bladder cancer (NMIBC). The other cohort included 468 patients with bladder cancer undergoing radical cystectomy (Cx). Urine supernatants (NMIBC n=216, Cx n=27) and plasma samples (NMIBC n=39, Cx n=27) from patients harbouring mutations were subsequently screened using ddPCR assays. Progression-free survival, recurrence-free survival, and overall survival were measured. Fisher's exact test, the Wilcoxon rank-sum test and Cox regression analysis were applied. In total, 36% of the NMIBC patients (129/363) and 11% of the Cx patients (44/403) harboured at least one FGFR3 or PIK3CA mutation. Screening of DNA from serial urine supernatants from the NMIBC cohort revealed that high levels of tumour DNA (tDNA) were associated with later disease progression in NMIBC (p=0.003). Furthermore, high levels of tDNA in plasma samples were associated with recurrence in the Cx cohort (p=0.016). A positive correlation between tDNA levels in urine and plasma was observed (correlation coefficient 0.6). The retrospective study design and low volumes of plasma available for analysis were limitations of the study. Increased levels of FGFR3 and PIK3CA mutated DNA in urine and plasma are indicative of later progression and metastasis in bladder cancer. Urine and plasma from patients with bladder cancer may be monitored for diagnosis of progression and metastasis using mutation assays. Copyright © 2016 European Association of Urology. Published by Elsevier B.V. All rights reserved.

[Application of MALDI-TOF-MS in gene testing for non-syndromic hearing loss].

PubMed

Zeng, Yun; Jiang, Dan; Feng, Da-fei; Jin, Dong-dong; Wu, Xiao-hui; Ding, Yan-li; Zou, Jing

2013-12-01

To investigate the feasibility of Matrix-Assisted Laser Desorption-Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) , according to the genetic test of non-syndromic hearing loss (NSHL), and check using the direct sequencing. Peripheral blood was collected from 454 NSHL patients. DNA samples were extracted and 20 loci of the four common disease-causing genes were analysed by MALDI-TOF-MS, including GJB2 (35delG, 167delT, 176_191del16, 235delC, 299_300delAT ), GJB3 (538C→T, 547G→A), SLC26A4 (281C→T, 589G→A, IVS7-2A→G, 1174A→T, 1226G→A, 1229C→T, IVS15+5G→A, 1975G→C, 2027T→A, 2162C→T, 2168A→G), and mitochondrial 12S rRNA (1494C→T, 1555A→G). Direct sequencing was also used to analyse the aforementioned 20 loci in order to validate the accuracy of MALDI-TOF-MS. Among the 454 patients, 166 cases (36.56%) of disease-causing mutations were detected, which included 69 cases (21.15%) of GJB2 gene mutation, four cases (0.88%) of GJB3 gene mutation, 64 cases (14.10%) of SLC26A4 gene mutation, and three cases (0.66%) of mitochondrial 12S rRNA gene mutation. Moreover, the results obtained from direct sequencing and MALDI-TOF-MS were consistent, and the results showed that the two methods were consistent. The MALDI-TOF-MS detection method was designed based on the hearing loss-related mutation hotspots seen in the Chinese population, and it has a high detection rate for NSHL related mutations. In comparison to the conventional detection methods, MALDI-TOF-MS has the following advantages: more detection sites, greater coverage, accurate, high throughput and low cost. Therefore, this method is capable of satisfying the needs of clinical detection for hearing impairment and it is suitable for large-scale implementation.

Linkage and mutational analysis of familial Alzheimer disease kindreds for the APP gene region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kamino, K.; Anderson, L.; O'dahl, S.

1992-11-01

A large number of familial Alzheimer disease (FAD) kindreds were examined to determine whether mutations in the amyloid precursor protein (APP) gene could be responsible for the disease. Previous studies have identified three mutations at APP codon 717 which are pathogenic for Alzheimer disease (AD). Samples from affected subjects were examined for mutations in exons 16 and 17 of the APP gene. A combination of direct sequencing and single-strand conformational polymorphism analysis was used. Sporadic AD and normal controls were also examined by the same methods. Five sequence variants were identified. One variant at APP codon 693 resulted in amore » Glu[yields]Gly change. This is the same codon as the hereditary cerebral hemorrhage with amyloidosis-Dutch type Glu[yields]Gln mutation. Another single-base change at APP codon 708 did not alter the amino acid encoded at this site. Two point mutations and a 6-bp deletion were identified in the intronic sequences surrounding exon 17. None of the variants could be unambigously determined to be responsible for FAD. The larger families were also analyzed by testing for linkage of FAD to a highly polymorphic short tandem repeat marker (D21S210) that is tightly linked to APP. Highly negative LOD scores were obtained for the family groups tested, and linkage was formally excluded beyond [theta] = .10 for the Volga German kindreds, [theta] = .20 for early-onset non-Volga Germans, and [theta] = .10 for late-onset families. LOD scores for linkage of FAD to markers centromeric to APP (D21S1/S11, D21S13, and D21S215) were also negative in the three family groups. These studies show that APP mutations account for AD in only a small fraction of FAD kindreds. 49 refs., 6 figs., 4 tabs.« less

Teaching the fluctuation test in silico by using mutate: a program to distinguish between the adaptive and spontaneous mutation hypotheses.

PubMed

Carvajal-Rodríguez, Antonio

2012-07-01

Mutate is a program developed for teaching purposes to impart a virtual laboratory class for undergraduate students of Genetics in Biology. The program emulates the so-called fluctuation test whose aim is to distinguish between spontaneous and adaptive mutation hypotheses in bacteria. The plan is to train students in certain key multidisciplinary aspects of current genetics such as sequence databases, DNA mutations, and hypothesis testing, while introducing the fluctuation test. This seminal experiment was originally performed studying Escherichia coli resistance to the infection by bacteriophage T1. The fluctuation test initiated the modern bacterial genetics that 25 years later ushered in the era of the recombinant DNA. Nowadays we know that some deletions in fhuA, the gene responsible for E. coli membrane receptor of T1, could cause the E. coli resistance to this phage. For the sake of simplicity, we will introduce the assumption that a single mutation generates the resistance to T1. During the practical, the students use the program to download some fhuA gene sequences, manually introduce some stop codon mutations, and design a fluctuation test to obtain data for distinguishing between preadaptative (spontaneous) and induced (adaptive) mutation hypotheses. The program can be launched from a browser or, if preferred, its executable file can be downloaded from http://webs.uvigo.es/acraaj/MutateWeb/Mutate.html. It requires the Java 5.0 (or higher) Runtime Environment (freely available at http://www.java.com). Copyright © 2012 Wiley Periodicals, Inc.

Multigenerational Inheritance of Long QT Syndrome Type 2 in a Japanese Family.

PubMed

Ichikawa, Mari; Ohno, Seiko; Fujii, Yusuke; Ozawa, Junichi; Sonoda, Keiko; Fukuyama, Megumi; Kato, Koichi; Kimura, Hiromi; Itoh, Hideki; Hayashi, Hideki; Horie, Minoru

2016-01-01

Congenital long QT syndrome (LQTS) is an important cause of sudden cardiac death in young people without any other structural disease. Mutations in the genes encoding the cardiac ion channels or associated proteins have been shown to result in ion channel dysfunction and thereby causing LQTS. We investigated a Japanese family with LQTS for four generations, with the female family members showing severe symptoms. We performed genetic tests for LQTS-related genes and identified a heterozygous KCNH2 mutation (p.K638del). In the family, the KCNH2 mutation had a very high multigenerational inheritance, and female genotype positives showed more severe phenotypes.

CpG Island Methylator Phenotype-Low (CIMP-Low) in Colorectal Cancer: Possible Associations with Male Sex and KRAS Mutations

PubMed Central

Ogino, Shuji; Kawasaki, Takako; Kirkner, Gregory J.; Loda, Massimo; Fuchs, Charles S.

2006-01-01

The CpG island methylator phenotype (CIMP or CIMP-high) with extensive promoter methylation seems to be a distinct epigenotype of colorectal cancer. However, no study has comprehensively examined features of colorectal cancer with less extensive promoter methylation (designated as “CIMP-low”). Using real-time polymerase chain reaction (MethyLight), we quantified DNA methylation in five CIMP-specific gene promoters [CACNA1G, CDKN2A (p16), CRABP1, MLH1, and NEUROG1] in 840 relatively unbiased, population-based colorectal cancer samples, obtained from two large prospective cohort studies. CIMP-low (defined as 1/5 to 3/5 methylated promoters) colorectal cancers were significantly more common among men (38 versus 30% in women, P = 0.01) and among KRAS-mutated tumors (44 versus 30% in KRAS/BRAF wild-type tumors, P = 0.0003; 19% in BRAF-mutated tumors, P < 0.0001). In addition, KRAS mutations were significantly more common in CIMP-low tumors (47%) than in CIMP-high tumors (with ≥4/5 methylated promoters, 12%, P < 0.0001) and CIMP-0 tumors (with 0/5 methylated promoters, 37%, P = 0.007). The associations of CIMP-low tumors with male sex and KRAS mutations still existed after tumors were stratified by microsatellite instability status. In conclusion, CIMP-low colorectal cancer is associated with male sex and KRAS mutations. The hypothesis that CIMP-low tumors are different from CIMP-high and CIMP-0 tumors needs to be tested further. PMID:17065427

CpG island methylator phenotype-low (CIMP-low) in colorectal cancer: possible associations with male sex and KRAS mutations.

PubMed

Ogino, Shuji; Kawasaki, Takako; Kirkner, Gregory J; Loda, Massimo; Fuchs, Charles S

2006-11-01

The CpG island methylator phenotype (CIMP or CIMP-high) with extensive promoter methylation seems to be a distinct epigenotype of colorectal cancer. However, no study has comprehensively examined features of colorectal cancer with less extensive promoter methylation (designated as "CIMP-low"). Using real-time polymerase chain reaction (MethyLight), we quantified DNA methylation in five CIMP-specific gene promoters [CACNA1G, CDKN2A (p16), CRABP1, MLH1, and NEUROG1] in 840 relatively unbiased, population-based colorectal cancer samples, obtained from two large prospective cohort studies. CIMP-low (defined as 1/5 to 3/5 methylated promoters) colorectal cancers were significantly more common among men (38 versus 30% in women, P = 0.01) and among KRAS-mutated tumors (44 versus 30% in KRAS/BRAF wild-type tumors, P = 0.0003; 19% in BRAF-mutated tumors, P < 0.0001). In addition, KRAS mutations were significantly more common in CIMP-low tumors (47%) than in CIMP-high tumors (with > or =4/5 methylated promoters, 12%, P < 0.0001) and CIMP-0 tumors (with 0/5 methylated promoters, 37%, P = 0.007). The associations of CIMP-low tumors with male sex and KRAS mutations still existed after tumors were stratified by microsatellite instability status. In conclusion, CIMP-low colorectal cancer is associated with male sex and KRAS mutations. The hypothesis that CIMP-low tumors are different from CIMP-high and CIMP-0 tumors needs to be tested further.

Extensive scanning of the calpain-3 gene broadens the spectrum of LGMD2A phenotypes

PubMed Central

Piluso, G; Politano, L; Aurino, S; Fanin, M; Ricci, E; Ventriglia, V; Belsito, A; Totaro, A; Saccone, V; Topaloglu, H; Nascimbeni, A; Fulizio, L; Broccolini, A; Canki-Klain, N; Comi, L; Nigro, G; Angelini, C; Nigro, V

2005-01-01

Background: The limb girdle muscular dystrophies (LGMD) are a heterogeneous group of Mendelian disorders highlighted by weakness of the pelvic and shoulder girdle muscles. Seventeen autosomal loci have been so far identified and genetic tests are mandatory to distinguish among the forms. Mutations at the calpain 3 locus (CAPN3) cause LGMD type 2A. Objective: To obtain unbiased information on the consequences of CAPN3 mutations. Patients: 530 subjects with different grades of symptoms and 300 controls. Methods: High throughput denaturing HPLC analysis of DNA pools. Results: 141 LGMD2A cases were identified, carrying 82 different CAPN3 mutations (45 novel), along with 18 novel polymorphisms/variants. Females had a more favourable course than males. In 94% of the more severely affected patient group, the defect was also discovered in the second allele. This proves the sensitivity of the approach. CAPN3 mutations were found in 35.1% of classical LGMD phenotypes. Mutations were also found in 18.4% of atypical patients and in 12.6% of subjects with high serum creatine kinase levels. Conclusions: A non-invasive and cost–effective strategy, based on the high throughput denaturing HPLC analysis of DNA pools, was used to obtain unbiased information on the consequences of CAPN3 mutations in the largest genetic study ever undertaken. This broadens the spectrum of LGMD2A phenotypes and sets the carrier frequency at 1:103. PMID:16141003

A Comprehensive Strategy for Accurate Mutation Detection of the Highly Homologous PMS2.

PubMed

Li, Jianli; Dai, Hongzheng; Feng, Yanming; Tang, Jia; Chen, Stella; Tian, Xia; Gorman, Elizabeth; Schmitt, Eric S; Hansen, Terah A A; Wang, Jing; Plon, Sharon E; Zhang, Victor Wei; Wong, Lee-Jun C

2015-09-01

Germline mutations in the DNA mismatch repair gene PMS2 underlie the cancer susceptibility syndrome, Lynch syndrome. However, accurate molecular testing of PMS2 is complicated by a large number of highly homologous sequences. To establish a comprehensive approach for mutation detection of PMS2, we have designed a strategy combining targeted capture next-generation sequencing (NGS), multiplex ligation-dependent probe amplification, and long-range PCR followed by NGS to simultaneously detect point mutations and copy number changes of PMS2. Exonic deletions (E2 to E9, E5 to E9, E8, E10, E14, and E1 to E15), duplications (E11 to E12), and a nonsense mutation, p.S22*, were identified. Traditional multiplex ligation-dependent probe amplification and Sanger sequencing approaches cannot differentiate the origin of the exonic deletions in the 3' region when PMS2 and PMS2CL share identical sequences as a result of gene conversion. Our approach allows unambiguous identification of mutations in the active gene with a straightforward long-range-PCR/NGS method. Breakpoint analysis of multiple samples revealed that recurrent exon 14 deletions are mediated by homologous Alu sequences. Our comprehensive approach provides a reliable tool for accurate molecular analysis of genes containing multiple copies of highly homologous sequences and should improve PMS2 molecular analysis for patients with Lynch syndrome. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Familial Mediterranean fever associated pyrin mutations in Greece

PubMed Central

Konstantopoulos, K; Kanta, A; Deltas, C; Atamian, V; Mavrogianni, D; Tzioufas, A; Kollainis, I; Ritis, K; Moutsopoulos, H

2003-01-01

Patients and methods: 62 patients fulfilling the Tel Hashomer diagnostic criteria for definite (33) or probable (29) FMF diagnosis were studied. Eight point mutations of pyrin gene were tested by standard methods. Of the 62 patients tested, 48 were Greek, four were Jewish, seven were Armenian, and three were Arab. Results: 42 patients were found to be homozygotes for pyrin mutations; 11 patients were found to carry only one of the tested mutations; in nine patients no mutations were detected. Conclusion: Molecular detection of pyrin gene mutations seems useful in confirming suspected cases, and in detecting asymptomatic cases, of Mediterranean fever in Greece. It may also be used as a screening tool within affected families. PMID:12695165

GNQ-209P Mutation in Metastatic Uveal Melanoma and Treatment Outcome.

PubMed

Abdel Karim, Nagla; Eldessouki, Ihab; Taftaf, Ahmad; Ayham, Deeb; Gaber, Ola; Makramalla, Abouelmagd; Correa, Zelia M

2018-01-01

Metastatic prognosis in uveal melanoma is assessed by gene expression profiling (GEP) testing of the tumor cells, usually obtained by fine needle aspiration (FNA). GEP has demonstrated high accuracy in distinguishing class I and II tumors, both having different metastatic potential. Transcriptomic studies identified distinct mutations including somatic mutations in GNAQ and GNA11 , detected in more than 80%, and contribute to the upregulation of the mitogen-activated protein kinase (MAPK) pathway and the development of uveal melanoma (UM). The role of these mutations in treatment selection and possible benefit from targeted therapy are somewhat unclear. However, until the discovery of novel agents, local versus systemic therapies remain options for treatment that can still be considered for disease control in certain cases. We report a series of patients with metastatic UM with distinct mutational profiles. One had significant liver metastases with proven GNQ-209P mutation on tissue biopsy while peripheral blood molecular profiling did not show these mutations. The other three cases had no GNQ-209P mutation. All cases received nab-paclitaxel (Abraxane) as a treatment drug, and we record their responses to treatment and their molecular-profiling results.

Prevalence of BRCA1 and BRCA2 mutations in women with breast carcinoma In Situ and referred for genetic testing.

PubMed

Hall, Michael J; Reid, Julia E; Wenstrup, Richard J

2010-12-01

Ductal and lobular carcinoma in situ (CIS) accounted for 62,280 (24.5%) of all new breast cancer diagnoses in 2009. BRCA1/2 mutations confer an extremely high risk of breast cancer, and management guidelines for BRCA1/2 mutation carriers advise close follow-up, intensive screening, and consideration of prophylactic surgery to lower this risk. The limited relevant previous data are not definitive in establishing the prevalence of BRCA1/2 mutations in breast CIS patients, creating uncertainty as to whether referral for cancer risk assessment and genetic testing is appropriate for this group. Therefore, we conducted a cross-sectional analysis of the Myriad Genetics BRCA1/2 database to determine the prevalence of these mutations in breast CIS patients. All statistical tests were 2-sided, and confidence intervals (CI) are reported at the 95% level (α = 0.05). The source population was 64,717 consecutive women who were not Ashkenazi Jewish, underwent BRCA1/2 testing, and provided a personal and family history of invasive breast and ovarian cancer; 7,295 (11.3%) reported a diagnosis of CIS (ductal or lobular) and had an overall 5.9% prevalence of mutated BRCA1/2 (mBRCA). Subgrouped by history (personal or family) of invasive breast and/or ovarian cancer, these CIS patients had the following prevalences of mBRCA: (1) no personal or family history, 2.3%; (2) personal history, 5.2%; (3) family history, 5%; and (4) personal and family history, 10.3%. mBRCA risk was significantly higher in women with early-onset (<50 years old) CIS than with late-onset (≥ 50 years old) CIS [odds ratio (OR) = 1.5; 95% CI = 1.1-2.1). Disease onset at less than 40 years age was associated with an even higher mBRCA risk (OR = 1.8; 95% CI = 1.3-2.3). By far the largest analysis of BRCA1/2 mutation prevalence in non-Ashkenazi Jewish breast CIS patients, this study shows that early-onset CIS is associated with mBRCA1/2 in patients referred for genetic testing. When a family history of breast and/or ovarian cancer are also present, testing women with early-onset CIS may increase both the likelihood of detecting BRCA1/2 mutations and opportunities for carriers to consider additional cancer prevention strategies. ©2010 AACR.

Recurrent mutation testing of BRCA1 and BRCA2 in Asian breast cancer patients identify carriers in those with presumed low risk by family history.

PubMed

Kang, Peter Choon Eng; Phuah, Sze Yee; Sivanandan, Kavitta; Kang, In Nee; Thirthagiri, Eswary; Liu, Jian Jun; Hassan, Norhashimah; Yoon, Sook-Yee; Thong, Meow Keong; Hui, Miao; Hartman, Mikael; Yip, Cheng Har; Mohd Taib, Nur Aishah; Teo, Soo Hwang

2014-04-01

Although the breast cancer predisposition genes BRCA1 and BRCA2 were discovered more than 20 years ago, there remains a gap in the availability of genetic counselling and genetic testing in Asian countries because of cost, access and inaccurate reporting of family history of cancer. In order to improve access to testing, we developed a rapid test for recurrent mutations in our Asian populations. In this study, we designed a genotyping assay with 55 BRCA1 and 44 BRCA2 mutations previously identified in Asian studies, and validated this assay in 267 individuals who had previously been tested by full sequencing. We tested the prevalence of these mutations in additional breast cancer cases. Using this genotyping approach, we analysed recurrent mutations in 533 Malaysian breast cancer cases with <10 % a priori risk, and found 1 BRCA1 (0.2 %) and 5 BRCA2 (0.9 %) carriers. Testing in a hospital-based unselected cohort of 532 Singaporean breast cancer cases revealed 6 BRCA1 (1.1 %) and 3 BRCA2 (0.6 %) carriers. Overall, 2 recurrent BRCA1 and 1 BRCA2 mutations in Malays, 3 BRCA1 and 2 BRCA2 mutations in Chinese and 1 BRCA1 mutation in Indians account for 60, 24 and 20 % of carrier families, respectively. By contrast, haplotype analyses suggest that a recurrent BRCA2 mutation (c.262_263delCT) found in 5 unrelated Malay families has at least 3 distinct haplotypes. Taken together, our data suggests that panel testing may help to identify carriers, particularly Asian BRCA2 carriers, who do not present with a priori strong family history characteristics.

Targeted prostate cancer screening in BRCA1 and BRCA2 mutation carriers: results from the initial screening round of the IMPACT study.

PubMed

Bancroft, Elizabeth K; Page, Elizabeth C; Castro, Elena; Lilja, Hans; Vickers, Andrew; Sjoberg, Daniel; Assel, Melissa; Foster, Christopher S; Mitchell, Gillian; Drew, Kate; Mæhle, Lovise; Axcrona, Karol; Evans, D Gareth; Bulman, Barbara; Eccles, Diana; McBride, Donna; van Asperen, Christi; Vasen, Hans; Kiemeney, Lambertus A; Ringelberg, Janneke; Cybulski, Cezary; Wokolorczyk, Dominika; Selkirk, Christina; Hulick, Peter J; Bojesen, Anders; Skytte, Anne-Bine; Lam, Jimmy; Taylor, Louise; Oldenburg, Rogier; Cremers, Ruben; Verhaegh, Gerald; van Zelst-Stams, Wendy A; Oosterwijk, Jan C; Blanco, Ignacio; Salinas, Monica; Cook, Jackie; Rosario, Derek J; Buys, Saundra; Conner, Tom; Ausems, Margreet G; Ong, Kai-ren; Hoffman, Jonathan; Domchek, Susan; Powers, Jacquelyn; Teixeira, Manuel R; Maia, Sofia; Foulkes, William D; Taherian, Nassim; Ruijs, Marielle; Helderman-van den Enden, Apollonia T; Izatt, Louise; Davidson, Rosemarie; Adank, Muriel A; Walker, Lisa; Schmutzler, Rita; Tucker, Kathy; Kirk, Judy; Hodgson, Shirley; Harris, Marion; Douglas, Fiona; Lindeman, Geoffrey J; Zgajnar, Janez; Tischkowitz, Marc; Clowes, Virginia E; Susman, Rachel; Ramón y Cajal, Teresa; Patcher, Nicholas; Gadea, Neus; Spigelman, Allan; van Os, Theo; Liljegren, Annelie; Side, Lucy; Brewer, Carole; Brady, Angela F; Donaldson, Alan; Stefansdottir, Vigdis; Friedman, Eitan; Chen-Shtoyerman, Rakefet; Amor, David J; Copakova, Lucia; Barwell, Julian; Giri, Veda N; Murthy, Vedang; Nicolai, Nicola; Teo, Soo-Hwang; Greenhalgh, Lynn; Strom, Sara; Henderson, Alex; McGrath, John; Gallagher, David; Aaronson, Neil; Ardern-Jones, Audrey; Bangma, Chris; Dearnaley, David; Costello, Philandra; Eyfjord, Jorunn; Rothwell, Jeanette; Falconer, Alison; Gronberg, Henrik; Hamdy, Freddie C; Johannsson, Oskar; Khoo, Vincent; Kote-Jarai, Zsofia; Lubinski, Jan; Axcrona, Ulrika; Melia, Jane; McKinley, Joanne; Mitra, Anita V; Moynihan, Clare; Rennert, Gad; Suri, Mohnish; Wilson, Penny; Killick, Emma; Moss, Sue; Eeles, Rosalind A

2014-09-01

Men with germline breast cancer 1, early onset (BRCA1) or breast cancer 2, early onset (BRCA2) gene mutations have a higher risk of developing prostate cancer (PCa) than noncarriers. IMPACT (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted screening in BRCA1/2 mutation carriers and controls) is an international consortium of 62 centres in 20 countries evaluating the use of targeted PCa screening in men with BRCA1/2 mutations. To report the first year's screening results for all men at enrollment in the study. We recruited men aged 40-69 yr with germline BRCA1/2 mutations and a control group of men who have tested negative for a pathogenic BRCA1 or BRCA2 mutation known to be present in their families. All men underwent prostate-specific antigen (PSA) testing at enrollment, and those men with PSA >3 ng/ml were offered prostate biopsy. PSA levels, PCa incidence, and tumour characteristics were evaluated. The Fisher exact test was used to compare the number of PCa cases among groups and the differences among disease types. We recruited 2481 men (791 BRCA1 carriers, 531 BRCA1 controls; 731 BRCA2 carriers, 428 BRCA2 controls). A total of 199 men (8%) presented with PSA >3.0 ng/ml, 162 biopsies were performed, and 59 PCas were diagnosed (18 BRCA1 carriers, 10 BRCA1 controls; 24 BRCA2 carriers, 7 BRCA2 controls); 66% of the tumours were classified as intermediate- or high-risk disease. The positive predictive value (PPV) for biopsy using a PSA threshold of 3.0 ng/ml in BRCA2 mutation carriers was 48%-double the PPV reported in population screening studies. A significant difference in detecting intermediate- or high-risk disease was observed in BRCA2 carriers. Ninety-five percent of the men were white, thus the results cannot be generalised to all ethnic groups. The IMPACT screening network will be useful for targeted PCa screening studies in men with germline genetic risk variants as they are discovered. These preliminary results support the use of targeted PSA screening based on BRCA genotype and show that this screening yields a high proportion of aggressive disease. In this report, we demonstrate that germline genetic markers can be used to identify men at higher risk of prostate cancer. Targeting screening at these men resulted in the identification of tumours that were more likely to require treatment. Copyright © 2014 European Association of Urology. All rights reserved.

Genetic mutations of young patients admitted to an emergency department for syncope during sport practice.

PubMed

Gómez Alcaraz, Jorge; Bustamante, José; Corral, Ervigio; Casado Florez, Maria Isabel; Vivas, David; Cañadas-Godoy, Victoria; González Del Castillo, Juan; González Armengol, Juan Jorge; López-Farré, Antonio; Martín Sánchez, Francisco Javier

2018-04-25

To study the frequency of genetic mutations related to genetic heart disease among young patients admitted for syncope during sport practice. A case series study that included patients≤45 years admitted for syncope during sport practice during 2010-2011. We collected demographic and clinical variables, genetic tests mutations and final clinical diagnosis. A genetic test was performed in 46 (76.7%) of 60 patients evaluated. The genetic test was positive in 12 (26%; 95% CI 15.6-40.3) patients; 10 (21.7%) had PKP2 mutation related to arrhythmogenic right ventricular dysplasia mutation, one (2.2%) KCNQ1 mutation and one (2.2%) SCN5A mutation related to channelopathies. The genetic test was positive in 11 (35.5%) cases of undetermined syncope and one (50%) case of cardiac syncope, being negative in all cases with neuromediated syncopes (P=.037). Gene mutations are common in young patients suffering from syncope during sports, especially in those with cardiac or undetermined aetiology. Copyright © 2018 Elsevier España, S.L.U. All rights reserved.

Comparison between two widely used laboratory methods in BRAF V600 mutation detection in a large cohort of clinical samples of cutaneous melanoma metastases to the lymph nodes.

PubMed

Jurkowska, Monika; Gos, Aleksandra; Ptaszyński, Konrad; Michej, Wanda; Tysarowski, Andrzej; Zub, Renata; Siedlecki, Janusz A; Rutkowski, Piotr

2015-01-01

The study compares detection rates of oncogenic BRAF mutations in a homogenous group of 236 FFPE cutaneous melanoma lymph node metastases, collected in one cancer center. BRAF mutational status was verified by two independent in-house PCR/Sanger sequencing tests, and the Cobas® 4800 BRAF V600 Mutation Test. The best of two sequencing approaches returned results for 230/236 samples. In 140 (60.9%), the mutation in codon 600 of BRAF was found. 91.4% of all mutated cases (128 samples) represented p.V600E. Both Sanger-based tests gave reproducible results although they differed significantly in the percentage of amplifiable samples: 230/236 to 109/143. Cobas generated results in all 236 cases, mutations changing codon V600 were detected in 144 of them (61.0%), including 5 not amplifiable and 5 negative in the standard sequencing. However, 6 cases positive in sequencing turned out to be negative in Cobas. Both tests provided us with the same BRAF V600 mutational status in 219 out of 230 cases with valid results (95.2%). The total BRAF V600 mutation detection rate didn't differ significantly between the two methodological approaches (60.9% vs. 61.0%). Sequencing was a reproducible method of V600 mutation detection and more powerful to detect mutations other than p.V600E, while Cobas test proved to be less susceptible to the poor DNA quality or investigator's bias. The study underlined an important role of pathologists in quality assurance of molecular diagnostics.

Prospective study of POLG mutations presenting in children with intractable epilepsy: prevalence and clinical features.

PubMed

Uusimaa, Johanna; Gowda, Vasantha; McShane, Anthony; Smith, Conrad; Evans, Julie; Shrier, Annie; Narasimhan, Manisha; O'Rourke, Anthony; Rajabally, Yusuf; Hedderly, Tammy; Cowan, Frances; Fratter, Carl; Poulton, Joanna

2013-06-01

To assess the frequency and clinical features of childhood-onset intractable epilepsy caused by the most common mutations in the POLG gene, which encodes the catalytic subunit of mitochondrial DNA polymerase gamma. Children presenting with nonsyndromic intractable epilepsy of unknown etiology but without documented liver dysfunction at presentation were eligible for this prospective, population-based study. Blood samples were analyzed for the three most common POLG mutations. If any of the three tested mutations were found, all the exons and the exon-intron boundaries of the POLG gene were sequenced. In addition, we retrospectively reviewed the notes of patients presenting with intractable epilepsy in which we had found POLG mutations. All available clinical data were collected by questionnaire and by reviewing the medical records. We analyzed 213 blood DNA samples from patients fulfilling the inclusion criteria of the prospective study. Among these, five patients (2.3%) were found with one of the three common POLG mutations as homozygous or compound heterozygous states. In addition, three patients were retrospectively identified. Seven of the eight patients had either raised cerebrospinal fluid (CSF) lactate (n = 3) or brain magnetic resonance imaging (MRI) changes (n = 4) at presentation with intractable epilepsy. Three patients later developed liver dysfunction, progressing to fatal liver failure in two without previous treatment with sodium valproate (VPA). Furthermore, it is worth mentioning that one patient presented first with an autism spectrum disorder before seizures emerged. Mutations in POLG are an important cause of early and juvenile onset nonsyndromic intractable epilepsy with highly variable associated manifestations including autistic features. This study emphasizes that genetic testing for POLG mutations in patients with nonsyndromic intractable epilepsies is very important for clinical diagnostics, genetic counseling, and treatment decisions because of the increased risk for VPA-induced liver failure in patients with POLG mutations. We recommend POLG gene testing for patients with intractable seizures and at least one elevated CSF lactate or suggestive brain MRI changes (predominantly abnormal T2 -weighted thalamic signal) with or without status epilepticus, epilepsia partialis continua, or liver manifestations typical for Alpers disease, especially when the disease course is progressive. Wiley Periodicals, Inc. © 2013 International League Against Epilepsy.

Prospective study of POLG mutations presenting in children with intractable epilepsy: Prevalence and clinical features

PubMed Central

Uusimaa, Johanna; Gowda, Vasantha; McShane, Anthony; Smith, Conrad; Evans, Julie; Shrier, Annie; Narasimhan, Manisha; O'Rourke, Anthony; Rajabally, Yusuf; Hedderly, Tammy; Cowan, Frances; Fratter, Carl; Poulton, Joanna

2013-01-01

Purpose To assess the frequency and clinical features of childhood-onset intractable epilepsy caused by the most common mutations in the POLG gene, which encodes the catalytic subunit of mitochondrial DNA polymerase gamma. Methods Children presenting with nonsyndromic intractable epilepsy of unknown etiology but without documented liver dysfunction at presentation were eligible for this prospective, population-based study. Blood samples were analyzed for the three most common POLG mutations. If any of the three tested mutations were found, all the exons and the exon–intron boundaries of the POLG gene were sequenced. In addition, we retrospectively reviewed the notes of patients presenting with intractable epilepsy in which we had found POLG mutations. All available clinical data were collected by questionnaire and by reviewing the medical records. Key Findings We analyzed 213 blood DNA samples from patients fulfilling the inclusion criteria of the prospective study. Among these, five patients (2.3%) were found with one of the three common POLG mutations as homozygous or compound heterozygous states. In addition, three patients were retrospectively identified. Seven of the eight patients had either raised cerebrospinal fluid (CSF) lactate (n = 3) or brain magnetic resonance imaging (MRI) changes (n = 4) at presentation with intractable epilepsy. Three patients later developed liver dysfunction, progressing to fatal liver failure in two without previous treatment with sodium valproate (VPA). Furthermore, it is worth mentioning that one patient presented first with an autism spectrum disorder before seizures emerged. Significance Mutations in POLG are an important cause of early and juvenile onset nonsyndromic intractable epilepsy with highly variable associated manifestations including autistic features. This study emphasizes that genetic testing for POLG mutations in patients with nonsyndromic intractable epilepsies is very important for clinical diagnostics, genetic counseling, and treatment decisions because of the increased risk for VPA-induced liver failure in patients with POLG mutations. We recommend POLG gene testing for patients with intractable seizures and at least one elevated CSF lactate or suggestive brain MRI changes (predominantly abnormal T2-weighted thalamic signal) with or without status epilepticus, epilepsia partialis continua, or liver manifestations typical for Alpers disease, especially when the disease course is progressive. PMID:23448099

Enhancing the Predictive Power of Mutations in the C-Terminus of the KCNQ1-Encoded Kv7.1 Voltage-Gated Potassium Channel.

PubMed

Kapplinger, Jamie D; Tseng, Andrew S; Salisbury, Benjamin A; Tester, David J; Callis, Thomas E; Alders, Marielle; Wilde, Arthur A M; Ackerman, Michael J

2015-04-01

Despite the overrepresentation of Kv7.1 mutations among patients with a robust diagnosis of long QT syndrome (LQTS), a background rate of innocuous Kv7.1 missense variants observed in healthy controls creates ambiguity in the interpretation of LQTS genetic test results. A recent study showed that the probability of pathogenicity for rare missense mutations depends in part on the topological location of the variant in Kv7.1's various structure-function domains. Since the Kv7.1's C-terminus accounts for nearly 50 % of the overall protein and nearly 50 % of the overall background rate of rare variants falls within the C-terminus, further enhancement in mutation calling may provide guidance in distinguishing pathogenic long QT syndrome type 1 (LQT1)-causing mutations from rare non-disease-causing variants in the Kv7.1's C-terminus. Therefore, we have used conservation analysis and a large case-control study to generate topology-based estimative predictive values to aid in interpretation, identifying three regions of high conservation within the Kv7.1's C-terminus which have a high probability of LQT1 pathogenicity.

Diagnosis of Fanconi Anemia: Mutation Analysis by Multiplex Ligation-Dependent Probe Amplification and PCR-Based Sanger Sequencing

PubMed Central

Gille, Johan J. P.; Floor, Karijn; Kerkhoven, Lianne; Ameziane, Najim; Joenje, Hans; de Winter, Johan P.

2012-01-01

Fanconi anemia (FA) is a rare inherited disease characterized by developmental defects, short stature, bone marrow failure, and a high risk of malignancies. FA is heterogeneous: 15 genetic subtypes have been distinguished so far. A clinical diagnosis of FA needs to be confirmed by testing cells for sensitivity to cross-linking agents in a chromosomal breakage test. As a second step, DNA testing can be employed to elucidate the genetic subtype of the patient and to identify the familial mutations. This knowledge allows preimplantation genetic diagnosis (PGD) and enables prenatal DNA testing in future pregnancies. Although simultaneous testing of all FA genes by next generation sequencing will be possible in the near future, this technique will not be available immediately for all laboratories. In addition, in populations with strong founder mutations, a limited test using Sanger sequencing and MLPA will be a cost-effective alternative. We describe a strategy and optimized conditions for the screening of FANCA, FANCB, FANCC, FANCE, FANCF, and FANCG and present the results obtained in a cohort of 54 patients referred to our diagnostic service since 2008. In addition, the follow up with respect to genetic counseling and carrier screening in the families is discussed. PMID:22778927

Molecular methods for somatic mutation testing in lung adenocarcinoma: EGFR and beyond

PubMed Central

Rogers, Toni-Maree; Fellowes, Andrew; Bell, Anthony; Fox, Stephen

2015-01-01

Somatic mutational profiling in cancer has revolutionized the practice of clinical oncology. The discovery of driver mutations in non-small cell lung cancer (NSCLC) is an example of this. Molecular testing of lung adenocarcinoma is now considered standard of care and part of the diagnostic algorithm. This article provides an overview of the workflow of molecular testing in a clinical diagnostic laboratory discussing in particular novel assays that are currently in use for somatic mutation detection in NSCLC focussing on epidermal growth factor receptor (EGFR) mutations and anaplastic lymphoma kinase (ALK), ROS1 and RET rearrangements. PMID:25870795

Quantitative PCR high-resolution melting (qPCR-HRM) curve analysis, a new approach to simultaneously screen point mutations and large rearrangements: application to MLH1 germline mutations in Lynch syndrome.

PubMed

Rouleau, Etienne; Lefol, Cédrick; Bourdon, Violaine; Coulet, Florence; Noguchi, Tetsuro; Soubrier, Florent; Bièche, Ivan; Olschwang, Sylviane; Sobol, Hagay; Lidereau, Rosette

2009-06-01

Several techniques have been developed to screen mismatch repair (MMR) genes for deleterious mutations. Until now, two different techniques were required to screen for both point mutations and large rearrangements. For the first time, we propose a new approach, called "quantitative PCR (qPCR) high-resolution melting (HRM) curve analysis (qPCR-HRM)," which combines qPCR and HRM to obtain a rapid and cost-effective method suitable for testing a large series of samples. We designed PCR amplicons to scan the MLH1 gene using qPCR HRM. Seventy-six patients were fully scanned in replicate, including 14 wild-type patients and 62 patients with known mutations (57 point mutations and five rearrangements). To validate the detected mutations, we used sequencing and/or hybridization on a dedicated MLH1 array-comparative genomic hybridization (array-CGH). All point mutations and rearrangements detected by denaturing high-performance liquid chromatography (dHPLC)+multiplex ligation-dependent probe amplification (MLPA) were successfully detected by qPCR HRM. Three large rearrangements were characterized with the dedicated MLH1 array-CGH. One variant was detected with qPCR HRM in a wild-type patient and was located within the reverse primer. One variant was not detected with qPCR HRM or with dHPLC due to its proximity to a T-stretch. With qPCR HRM, prescreening for point mutations and large rearrangements are performed in one tube and in one step with a single machine, without the need for any automated sequencer in the prescreening process. In replicate, its reagent cost, sensitivity, and specificity are comparable to those of dHPLC+MLPA techniques. However, qPCR HRM outperformed the other techniques in terms of its rapidity and amount of data provided.

Japanese Society of Medical Oncology Clinical Guidelines: Molecular Testing for Colorectal Cancer Treatment, Third Edition.

PubMed

Yamazaki, Kentaro; Taniguchi, Hiroya; Yoshino, Takayuki; Akagi, Kiwamu; Ishida, Hideyuki; Ebi, Hiromichi; Nakatani, Kaname; Muro, Kei; Yatabe, Yasushi; Yamaguchi, Kensei; Tsuchihara, Katsuya

2018-06-01

The Japanese Society of Medical Oncology (JSMO) previously published 2 editions of the clinical guidelines: "Japanese guidelines for testing of KRAS gene mutation in colorectal cancer" in 2008 and "Japanese Society of Medical Oncology Clinical Guidelines: RAS (KRAS/NRAS) mutation testing in colorectal cancer patients" in 2014. These guidelines have contributed to the proper use of KRAS and RAS mutation testing, respectively. Recently, clinical utility, particularly for colorectal cancer (CRC) patients with BRAF V600E mutation or DNA mismatch-repair (MMR) deficiency, has been established. Therefore, the guideline members decided these genetic alterations should also be involved. The aim of this revision is to properly carry out testing for BRAF V600E mutation and MMR deficiency in addition to RAS mutation. The revised guidelines include the basic requirements for testing for these genetic alterations based on recent scientific evidence. Furthermore, because clinical utility of comprehensive genetic testing using next-generation sequencing and somatic gene testing of analyzing circulating tumor DNA has increasingly evolved with recent advancements in testing technology, we noted the current situation and prospects for these testing technologies and their clinical implementation in the revised guidelines. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Improving newborn screening for cystic fibrosis using next-generation sequencing technology: a technical feasibility study.

PubMed

Baker, Mei W; Atkins, Anne E; Cordovado, Suzanne K; Hendrix, Miyono; Earley, Marie C; Farrell, Philip M

2016-03-01

Many regions have implemented newborn screening (NBS) for cystic fibrosis (CF) using a limited panel of cystic fibrosis transmembrane regulator (CFTR) mutations after immunoreactive trypsinogen (IRT) analysis. We sought to assess the feasibility of further improving the screening using next-generation sequencing (NGS) technology. An NGS assay was used to detect 162 CFTR mutations/variants characterized by the CFTR2 project. We used 67 dried blood spots (DBSs) containing 48 distinct CFTR mutations to validate the assay. NGS assay was retrospectively performed on 165 CF screen-positive samples with one CFTR mutation. The NGS assay was successfully performed using DNA isolated from DBSs, and it correctly detected all CFTR mutations in the validation. Among 165 screen-positive infants with one CFTR mutation, no additional disease-causing mutation was identified in 151 samples consistent with normal sweat tests. Five infants had a CF-causing mutation that was not included in this panel, and nine with two CF-causing mutations were identified. The NGS assay was 100% concordant with traditional methods. Retrospective analysis results indicate an IRT/NGS screening algorithm would enable high sensitivity, better specificity and positive predictive value (PPV). This study lays the foundation for prospective studies and for introducing NGS in NBS laboratories.

Characterization of sulfonylurea-resistant Schoenoplectus juncoides having a target-site Asp(376)Glu mutation in the acetolactate synthase.

PubMed

Sada, Yoshinao; Ikeda, Hajime; Yamato, Seiji; Kizawa, Satoru

2013-09-01

Schoenoplectus juncoides, a noxious weed for paddy rice, is known to become resistant to sulfonylurea (SU) herbicides by a target-site mutation in either of the two acetolactate synthase (ALS) genes (ALS1 and ALS2). SU-resistant S. juncoides plants having an Asp376Glu mutation in ALS2 were found from a paddy rice field in Japan, but their resistance profile has not been quantitatively investigated. In this study, dose-response of the SU-resistant accession was compared with that of a SU-susceptible accession at in vivo whole-plant level as well as at in vitro enzymatic level. In whole-plant tests, resistance factors (RFs) based on 50% growth reduction (GR50) for imazosulfuron (ISF), bensulfuron-methyl (BSM), metsulfuron-methyl (MSM), bispyribac-sodium (BPS), and imazaquin (IMQ) were 176, 40, 14, 5.2 and 1.5, respectively. Thus, the accession having an Asp376Glu mutation in ALS2 was highly resistant to the three SU herbicides and moderately resistant to BPS, but was not substantially resistant to IMQ. This is slightly different from the earlier results reported from other weeds with an Asp376Glu mutation, in which the mutation confers resistance to broadly all the chemical classes of ALS-inhibiting herbicides. In enzymatic tests, ALS2 of S. juncoides was expressed in E. coli; the resultant ALS2 was subjected to an in vitro assay. RFs of the mutated ALS2 based on 50% enzymatic inhibition (I50) for ISF, BSM, MSM, BPS, and IMQ were 3699, 2438, 322, 80, and 4.8, respectively. The RFs of ALS2 were highly correlated with those of the whole-plant; this suggests that the Asp376Glu mutation in ALS2 is a molecular basis for the whole-plant resistance. The presence of two ALS genes in S. juncoides can at least partially explain why the whole-plant RFs were less than those of the expressed ALS2 enzymes. Copyright © 2013 Elsevier Inc. All rights reserved.

Murine knockin model for progranulin-deficient frontotemporal dementia with nonsense-mediated mRNA decay

PubMed Central

Nguyen, Thi A.; Zhang, Jiasheng; Devireddy, Swathi; Zhou, Ping; Karydas, Anna M.; Xu, Xialian; Miller, Bruce L.; Rigo, Frank; Ferguson, Shawn M.; Walther, Tobias C.; Farese, Robert V.

2018-01-01

Frontotemporal dementia (FTD) is the most common neurodegenerative disorder in individuals under age 60 and has no treatment or cure. Because many cases of FTD result from GRN nonsense mutations, an animal model for this type of mutation is highly desirable for understanding pathogenesis and testing therapies. Here, we generated and characterized GrnR493X knockin mice, which model the most common human GRN mutation, a premature stop codon at arginine 493 (R493X). Homozygous GrnR493X mice have markedly reduced Grn mRNA levels, lack detectable progranulin protein, and phenocopy Grn knockout mice, with CNS microgliosis, cytoplasmic TDP-43 accumulation, reduced synaptic density, lipofuscinosis, hyperinflammatory macrophages, excessive grooming behavior, and reduced survival. Inhibition of nonsense-mediated mRNA decay (NMD) by genetic, pharmacological, or antisense oligonucleotide-based approaches showed that NMD contributes to the reduced mRNA levels in GrnR493X mice and cell lines and in fibroblasts from patients containing the GRNR493X mutation. Moreover, the expressed truncated R493X mutant protein was functional in several assays in progranulin-deficient cells. Together, these findings establish a murine model for in vivo testing of NMD inhibition or other therapies as potential approaches for treating progranulin deficiency caused by the R493X mutation. PMID:29511098

Identification of proteins that interact with TANK binding kinase 1 and testing for mutations associated with glaucoma.

PubMed

Seo, Seongjin; Solivan-Timpe, Frances; Roos, Ben R; Robin, Alan L; Stone, Edwin M; Kwon, Young H; Alward, Wallace L M; Fingert, John H

2013-02-01

Copy number variations (duplications) of TANK binding kinase 1 (TBK1) have been associated with normal tension glaucoma (NTG), a common cause of blindness worldwide. Mutations in other genes involved in autophagy (TLR4 and OPTN) have been associated with NTG. Here we report searching for additional proteins involved in autophagy that may also have roles in NTG. HEK-293T cells were transfected to produce synthetic TBK1 protein with FLAG and S tags. Proteins that associate with TBK1 were isolated from HEK-293T lysates using tandem affinity purification (TAP) and polyacrylamide gel electrophoresis (PAGE). Isolated proteins were identified with mass spectrometry. A cohort of 148 NTG patients and 77 controls from Iowa were tested for glaucoma-causing mutations in genes that encode identified proteins that interact with TBK1 using high resolution melt (HRM) analysis and DNA sequencing. TAP studies show that three proteins expressed in HEK-293T cells (NAP1, TANK and TBKBP1) interact with TBK1. Testing cohorts of NTG and normal controls for disease-causing mutations in TANK, identified a total of nine unique variants including three non-synonymous changes, one synonymous changes and five intronic changes. When analyzed alone or as a group, the non-synonymous TBK1 coding sequence changes were not associated with either NTG or primary open angle glaucoma. TAP showed that NAP1, TANK and TBKBP1 interact with TBK1 and are good candidates for contributing to NTG. A mutation screen of TANK detected three non-synonymous variants. Although, it remains possible that one or more of these TANK mutations may have a role in NTG, the data in this report do not provide statistical support for an association between TANK variants and NTG.

PIK3CA oncogenic mutations represent a major mechanism of resistance to trastuzumab in HER2/neu overexpressing uterine serous carcinomas

PubMed Central

Black, Jonathan D; Lopez, Salvatore; Cocco, Emiliano; Bellone, Stefania; Altwerger, Gary; Schwab, Carlton L; English, Diana P; Bonazzoli, Elena; Predolini, Federica; Ferrari, Francesca; Ratner, Elena; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E; Santin, Alessandro D

2015-01-01

Objectives: We evaluated the role of PIK3CA-mutations as mechanism of resistance to trastuzumab in primary HER2/neu-amplified uterine-serous-carcinoma (USC) cell lines. Methods: Fifteen whole-exome-sequenced USC cell lines were tested for HER2/neu-amplification and PIK3CA-mutations. Four HER2/neu-amplified USC (2-harbouring wild-type-PIK3CA-genes and 2-harbouring oncogenic-PIK3CA-mutations) were evaluated in in vitro dose-titration-proliferation-assays, cell-viability and HER2 and S6-protein-phosphorylation after exposure to trastuzumab. USC harbouring wild-type-PIK3CA were transfected with plasmids encoding oncogenic PIK3CA-mutations (i.e., H1047R/R93Q) and exposed to trastuzumab. Finally, trastuzumab efficacy was tested by using two USC xenograft mouse models. Results: Seven out of fifteen (46%) of the USC cell lines were HER2/neu-amplified by fluorescence in situ hybridisation. Within these tumours four out of seven (57%) were found to harbour oncogenic PIK3CA-mutations vs two out of eight (25%) of the HER2/neu not amplified cell lines (P=0.01). HER2/neu-amplified/PIK3CA-mutated USC were highly resistant to trastuzumab when compared with HER2/neu-amplified/wild-type-PIK3CA cell lines (P=0.02). HER2/neu-amplified/PIK3CA wild-type cell lines transfected with oncogenic PIK3CA-mutations increased their resistance to trastuzumab (P<0.0001). Trastuzumab was effective in reducing tumour growth (P=0.001) and improved survival (P=0.0001) in mouse xenografts harbouring HER2-amplified/PIK3CA wild-type USC but not in HER2-amplified/PIK3CA-mutated tumours. Conclusions: Oncogenic PIK3CA mutations are common in HER2/neu-amplified USC and may constitute a major mechanism of resistance to trastuzumab treatment. PMID:26325104

PIK3CA oncogenic mutations represent a major mechanism of resistance to trastuzumab in HER2/neu overexpressing uterine serous carcinomas.

PubMed

Black, Jonathan D; Lopez, Salvatore; Cocco, Emiliano; Bellone, Stefania; Altwerger, Gary; Schwab, Carlton L; English, Diana P; Bonazzoli, Elena; Predolini, Federica; Ferrari, Francesca; Ratner, Elena; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E; Santin, Alessandro D

2015-09-29

We evaluated the role of PIK3CA-mutations as mechanism of resistance to trastuzumab in primary HER2/neu-amplified uterine-serous-carcinoma (USC) cell lines. Fifteen whole-exome-sequenced USC cell lines were tested for HER2/neu-amplification and PIK3CA-mutations. Four HER2/neu-amplified USC (2-harbouring wild-type-PIK3CA-genes and 2-harbouring oncogenic-PIK3CA-mutations) were evaluated in in vitro dose-titration-proliferation-assays, cell-viability and HER2 and S6-protein-phosphorylation after exposure to trastuzumab. USC harbouring wild-type-PIK3CA were transfected with plasmids encoding oncogenic PIK3CA-mutations (i.e., H1047R/R93Q) and exposed to trastuzumab. Finally, trastuzumab efficacy was tested by using two USC xenograft mouse models. Seven out of fifteen (46%) of the USC cell lines were HER2/neu-amplified by fluorescence in situ hybridisation. Within these tumours four out of seven (57%) were found to harbour oncogenic PIK3CA-mutations vs two out of eight (25%) of the HER2/neu not amplified cell lines (P=0.01). HER2/neu-amplified/PIK3CA-mutated USC were highly resistant to trastuzumab when compared with HER2/neu-amplified/wild-type-PIK3CA cell lines (P=0.02). HER2/neu-amplified/PIK3CA wild-type cell lines transfected with oncogenic PIK3CA-mutations increased their resistance to trastuzumab (P<0.0001). Trastuzumab was effective in reducing tumour growth (P=0.001) and improved survival (P=0.0001) in mouse xenografts harbouring HER2-amplified/PIK3CA wild-type USC but not in HER2-amplified/PIK3CA-mutated tumours. Oncogenic PIK3CA mutations are common in HER2/neu-amplified USC and may constitute a major mechanism of resistance to trastuzumab treatment.

Genetic study of the BRAF gene reveals new variants and high frequency of the V600E mutation among Iranian ameloblastoma patients.

PubMed

Soltani, Maryam; Tabatabaiefar, Mohammad Amin; Mohsenifar, Zhaleh; Pourreza, Mohammad Reza; Moridnia, Abbas; Shariati, Laleh; Razavi, Seyyed Mohammad

2018-01-01

Ameloblastoma is a benign, slow-growing and locally invasive tumor. It is one of the most prevalent odontogenic tumors, with an incidence rate of 1% of all oral tumors and approximately 18% of odontogenic tumors. A group of genes have been investigated in patients with ameloblastoma. The BRAF V600E mutation has been implicated as the most common mutation in ameloblastoma. The presence or absence of this mutation has been associated with several clinicopathological properties, including location, age at diagnosis, histology, and prognosis. Although some populations have been investigated so far, little data are available on the Iranian population. The current research was launched to study the BRAF V600E mutation among a cohort of Iranian patients with ameloblastoma. In this clinicopathological and molecular biology study, a total of 19 formalin-fixed, paraffin-embedded tissues were studied. DNA extraction was performed, followed by PCR-sequencing of exons 10 and 15 of the BRAF gene to identify mutations. In silico analysis was performed for the identified variants. Results were analyzed by T test, Chi-square, and Fisher's exact test. Totally, 12 of 19 samples (63%) harbored the p. V600E hotspot mutation. In addition, we identified several variants, two of which were novel. The c.1769T>G (p. V590G) and c.1751C>T (p.L584F) as the novel variants showed a possible damaging effect by in silico analysis. No variant was found within exon 10. Our study confirms the role of BRAF mutations in ameloblastoma in the Iranian patients studied. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

[Clinical investigation and mutation analysis of a child with citrin deficiency complicated with purpura, convulsive seizures and methioninemia].

PubMed

Wen, Peng-qiang; Wang, Guo-bing; Chen, Zhan-ling; Liu, Xiao-hong; Cui, Dong; Shang, Yue; Li, Cheng-rong

2013-12-01

To analyze the clinical features and SLC25A13 gene mutations of a child with citrin deficiency complicated with purpura, convulsive seizures and methioninemia. The patient was subjected to physical examination and routine laboratory tests. Blood amino acids and acylcarnitines, and urine organic acids and galactose were analyzed respectively with tandem mass spectrometry and gas chromatographic mass spectrometry. SLC25A13 gene mutation screening was conducted by high resolution melt (HRM) analysis. The petechiae on the patient's face and platelet count (27×10(9)/L, reference range 100×10(9)/L-300×10(9)/L) supported the diagnosis of immunologic thrombocytopenic purpura (ITP). Laboratory tests found that the patient have abnormal coagulation, cardiac enzyme, liver function and liver enzymes dysfunction. Tandem mass spectrometry also found methionine to be increased (286 μmol/L, reference ranges 8-35 μmol/L). The patient did not manifest any galactosemia, citrullinemia and tyrosinemia. Analysis of SLC25A13 gene mutation found that the patient has carried IVS16ins3kb, in addition with abnormal HRM result for exon 6. Direct sequencing of exon 6 revealed a novel mutation c.495delA. The same mutation was not detected in 100 unrelated healthy controls. Further analysis of her family has confirmed that the c.495delA mutation has derived from her farther, and that the IVS16ins3kb was derived from her mother. The clinical features and metabolic spectrum of citrin deficiency can be variable. The poor prognosis and severity of clinical symptoms of the patient may be attributed to the novel c.495delA mutation.

Co-amplification at lower denaturation-temperature PCR combined with unlabled-probe high-resolution melting to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma cases.

PubMed

Wu, Jiong; Zhou, Yan; Zhang, Chun-Yan; Song, Bin-Bin; Wang, Bei-Li; Pan, Bai-Shen; Lou, Wen-Hui; Guo, Wei

2014-01-01

The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. It was found that the sensitivity of Sanger reached 0.5% with COLD- PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.

Hereditary Gastrointestinal Cancer Syndromes

PubMed Central

Lynch, Jane F.; Shaw, Trudy G.

2011-01-01

ABSTRACT The rapid growth of molecular genetics and its attendant germline mutation discoveries has enabled identification of persons who are at an inordinately high cancer risk and, therefore, ideal candidates for prevention. However, one must fully appreciate the extensive genotypic and phenotypic heterogeneity that exists in hereditary cancer. Once the causative germline mutation has been identified in a patient, high-risk members of the family can be similarly tested and identified and provided highly targeted surveillance and management opportunities. DNA testing can change the individual's presumed risk status and affect decision making by patients and their physicians regarding surveillance and management. Our purpose is to describe familial/hereditary cancers of the gastrointestinal tract, including familial Barrett's esophagus, hereditary diffuse gastric cancer, gastrointestinal stromal tumors, familial adenomatous polyposis and desmoid tumors, Lynch syndrome, small bowel cancer, and familial pancreatic cancer. We use our discussion of Lynch syndrome as a model for diagnostic and clinical translation strategies for all hereditary gastrointestinal tract cancers, which clearly can then be extended to cancer of all anatomic sites. Highly pertinent questions from the patient's perspective include the following: What kind of counseling will be provided to a patient with a Lynch syndrome mutation, and should that counseling be mandatory? Does the proband have the responsibility to inform relatives about the familial mutation, even if the relatives do not want to know whether they carry it? Is the patient is responsible for notifying family members that a parent or sibling has Lynch syndrome? Can notification be forced and, if so, under what circumstances? These questions point out the need for criteria regarding which family members to inform and how to inform them. PMID:22368732

20 ans après: a second mutation in MAOA identified by targeted high-throughput sequencing in a family with altered behavior and cognition

PubMed Central

Piton, Amélie; Poquet, Hélène; Redin, Claire; Masurel, Alice; Lauer, Julia; Muller, Jean; Thevenon, Julien; Herenger, Yvan; Chancenotte, Sophie; Bonnet, Marlène; Pinoit, Jean-Michel; Huet, Frédéric; Thauvin-Robinet, Christel; Jaeger, Anne-Sophie; Le Gras, Stéphanie; Jost, Bernard; Gérard, Bénédicte; Peoc'h, Katell; Launay, Jean-Marie; Faivre, Laurence; Mandel, Jean-Louis

2014-01-01

Intellectual disability (ID) is characterized by an extraordinary genetic heterogeneity, with >250 genes that have been implicated in monogenic forms of ID. Because this complexity precluded systematic testing for mutations and because clinical features are often non-specific, for some of these genes only few cases or families have been unambiguously documented. It is the case of the X-linked gene encoding monoamine oxidase A (MAOA), for which only one nonsense mutation has been identified in Brunner syndrome, characterized in a single family by mild non-dysmorphic ID and impulsive, violent and aggressive behaviors. We have performed targeted high-throughput sequencing of 220 genes, including MAOA, in patients with undiagnosed ID. We identified a c.797_798delinsTT (p.C266F) missense mutation in MAOA in a boy with autism spectrum disorder, attention deficit and autoaggressive behavior. Two maternal uncles carry the mutation and have severe ID, with a history of maltreatment in early childhood. This novel missense mutation decreases MAOA enzymatic activity, leading to abnormal levels of urinary monoamines. The identification of this new point mutation confirms, for the first time since 1993, the monogenic implication of the MAOA gene in ID of various degrees, autism and behavioral disturbances. The variable expressivity of the mutation observed in male patients of this family may involve gene–environment interactions, and the identification of a perturbation in monoamine metabolism should be taken into account when prescribing psychoactive drugs in such patients. PMID:24169519

20 ans après: a second mutation in MAOA identified by targeted high-throughput sequencing in a family with altered behavior and cognition.

PubMed

Piton, Amélie; Poquet, Hélène; Redin, Claire; Masurel, Alice; Lauer, Julia; Muller, Jean; Thevenon, Julien; Herenger, Yvan; Chancenotte, Sophie; Bonnet, Marlène; Pinoit, Jean-Michel; Huet, Frédéric; Thauvin-Robinet, Christel; Jaeger, Anne-Sophie; Le Gras, Stéphanie; Jost, Bernard; Gérard, Bénédicte; Peoc'h, Katell; Launay, Jean-Marie; Faivre, Laurence; Mandel, Jean-Louis

2014-06-01

Intellectual disability (ID) is characterized by an extraordinary genetic heterogeneity, with >250 genes that have been implicated in monogenic forms of ID. Because this complexity precluded systematic testing for mutations and because clinical features are often non-specific, for some of these genes only few cases or families have been unambiguously documented. It is the case of the X-linked gene encoding monoamine oxidase A (MAOA), for which only one nonsense mutation has been identified in Brunner syndrome, characterized in a single family by mild non-dysmorphic ID and impulsive, violent and aggressive behaviors. We have performed targeted high-throughput sequencing of 220 genes, including MAOA, in patients with undiagnosed ID. We identified a c.797_798delinsTT (p.C266F) missense mutation in MAOA in a boy with autism spectrum disorder, attention deficit and autoaggressive behavior. Two maternal uncles carry the mutation and have severe ID, with a history of maltreatment in early childhood. This novel missense mutation decreases MAOA enzymatic activity, leading to abnormal levels of urinary monoamines. The identification of this new point mutation confirms, for the first time since 1993, the monogenic implication of the MAOA gene in ID of various degrees, autism and behavioral disturbances. The variable expressivity of the mutation observed in male patients of this family may involve gene-environment interactions, and the identification of a perturbation in monoamine metabolism should be taken into account when prescribing psychoactive drugs in such patients.

Mutations in the mitochondrial thioredoxin reductase gene TXNRD2 cause dilated cardiomyopathy.

PubMed

Sibbing, Dirk; Pfeufer, Arne; Perisic, Tamara; Mannes, Alexander M; Fritz-Wolf, Karin; Unwin, Sarah; Sinner, Moritz F; Gieger, Christian; Gloeckner, Christian Johannes; Wichmann, Heinz-Erich; Kremmer, Elisabeth; Schäfer, Zasie; Walch, Axel; Hinterseer, Martin; Näbauer, Michael; Kääb, Stefan; Kastrati, Adnan; Schömig, Albert; Meitinger, Thomas; Bornkamm, Georg W; Conrad, Marcus; von Beckerath, Nicolas

2011-05-01

Cardiac energy requirement is met to a large extent by oxidative phosphorylation in mitochondria that are highly abundant in cardiac myocytes. Human mitochondrial thioredoxin reductase (TXNRD2) is a selenocysteine-containing enzyme essential for mitochondrial oxygen radical scavenging. Cardiac-specific deletion of Txnrd2 in mice results in dilated cardiomyopathy (DCM). The aim of this study was to investigate whether TXNRD2 mutations explain a fraction of monogenic DCM cases. Sequencing and subsequent genotyping of TXNRD2 in patients diagnosed with DCM (n = 227) and in DCM-free (n = 683) individuals from the general population sample KORA S4 was performed. The functional impact of observed mutations on Txnrd2 function was tested in mouse fibroblasts. We identified two novel amino acid residue-altering TXNRD2 mutations [175G > A (Ala59Thr) and 1124G > A (Gly375Arg)] in three heterozygous carriers among 227 patients that were not observed in the 683 DCM-free individuals. Both DCM-associated mutations result in amino acid substitutions of highly conserved residues in helices contributing to the flavin-adenine dinucleotide (FAD)-binding domain of TXNRD2. Functional analysis of both mutations in Txnrd2(-/-) mouse fibroblasts revealed that contrasting to wild-type (wt) Txnrd2, neither mutant did restore Txnrd2 function. Mutants even impaired the survival of Txnrd2 wt cells under oxidative stress by a dominant-negative mechanism. For the first time, we describe mutations in DCM patients in a gene involved in the regulation of cellular redox state. TXNRD2 mutations may explain a fraction of human DCM disease burden.

Cost-effectiveness analysis of EGFR mutation testing in patients with non-small cell lung cancer (NSCLC) with gefitinib or carboplatin-paclitaxel.

PubMed

Arrieta, Oscar; Anaya, Pablo; Morales-Oyarvide, Vicente; Ramírez-Tirado, Laura Alejandra; Polanco, Ana C

2016-09-01

Assess the cost-effectiveness of an EGFR-mutation testing strategy for advanced NSCLC in first-line therapy with either gefitinib or carboplatin-paclitaxel in Mexican institutions. Cost-effectiveness analysis using a discrete event simulation (DES) model to simulate two therapeutic strategies in patients with advanced NSCLC. Strategy one included patients tested for EGFR-mutation and therapy given accordingly. Strategy two included chemotherapy for all patients without testing. All results are presented in 2014 US dollars. The analysis was made with data from the Mexican frequency of EGFR-mutation. A univariate sensitivity analysis was conducted on EGFR prevalence. Progression-free survival (PFS) transition probabilities were estimated on data from the IPASS and simulated with a Weibull distribution, run with parallel trials to calculate a probabilistic sensitivity analysis. PFS of patients in the testing strategy was 6.76 months (95 % CI 6.10-7.44) vs 5.85 months (95 % CI 5.43-6.29) in the non-testing group. The one-way sensitivity analysis showed that PFS has a direct relationship with EGFR-mutation prevalence, while the ICER and testing cost have an inverse relationship with EGFR-mutation prevalence. The probabilistic sensitivity analysis showed that all iterations had incremental costs and incremental PFS for strategy 1 in comparison with strategy 2. There is a direct relationship between the ICER and the cost of EGFR testing, with an inverse relationship with the prevalence of EGFR-mutation. When prevalence is >10 % ICER remains constant. This study could impact Mexican and Latin American health policies regarding mutation detection testing and treatment for advanced NSCLC.

Impact of genetic risk information and type of disease on perceived risk, anticipated affect, and expected consequences of genetic tests.

PubMed

Cameron, Linda D; Sherman, Kerry A; Marteau, Theresa M; Brown, Paul M

2009-05-01

Genetic tests vary in their prediction of disease occurrence, with some mutations conferring relatively low risk and others indicating near certainty. The authors assessed how increments in absolute risk of disease influence risk perceptions, interest, and expected consequences of genetic tests for diseases of varying severity. Adults (N = 752), recruited from New Zealand, Australia, and the United Kingdom for an online analogue study, were randomly assigned to receive information about a test of genetic risk for diabetes, heart disease, colon cancer, or lung cancer. The lifetime risk varied across conditions by 10% increments, from 20% to 100%. Participants completed measures of perceived likelihood of disease for individuals with mutations, risk-related affect, interest, and testing consequences. Analyses revealed two increment clusters yielding differences in likelihood perceptions: A "moderate-risk" cluster (20%-70%), and a "high-risk" cluster (80%-100%). Risk increment influenced anticipated worry, feelings of risk, testing-induced distress, and family obligations, with nonlinear patterns including disproportionately high responses for the 50% increment. Risk increment did not alter testing interest or perceived benefits. These patterns of effects held across the four diseases. Magnitude of risk from genetic testing has a nonlinear influence on risk-related appraisals and affect but is unrelated to test interest.

Mutational spectra of the lacI transgene isolated from Big Blue{reg_sign} mice exposed to three carcinogenic aromatic amines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Staedtler, F.; Locher, F.; Sreenan, G.

1997-10-01

In order to evaluate the in vivo genotoxic potential of three putative genotoxic mouse liver carcinogens, high doses of 4-chloro-o-phenylenediamine, 2-nitro-p-phenylenediamine and 2, 4-diaminotoluene were tested short term in the Big Blue{reg_sign} transgenic mouse mutation assay. Small statistically significant increases in the lacI mutant frequencies in the liver by factors 1.7 to 2.0 were found. A representative number of 347 lacI mutants isolated from liver tissue of male and female animals were analyses by DNA sequencing. The mutational spectra were examined with the Adams-Skopek algorithm. The spontaneous mutational spectra from untreated male and female animals were similar and consistent withmore » spectral Big Blue{reg_sign} control data stored in the lacI database. Most of the background mutations were located in the 5{prime} portion of the coding region of the lacI gene. Single base substitutions were most prominent. G:C to A:T transitions and G:C to T:A transversions occurred predominatly and were preferentially located at CpG sites. Despite the increases observed in the mutant frequencies of the treated animals, the corresponding mutational spectra did not differ from the controls. However, it is possible that certain classes of point mutations were substantially increased but not detected due to the limited number of sequenced mutants. In two animals treated with 2, 4- diaminotoluene unusually high mutant frequencies and the multiple occurrence of certain mutations in the liver was observed. From one of these animals six lacI mutants isolated from colon tissue were all different. Since 2, 4-diaminotoluene was shown to induce liver cell proliferation these results may reflect clonal expansion of single mutated liver cells.« less

Clarithromycin-Based Triple Therapy is Still Useful as an Initial Treatment for Helicobacter pylori Infection in the Dominican Republic.

PubMed

Miftahussurur, Muhammad; Cruz, Modesto; Subsomwong, Phawinee; Jiménez Abreu, José A; Hosking, Celso; Nagashima, Hiroyuki; Akada, Junko; Yamaoka, Yoshio

2017-05-01

Abstract Helicobacter pylori antibiotic susceptibility in the Dominican Republic has not been monitored. We assessed H. pylori antibiotic susceptibility in the Dominican Republic, and analyzed H. pylori mutations associated with antibiotic resistance. We recruited 158 dyspeptic patients in Santo Domingo and used agar dilution to test susceptibility to five antibiotics. Polymerase chain reaction-based sequencing was used to assess gyrA , gyrB , rdxA , frxA , and 23S rRNA mutations; next-generation sequencing was used to identify other metronidazole resistance-associated genes. Among 64 H. pylori strains isolated, we identified two (3.1%), one (1.6%), and no strains with clarithromycin, amoxicillin, and tetracycline resistance, respectively. Moreover, high frequency of metronidazole resistance (53/64, 82.8%) was observed, whereas levofloxacin resistance is emerging (23/64, 35.9%). We identified many rdxA and frxA mutations in metronidazole-resistant strains, but no synergistic effect was apparent. We revealed novel mutations in dppA , dppB , fdxA , and fdxB , irrespective of rdxA and frxA mutations. Novel mutations at Ser-14 of trx1 and Arg-221 of dapF were associated with different levels of metronidazole resistance. Most levofloxacin-resistant strains had a substitution at Asn-87 of gyrA , including the strain with the highest levofloxacin resistance, whereas only three substitutions were found at Ser-479 of gyrB with no synergistic effect. Besides the 23S rRNA A2142G mutation, we observed another mutation at T1958G in both clarithromycin-resistant strains. We confirmed high metronidazole and levofloxacin resistance associated with genetic mutations in the Dominican Republic. However, prevalence of clarithromycin resistance was low, suggesting that standard clarithromycin-based triple therapy remains useful as initial treatment of H. pylori infection.

Clarithromycin-Based Triple Therapy is Still Useful as an Initial Treatment for Helicobacter pylori Infection in the Dominican Republic

PubMed Central

Miftahussurur, Muhammad; Cruz, Modesto; Subsomwong, Phawinee; Jiménez Abreu, José A.; Hosking, Celso; Nagashima, Hiroyuki; Akada, Junko; Yamaoka, Yoshio

2017-01-01

Helicobacter pylori antibiotic susceptibility in the Dominican Republic has not been monitored. We assessed H. pylori antibiotic susceptibility in the Dominican Republic, and analyzed H. pylori mutations associated with antibiotic resistance. We recruited 158 dyspeptic patients in Santo Domingo and used agar dilution to test susceptibility to five antibiotics. Polymerase chain reaction–based sequencing was used to assess gyrA, gyrB, rdxA, frxA, and 23S rRNA mutations; next-generation sequencing was used to identify other metronidazole resistance–associated genes. Among 64 H. pylori strains isolated, we identified two (3.1%), one (1.6%), and no strains with clarithromycin, amoxicillin, and tetracycline resistance, respectively. Moreover, high frequency of metronidazole resistance (53/64, 82.8%) was observed, whereas levofloxacin resistance is emerging (23/64, 35.9%). We identified many rdxA and frxA mutations in metronidazole-resistant strains, but no synergistic effect was apparent. We revealed novel mutations in dppA, dppB, fdxA, and fdxB, irrespective of rdxA and frxA mutations. Novel mutations at Ser-14 of trx1 and Arg-221 of dapF were associated with different levels of metronidazole resistance. Most levofloxacin-resistant strains had a substitution at Asn-87 of gyrA, including the strain with the highest levofloxacin resistance, whereas only three substitutions were found at Ser-479 of gyrB with no synergistic effect. Besides the 23S rRNA A2142G mutation, we observed another mutation at T1958G in both clarithromycin-resistant strains. We confirmed high metronidazole and levofloxacin resistance associated with genetic mutations in the Dominican Republic. However, prevalence of clarithromycin resistance was low, suggesting that standard clarithromycin-based triple therapy remains useful as initial treatment of H. pylori infection. PMID:28193745

Significant clinical impact of recurrent BRCA1 and BRCA2 mutations in Mexico.

PubMed

Villarreal-Garza, Cynthia; Alvarez-Gómez, Rosa María; Pérez-Plasencia, Carlos; Herrera, Luis A; Herzog, Josef; Castillo, Danielle; Mohar, Alejandro; Castro, Clementina; Gallardo, Lenny N; Gallardo, Dolores; Santibáñez, Miguel; Blazer, Kathleen R; Weitzel, Jeffrey N

2015-02-01

Frequent recurrent mutations in the breast and ovarian cancer susceptibility (BRCA) genes BRCA1 and BRCA2 among Hispanics, including a large rearrangement Mexican founder mutation (BRCA1 exon 9-12 deletion [ex9-12del]), suggest that an ancestry-informed BRCA-testing strategy could reduce disparities and promote cancer prevention by enabling economic screening for hereditary breast and ovarian cancer in Mexico. In a multistage approach, 188 patients with cancer who were unselected for family cancer history (92 with ovarian cancer and 96 with breast cancer) were screened for BRCA mutations using a Hispanic mutation panel (HISPANEL) of 115 recurrent mutations in a multiplex assay (114 were screened on a mass spectroscopy platform, and a polymerase chain reaction assay was used to screen for the BRCA1 ex9-12del mutation). This was followed by sequencing of all BRCA exons and adjacent intronic regions and a BRCA1 multiplex ligation-dependent probe amplification assay (MLPA) for HISPANEL-negative patients. BRCA mutation prevalence was calculated and correlated with histology and tumor receptor status, and HISPANEL sensitivity was estimated. BRCA mutations were detected in 26 of 92 patients (28%) with ovarian cancer, in 14 of 96 patients (15%) with breast cancer overall, and in 9 of 33 patients (27%) who had tumors that were negative for estrogen receptor, progesterone receptor, and human epithelial growth factor 2 (triple-negative breast cancer). Most patients with breast cancer were diagnosed with locally advanced disease. The Mexican founder mutation (BRCA1 ex9-12del) accounted for 35% of BRCA-associated ovarian cancers and 29% of BRCA-associated breast cancers. At 2% of the sequencing and MLPA cost, HISPANEL detected 68% of all BRCA mutations. In this study, a remarkably high prevalence of BRCA mutations was observed among patients with ovarian cancer and breast cancer who were not selected for family history, and the BRCA1 ex9-12del mutation explained 33% of the total. The remarkable frequency of BRCA1 ex9-12del in Mexico City supports a nearby origin of this Mexican founder mutation and may constitute a regional public health problem. The HISPANEL mutation panel presents a translational opportunity for cost-effective genetic testing to enable breast and ovarian cancer prevention. © 2014 American Cancer Society.

Tumor mismatch repair immunohistochemistry and DNA MLH1 methylation testing of patients with endometrial cancer diagnosed at age younger than 60 years optimizes triage for population-level germline mismatch repair gene mutation testing.

PubMed

Buchanan, Daniel D; Tan, Yen Y; Walsh, Michael D; Clendenning, Mark; Metcalf, Alexander M; Ferguson, Kaltin; Arnold, Sven T; Thompson, Bryony A; Lose, Felicity A; Parsons, Michael T; Walters, Rhiannon J; Pearson, Sally-Ann; Cummings, Margaret; Oehler, Martin K; Blomfield, Penelope B; Quinn, Michael A; Kirk, Judy A; Stewart, Colin J; Obermair, Andreas; Young, Joanne P; Webb, Penelope M; Spurdle, Amanda B

2014-01-10

Clinicopathologic data from a population-based endometrial cancer cohort, unselected for age or family history, were analyzed to determine the optimal scheme for identification of patients with germline mismatch repair (MMR) gene mutations. Endometrial cancers from 702 patients recruited into the Australian National Endometrial Cancer Study (ANECS) were tested for MMR protein expression using immunohistochemistry (IHC) and for MLH1 gene promoter methylation in MLH1-deficient cases. MMR mutation testing was performed on germline DNA of patients with MMR-protein deficient tumors. Prediction of germline mutation status was compared for combinations of tumor characteristics, age at diagnosis, and various clinical criteria (Amsterdam, Bethesda, Society of Gynecologic Oncology, ANECS). Tumor MMR-protein deficiency was detected in 170 (24%) of 702 cases. Germline testing of 158 MMR-deficient cases identified 22 truncating mutations (3% of all cases) and four unclassified variants. Tumor MLH1 methylation was detected in 99 (89%) of 111 cases demonstrating MLH1/PMS2 IHC loss; all were germline MLH1 mutation negative. A combination of MMR IHC plus MLH1 methylation testing in women younger than 60 years of age at diagnosis provided the highest positive predictive value for the identification of mutation carriers at 46% versus ≤ 41% for any other criteria considered. Population-level identification of patients with MMR mutation-positive endometrial cancer is optimized by stepwise testing for tumor MMR IHC loss in patients younger than 60 years, tumor MLH1 methylation in individuals with MLH1 IHC loss, and germline mutations in patients exhibiting loss of MSH6, MSH2, or PMS2 or loss of MLH1/PMS2 with absence of MLH1 methylation.

Germline mutation prevalence in individuals with pancreatic cancer and a history of previous malignancy.

PubMed

Dudley, Beth; Karloski, Eve; Monzon, Federico A; Singhi, Aatur D; Lincoln, Stephen E; Bahary, Nathan; Brand, Randall E

2018-04-15

Approximately 10% of pancreatic adenocarcinoma (PC) cases are attributed to hereditary causes. Individuals with PC and a personal history of another cancer associated with hereditary breast and ovarian cancer (HBOC) or Lynch syndrome (LS) may be more likely to carry germline mutations. Participants with PC and a history of cancer were selected from a pancreatic disease registry. Of 1296 individuals with PC, 149 had a relevant history of cancer. If banked DNA was available, a multigene panel was performed for individuals who had not 1) previously had a mutation identified through clinical testing or 2) undergone clinical multigene panel testing with no mutations detected. Twenty-two of 124 individuals with PC and another HBOC- or LS-related cancer who underwent genetic testing had a mutation identified in a PC susceptibility gene (18%). If prostate cancer is excluded, the mutation prevalence increased to 23% (21/93). Mutation carriers were more likely to have more than 1 previous cancer diagnosis (P = .001), to have had clinical genetic testing (P = .001), and to meet National Comprehensive Cancer Network (NCCN) genetic testing criteria (P < .001). Approximately 23% of mutation carriers did not meet NCCN HBOC or LS testing guidelines based on their personal cancer history and reported cancer history in first-degree relatives. At least 18% of individuals with PC and a personal history of other HBOC- or LS-related cancers carry mutations in a PC susceptibility gene based on our data, suggesting that criteria for genetic testing in individuals with PC should include consideration of previous cancer history. Cancer 2018;124:1691-700. © 2018 American Cancer Society. © 2018 American Cancer Society.

Germline mosaicism of PHOX2B mutation accounts for familial recurrence of congenital central hypoventilation syndrome (CCHS).

PubMed

Rand, Casey M; Yu, Min; Jennings, Lawrence J; Panesar, Kelvin; Berry-Kravis, Elizabeth M; Zhou, Lili; Weese-Mayer, Debra E

2012-09-01

Congenital central hypoventilation syndrome (CCHS), a rare disorder characterized by alveolar hypoventilation and autonomic dysregulation, is caused by mutations in the PHOX2B gene. Most mutations occur de novo, but recent evidence suggests that up to 25% are inherited from asymptomatic parents with somatic mosaicism for these mutations. However, to date, germline mosaicism has not been reported. This report describes a family with recurrence of PHOX2B mutation-confirmed CCHS due to germline mosaicism. The first occurrence was a baby girl, noted on day 2 of life to have multiple episodes of apnea, bradycardia, and cyanosis while breathing room air. PHOX2B gene testing confirmed the diagnosis of CCHS with a heterozygous polyalanine repeat expansion mutation (PARM); genotype 20/27 (normal 20/20). Both parents tested negative for this mutation using fragment analysis (limit of detection<1%). Upon subsequent pregnancy [paternity confirmed using short tandem repeat (STR) analysis], amniocentesis testing identified the PHOX2B 20/27 genotype, confirmed with repeat testing. Elective abortion was performed at 21.5 weeks gestation. Testing of abortus tissue confirmed amniocentesis testing. The PHOX2B 20/27 expansion was not observed in a paternal sperm sample. This case represents the first reported family with recurrence of PHOX2B mutation-confirmed CCHS without detection of a parental carrier state or mosaicism, confirming the previously hypothesized possibility of germline mosaicism for PHOX2B mutations. This is an important finding for genetic counseling of CCHS families, suggesting that even if somatic mosaicism is not detected in parental samples, there is still reason for careful genetic counseling and consideration of prenatal testing during subsequent pregnancies. Copyright © 2012 Wiley Periodicals, Inc.

Low Prevalence of CHEK2 Gene Mutations in Multiethnic Cohorts of Breast Cancer Patients in Malaysia

PubMed Central

Mohamad, Suriati; Isa, Nurismah Md; Muhammad, Rohaizak; Emran, Nor Aina; Kitan, Nor Mayah; Kang, Peter; Kang, In Nee; Taib, Nur Aishah Mohd; Teo, Soo Hwang; Akmal, Sharifah Noor

2015-01-01

CHEK2 is a protein kinase that is involved in cell-cycle checkpoint control after DNA damage. Germline mutations in CHEK2 gene have been associated with increase in breast cancer risk. The aim of this study is to identify the CHEK2 gene germline mutations among high-risk breast cancer patients and its contribution to the multiethnic population in Malaysia. We screened the entire coding region of CHEK2 gene on 59 high-risk breast cancer patients who tested negative for BRCA1/2 germline mutations from UKM Medical Centre (UKMMC), Hospital Kuala Lumpur (HKL) and Hospital Putrajaya (HPJ). Sequence variants identified were screened further in case-control cohorts consisting of 878 unselected invasive breast cancer patients (180 Malays, 526 Chinese and 172 Indian) and 270 healthy individuals (90 Malays, 90 Chinese and 90 Indian). By screening the entire coding region of the CHEK2 gene, two missense mutations, c.480A>G (p.I160M) and c.538C>T (p.R180C) were identified in two unrelated patients (3.4%). Further screening of these missense mutations on the case-control cohorts unveiled the variant p.I160M in 2/172 (1.1%) Indian cases and 1/90 (1.1%) Indian control, variant p.R180C in 2/526 (0.38%) Chinese cases and 0/90 Chinese control, and in 2/180 (1.1%) of Malay cases and 1/90 (1.1%) of Malay control. The results of this study suggest that CHEK2 mutations are rare among high-risk breast cancer patients and may play a minor contributing role in breast carcinogenesis among Malaysian population. PMID:25629968

Personalized Oncology Through Integrative High-Throughput Sequencing: A Pilot Study

PubMed Central

Roychowdhury, Sameek; Iyer, Matthew K.; Robinson, Dan R.; Lonigro, Robert J.; Wu, Yi-Mi; Cao, Xuhong; Kalyana-Sundaram, Shanker; Sam, Lee; Balbin, O. Alejandro; Quist, Michael J.; Barrette, Terrence; Everett, Jessica; Siddiqui, Javed; Kunju, Lakshmi P.; Navone, Nora; Araujo, John C.; Troncoso, Patricia; Logothetis, Christopher J.; Innis, Jeffrey W.; Smith, David C.; Lao, Christopher D.; Kim, Scott Y.; Roberts, J. Scott; Gruber, Stephen B.; Pienta, Kenneth J.; Talpaz, Moshe; Chinnaiyan, Arul M.

2012-01-01

Individual cancers harbor a set of genetic aberrations that can be informative for identifying rational therapies currently available or in clinical trials. We implemented a pilot study to explore the practical challenges of applying high-throughput sequencing in clinical oncology. We enrolled patients with advanced or refractory cancer who were eligible for clinical trials. For each patient, we performed whole-genome sequencing of the tumor, targeted whole-exome sequencing of tumor and normal DNA, and transcriptome sequencing (RNA-Seq) of the tumor to identify potentially informative mutations in a clinically relevant time frame of 3 to 4 weeks. With this approach, we detected several classes of cancer mutations including structural rearrangements, copy number alterations, point mutations, and gene expression alterations. A multidisciplinary Sequencing Tumor Board (STB) deliberated on the clinical interpretation of the sequencing results obtained. We tested our sequencing strategy on human prostate cancer xenografts. Next, we enrolled two patients into the clinical protocol and were able to review the results at our STB within 24 days of biopsy. The first patient had metastatic colorectal cancer in which we identified somatic point mutations in NRAS, TP53, AURKA, FAS, and MYH11, plus amplification and overexpression of cyclin-dependent kinase 8 (CDK8). The second patient had malignant melanoma, in which we identified a somatic point mutation in HRAS and a structural rearrangement affecting CDKN2C. The STB identified the CDK8 amplification and Ras mutation as providing a rationale for clinical trials with CDK inhibitors or MEK (mitogenactivated or extracellular signal–regulated protein kinase kinase) and PI3K (phosphatidylinositol 3-kinase) inhibitors, respectively. Integrative high-throughput sequencing of patients with advanced cancer generates a comprehensive, individual mutational landscape to facilitate biomarker-driven clinical trials in oncology. PMID:22133722

New MCM8 mutation associated with premature ovarian insufficiency and chromosomal instability in a highly consanguineous Tunisian family.

PubMed

Bouali, Nouha; Francou, Bruno; Bouligand, Jérôme; Imanci, Dilek; Dimassi, Sarra; Tosca, Lucie; Zaouali, Monia; Mougou, Soumaya; Young, Jacques; Saad, Ali; Guiochon-Mantel, Anne

2017-10-01

To identify the gene(s) involved in the etiology of premature ovarian insufficiency in a highly consanguineous Tunisian family. Genetic analysis of a large consanguineous family with several affected siblings. University hospital-based cytogenetics and molecular genetics laboratories. A highly consanguineous Tunisian family with several affected siblings born to healthy second-degree cousins. None. Targeted exome sequencing was performed by next-generation sequencing for affected family members. Mutations were validated by Sanger sequencing. Functional experiments were performed to explore the deleterious effects of the identified mutation. DNA damage was induced by increasing mitomycin C (MMC) concentrations on cultured peripheral lymphocytes. Analysis of the next-generation sequencing data revealed a new homozygous missense mutation in the minichromosome maintenance 8 gene (MCM8).This homozygous mutation (c. 482A>C; p.His161Pro) was predicted to be deleterious and segregated with the disease in the family. MCM8 participates in homologous recombination during meiosis and DNA double-stranded break repair by dimerizing with MCM9. Mcm8 knock out results in an early block in follicle development and small gonads. Given this, we tested the chromosomal breakage repair capacity of homozygous and heterozygous MCM8 p.His161Pro mutation on cultured peripheral lymphocytes exposed to increasing MMC concentrations. We found that chromosomal breakage after MMC exposure was significantly higher in cells from homozygously affected individuals than in those from a healthy control. Our findings provide additional support to the view that MCM8 mutations are involved in the primary ovarian insufficiency phenotype. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Low prevalence of CHEK2 gene mutations in multiethnic cohorts of breast cancer patients in Malaysia.

PubMed

Mohamad, Suriati; Isa, Nurismah Md; Muhammad, Rohaizak; Emran, Nor Aina; Kitan, Nor Mayah; Kang, Peter; Kang, In Nee; Taib, Nur Aishah Mohd; Teo, Soo Hwang; Akmal, Sharifah Noor

2015-01-01

CHEK2 is a protein kinase that is involved in cell-cycle checkpoint control after DNA damage. Germline mutations in CHEK2 gene have been associated with increase in breast cancer risk. The aim of this study is to identify the CHEK2 gene germline mutations among high-risk breast cancer patients and its contribution to the multiethnic population in Malaysia. We screened the entire coding region of CHEK2 gene on 59 high-risk breast cancer patients who tested negative for BRCA1/2 germline mutations from UKM Medical Centre (UKMMC), Hospital Kuala Lumpur (HKL) and Hospital Putrajaya (HPJ). Sequence variants identified were screened further in case-control cohorts consisting of 878 unselected invasive breast cancer patients (180 Malays, 526 Chinese and 172 Indian) and 270 healthy individuals (90 Malays, 90 Chinese and 90 Indian). By screening the entire coding region of the CHEK2 gene, two missense mutations, c.480A>G (p.I160M) and c.538C>T (p.R180C) were identified in two unrelated patients (3.4%). Further screening of these missense mutations on the case-control cohorts unveiled the variant p.I160M in 2/172 (1.1%) Indian cases and 1/90 (1.1%) Indian control, variant p.R180C in 2/526 (0.38%) Chinese cases and 0/90 Chinese control, and in 2/180 (1.1%) of Malay cases and 1/90 (1.1%) of Malay control. The results of this study suggest that CHEK2 mutations are rare among high-risk breast cancer patients and may play a minor contributing role in breast carcinogenesis among Malaysian population.

Results of the First Italian External Quality Assurance Scheme for somatic EGFR mutation testing in non-small-cell lung cancer.

PubMed

Normanno, Nicola; Pinto, Carmine; Taddei, Gianluigi; Gambacorta, Marcello; Castiglione, Francesca; Barberis, Massimo; Clemente, Claudio; Marchetti, Antonio

2013-06-01

The Italian Association of Medical Oncology (AIOM) and the Italian Society of Pathology and Cytology organized an external quality assessment (EQA) scheme for EGFR mutation testing in non-small-cell lung cancer. Ten specimens, including three small biopsies with known epidermal growth factor receptor (EGFR) mutation status, were validated in three referral laboratories and provided to 47 participating centers. The participants were requested to perform mutational analysis, using their usual method, and to submit results within a 4-week time frame. According to a predefined scoring system, two points were assigned to correct genotype and zero points to false-negative or false-positive results. The threshold to pass the EQA was set at higher than 18 of 20 points. Two rounds were preplanned. All participating centers submitted the results within the time frame. Polymerase chain reaction (PCR)/sequencing was the main methodology used (n = 37 laboratories), although a few centers did use pyrosequencing (n = 8) or real-time PCR (n = 2). A significant number of analytical errors were observed (n = 20), with a high frequency of false-positive results (n = 16). The lower scores were obtained for the small biopsies. Fourteen of 47 centers (30%) that did not pass the first round, having a score less than or equal to 18 points, used PCR/sequencing, whereas 10 of 10 laboratories, using pyrosequencing or real-time PCR, passed the first round. Eight laboratories passed the second round. Overall, 41of 47 centers (87%) passed the EQA. The results of the EQA for EGFR testing in non-small-cell lung cancer suggest that good quality EGFR mutational analysis is performed in Italian laboratories, although differences between testing methods were observed, especially for small biopsies.

KRAS Testing

PubMed Central

Shackelford, Rodney E.; Whitling, Nicholas A.; McNab, Patricia; Japa, Shanker

2012-01-01

Activating point mutations in codons 12, 13, and 61 of the KRAS proto-oncogene are common in colorectal, non–small cell lung, pancreatic, and thyroid cancers. Constitutively activated KRAS mutations are strongly associated with a resistance to anti–epidermal growth factor receptor (EGFR) therapies, such as panitumumab and cetuximab used for treating metastatic colorectal carcinoma and EGFR tyrosine inhibitors used for advanced non–small cell lung cancers. Since anti-EGFR therapies are costly and may exert deleterious effects on individuals without activating mutations, KRAS mutation testing is recommended prior to the initiation of anti-EGFR therapy for these malignancies. The goal of this review is to summarize the KRAS mutation testing methods. Testing is now routinely requested in the clinical practice to provide data to assign the most appropriate anticancer chemotherapy for each given patient. Review of the most relevant literature was performed. Several areas were considered: ordering of the test, selection of the sample to be tested, and review of the testing methodologies. We found that several different methods are used for clinical KRAS mutation testing. Each of the methodologies is described, and information is provided about their performance, cost, turnaround times, detection limits, sensitivities, and specificities. We also provided “tips” for the appropriate selection and preparation of the sample to be tested. This is an important aspect of KRAS testing for clinical use, as the results of the test will affect clinical decisions with consequences for the patient. PMID:23264846

A GYS1 gene mutation is highly associated with polysaccharide storage myopathy in Cob Normand draught horses.

PubMed

Herszberg, B; McCue, M E; Larcher, T; Mata, X; Vaiman, A; Chaffaux, S; Chérel, Y; Valberg, S J; Mickelson, J R; Guérin, G

2009-02-01

Glycogen storage diseases or glycogenoses are inherited diseases caused by abnormalities of enzymes that regulate the synthesis or degradation of glycogen. Deleterious mutations in many genes of the glyco(geno)lytic or the glycogenesis pathways can potentially cause a glycogenosis, and currently mutations in fourteen different genes are known to cause animal or human glycogenoses, resulting in myopathies and/or hepatic disorders. The genetic bases of two forms of glycogenosis are currently known in horses. A fatal neonatal polysystemic type IV glycogenosis, inherited recessively in affected Quarter Horse foals, is due to a mutation in the glycogen branching enzyme gene (GBE1). A second type of glycogenosis, termed polysaccharide storage myopathy (PSSM), is observed in adult Quarter Horses and other breeds. A severe form of PSSM also occurs in draught horses. A mutation in the skeletal muscle glycogen synthase gene (GYS1) was recently reported to be highly associated with PSSM in Quarter Horses and Belgian draught horses. This GYS1 point mutation appears to cause a gain-of-function of the enzyme and to result in the accumulation of a glycogen-like, less-branched polysaccharide in skeletal muscle. It is inherited as a dominant trait. The aim of this work was to test for possible associations between genetic polymorphisms in four candidate genes of the glycogen pathway or the GYS1 mutation in Cob Normand draught horses diagnosed with PSSM by muscle biopsy.

Case-control analysis of truncating mutations in DNA damage response genes connects TEX15 and FANCD2 with hereditary breast cancer susceptibility.

PubMed

Mantere, Tuomo; Tervasmäki, Anna; Nurmi, Anna; Rapakko, Katrin; Kauppila, Saila; Tang, Jiangbo; Schleutker, Johanna; Kallioniemi, Anne; Hartikainen, Jaana M; Mannermaa, Arto; Nieminen, Pentti; Hanhisalo, Riitta; Lehto, Sini; Suvanto, Maija; Grip, Mervi; Jukkola-Vuorinen, Arja; Tengström, Maria; Auvinen, Päivi; Kvist, Anders; Borg, Åke; Blomqvist, Carl; Aittomäki, Kristiina; Greenberg, Roger A; Winqvist, Robert; Nevanlinna, Heli; Pylkäs, Katri

2017-04-06

Several known breast cancer susceptibility genes encode proteins involved in DNA damage response (DDR) and are characterized by rare loss-of-function mutations. However, these explain less than half of the familial cases. To identify novel susceptibility factors, 39 rare truncating mutations, identified in 189 Northern Finnish hereditary breast cancer patients in parallel sequencing of 796 DDR genes, were studied for disease association. Mutation screening was performed for Northern Finnish breast cancer cases (n = 578-1565) and controls (n = 337-1228). Mutations showing potential cancer association were analyzed in additional Finnish cohorts. c.7253dupT in TEX15, encoding a DDR factor important in meiosis, associated with hereditary breast cancer (p = 0.018) and likely represents a Northern Finnish founder mutation. A deleterious c.2715 + 1G > A mutation in the Fanconi anemia gene, FANCD2, was over two times more common in the combined Finnish hereditary cohort compared to controls. A deletion (c.640_644del5) in RNF168, causative for recessive RIDDLE syndrome, had high prevalence in majority of the analyzed cohorts, but did not associate with breast cancer. In conclusion, truncating variants in TEX15 and FANCD2 are potential breast cancer risk factors, warranting further investigations in other populations. Furthermore, high frequency of RNF168 c.640_644del5 indicates the need for its testing in Finnish patients with RIDDLE syndrome symptoms.

PIK3CA-associated developmental disorders exhibit distinct classes of mutations with variable expression and tissue distribution

PubMed Central

Timms, Andrew E.; Conti, Valerio; Girisha, Katta M.; Martin, Beth; Olds, Carissa; Collins, Sarah; Park, Kaylee; Carter, Melissa; Krägeloh-Mann, Inge; Chitayat, David; Parikh, Aditi Shah; Bradshaw, Rachael; Torti, Erin; Braddock, Stephen; Burke, Leah; Ghedia, Sondhya; Stephan, Mark; Stewart, Fiona; Prasad, Chitra; Napier, Melanie; Saitta, Sulagna; Straussberg, Rachel; Gabbett, Michael; O’Connor, Bridget C.; Yin, Lim Jiin; Lai, Angeline Hwei Meeng; Martin, Nicole; McKinnon, Margaret; Addor, Marie-Claude; Schwartz, Charles E.; Lanoel, Agustina; Conway, Robert L.; Devriendt, Koenraad; Tatton-Brown, Katrina; Pierpont, Mary Ella; Painter, Michael; Worgan, Lisa; Reggin, James; Hennekam, Raoul; Pritchard, Colin C.; Aracena, Mariana; Gripp, Karen W.; Cordisco, Maria; Van Esch, Hilde; Garavelli, Livia; Curry, Cynthia; Goriely, Anne; Kayserilli, Hulya; Shendure, Jay; Graham, John; Guerrini, Renzo; Dobyns, William B.

2016-01-01

Mosaicism is increasingly recognized as a cause of developmental disorders with the advent of next-generation sequencing (NGS). Mosaic mutations of PIK3CA have been associated with the widest spectrum of phenotypes associated with overgrowth and vascular malformations. We performed targeted NGS using 2 independent deep-coverage methods that utilize molecular inversion probes and amplicon sequencing in a cohort of 241 samples from 181 individuals with brain and/or body overgrowth. We identified PIK3CA mutations in 60 individuals. Several other individuals (n = 12) were identified separately to have mutations in PIK3CA by clinical targeted-panel testing (n = 6), whole-exome sequencing (n = 5), or Sanger sequencing (n = 1). Based on the clinical and molecular features, this cohort segregated into three distinct groups: (a) severe focal overgrowth due to low-level but highly activating (hotspot) mutations, (b) predominantly brain overgrowth and less severe somatic overgrowth due to less-activating mutations, and (c) intermediate phenotypes (capillary malformations with overgrowth) with intermediately activating mutations. Sixteen of 29 PIK3CA mutations were novel. We also identified constitutional PIK3CA mutations in 10 patients. Our molecular data, combined with review of the literature, show that PIK3CA-related overgrowth disorders comprise a discontinuous spectrum of disorders that correlate with the severity and distribution of mutations. PMID:27631024

Favorable outcome of patients with lung adenocarcinoma harboring POLE mutations and expressing high PD-L1.

PubMed

Liu, Liang; Ruiz, Jimmy; O'Neill, Stacey S; Grant, Stefan C; Petty, W Jeffrey; Yang, Meng; Chen, Kexin; Topaloglu, Umit; Pasche, Boris; Zhang, Wei

2018-04-12

Mutations in polymerase ε (POLE) confer favorable prognosis and outcomes in various cancer types, but their role in non-small cell lung cancer (NSCLC) is unknown. Utilizing the data of 513 patients with adenocarcinoma (LUAD) and 497 patients with squamous cell carcinoma (LUSC) from The Cancer Genome Atlas (TCGA) cohort, we tested the prognostic value of POLE mutations and programmed cell death ligand 1 (PD-L1) expression in the two main subtypes of NSCLC. POLE mutation is a favorable biomarker for the improved overall survival (OS) of the LUSC patients (P = 0.033, 28 mutant vs. 469 wildtype patients), but not that of the LUAD patients (P = 0.12, 31 mutant vs. 482 wildtype patients). POLE-mutant LUAD patients with high expression of PD-L1 (Mut-High, n = 6) exhibited improved OS (P = 0.024) when compared to POLE-mutant patients with low PD-L1 expression (Mut-Low, n = 24) and other patients without POLE mutation (n = 476). This benefit was not due to the high content of the tumor infiltrating lymphocytes. Instead, the antitumor immune response was activated in Mut-High patients so that these patients were likely responding more effectively to immuno-oncology (IO) treatments; whereas genes involved with metabolic pathways were enriched in Mut-Low group, which may cause the decreased OS of these patients. Our study sheds light on the molecular basis of NSCLC and adds to our understanding of responses to chemotherapy and IO therapy.

[Observation and analysis on mutation of routine STR locus].

PubMed

Li, Qiu-yang; Feng, Wei-jun; Yang, Qin-gen

2005-05-01

To observe and analyze the characteristic of mutation at STR locus. 27 mutant genes observed in 1211 paternity testing cases were checked by PAGE-silver stained and PowerPlex 16 System Kit and validated by sequencing. Mutant genes locate on 15 loci. The pattern of mutation was accord with stepwise mutation model. The mutation ratio of male-to-female was 8:1 and correlated to the age of father. Mutation rate is correlated to the geometric mean of the number of homogeneous repeats of locus. The higher the mean, the higher the mutation rate. These loci are not so appropriate for use in paternity testing.

Genetic testing in steroid-resistant nephrotic syndrome: why, who, when and how?

PubMed

Preston, Rebecca; Stuart, Helen M; Lennon, Rachel

2017-11-27

Steroid-resistant nephrotic syndrome (SRNS) is a common cause of chronic kidney disease in childhood and has a significant risk of rapid progression to end-stage renal disease. The identification of over 50 monogenic causes of SRNS has revealed dysfunction in podocyte-associated proteins in the pathogenesis of proteinuria, highlighting their essential role in glomerular function. Recent technological advances in high-throughput sequencing have enabled indication-driven genetic panel testing for patients with SRNS. The availability of genetic testing, combined with the significant phenotypic variability of monogenic SRNS, poses unique challenges for clinicians when directing genetic testing. This highlights the need for clear clinical guidelines that provide a systematic approach for mutational screening in SRNS. The likelihood of identifying a causative mutation is inversely related to age at disease onset and is increased with a positive family history or the presence of extra-renal manifestations. An unequivocal molecular diagnosis could allow for a personalised treatment approach with weaning of immunosuppressive therapy, avoidance of renal biopsy and provision of accurate, well-informed genetic counselling. Identification of novel causative mutations will continue to unravel the pathogenic mechanisms of glomerular disease and provide new insights into podocyte biology and glomerular function.

Wee-1 Kinase Inhibition Overcomes Cisplatin Resistance Associated with High-Risk TP53 Mutations in Head and Neck Cancer through Mitotic Arrest Followed by Senescence

PubMed Central

Osman, Abdullah A.; Monroe, Marcus M.; Ortega Alves, Marcus V.; Patel, Ameeta A.; Katsonis, Panagiotis; Fitzgerald, Alison L.; Neskey, David M.; Frederick, Mitchell J.; Woo, Sang Hyeok; Caulin, Carlos; Hsu, Teng-Kuei; McDonald, Thomas O.; Kimmel, Marek; Meyn, Raymond E.; Lichtarge, Olivier; Myers, Jeffrey N.

2015-01-01

Although cisplatin has played a role in “standard-of-care” multimodality therapy for patients with advanced squamous cell carcinoma of the head and neck (HNSCC), the rate of treatment failure remains particularly high for patients receiving cisplatin whose tumors have mutations in the TP53 gene. We found that cisplatin treatment of HNSCC cells with mutant TP53 leads to arrest of cells in the G2 phase of the cell cycle, leading us to hypothesize that the wee-1 kinase inhibitor MK-1775 would abrogate the cisplatin-induced G2 block and thereby sensitize isogenic HNSCC cells with mutant TP53 or lacking p53 expression to cisplatin. We tested this hypothesis using clonogenic survival assays, flow cytometry, and in vivo tumor growth delay experiments with an orthotopic nude mouse model of oral tongue cancer. We also used a novel TP53 mutation classification scheme to identify which TP53 mutations are associated with limited tumor responses to cisplatin treatment. Clonogenic survival analyses indicate that nanomolar concentration of MK-1775 sensitizes HNSCC cells with high-risk mutant p53 to cisplatin. Consistent with its ability to chemosensitize, MK-1775 abrogated the cisplatin-induced G2 block in p53-defective cells leading to mitotic arrest associated with a senescence-like phenotype. Furthermore, MK-1775 enhanced the efficacy of cisplatin in vivo in tumors harboring TP53 mutations. These results indicate that HNSCC cells expressing high-risk p53 mutations are significantly sensitized to cisplatin therapy by the selective wee-1 kinase inhibitor, supporting the clinical evaluation of MK-1775 in combination with cisplatin for the treatment of patients with TP53 mutant HNSCC. PMID:25504633

Efficient genome editing of wild strawberry genes, vector development and validation.

PubMed

Zhou, Junhui; Wang, Guoming; Liu, Zhongchi

2018-03-25

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is an effective genome editing tool for plant and animal genomes. However, there are still few reports on the successful application of CRISPR-Cas9 to horticultural plants, especially with regard to germ-line transmission of targeted mutations. Here, we report high-efficiency genome editing in the wild strawberry Fragaria vesca and its successful application to mutate the auxin biosynthesis gene TAA1 and auxin response factor 8 (ARF8). In our CRISPR system, the Arabidopsis U6 promoter AtU6-26 and the wild strawberry U6 promoter FveU6-2 were each used to drive the expression of sgRNA, and both promoters were shown to lead to high-efficiency genome editing in strawberry. To test germ-line transmission of the edited mutations and new mutations induced in the next generation, the progeny of the primary (T0) transgenic plants carrying the CRISPR construct was analysed. New mutations were detected in the progeny plants at a high efficiency, including large deletions between the two PAM sites. Further, T1 plants harbouring arf8 homozygous knockout mutations grew considerably faster than wild-type plants. The results indicate that our CRISPR vectors can be used to edit the wild strawberry genome at a high efficiency and that both sgRNA design and appropriate U6 promoters contribute to the success of genomic editing. Our results open up exciting opportunities for engineering strawberry and related horticultural crops to improve traits of economic importance. © 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Targeted treatment of mutated EGFR-expressing non-small-cell lung cancer: focus on erlotinib with companion diagnostics

PubMed Central

Karachaliou, Niki; Rosell, Rafael

2014-01-01

Deeper understanding of the pathobiology of non-small-cell lung cancer (NSCLC) has led to the development of small molecules that target genetic mutations known to play critical roles in the progression to metastatic disease. The discovery of epidermal growth factor receptor (EGFR) mutations in 15%–20% of lung adenocarcinomas and the associated response to EGFR tyrosine kinase inhibitors have provided a successful avenue of attack in late-stage adenocarcinomas. Use of the EGFR tyrosine kinase inhibitors gefitinib, erlotinib, and afatinib is limited to patients who have adenocarcinomas with known activating EGFR mutations. However, the EGFR mutation testing landscape is varied and includes many screening and targeted methods, each with its own benefits and limitations. These tests can simplify the drug discovery process, make clinical trials more efficient and informative, and individualize cancer therapy. In practice, the choice of method should be determined by the nature of the sample to be tested, the testing laboratory’s expertise and access to equipment, and whether the detection of only known activating EGFR mutations, or of all possible mutations, is required. Development of companion diagnostic tests for this identification is advancing; nevertheless, the use of such tests merits greater attention. PMID:28210145

Degenerative myelopathy in German Shepherd Dog: comparison of two molecular assays for the identification of the SOD1:c.118G>A mutation.

PubMed

Capucchio, Maria Teresa; Spalenza, Veronica; Biasibetti, Elena; Bottero, Maria Teresa; Rasero, Roberto; Dalmasso, Alessandra; Sacchi, Paola

2014-02-01

Degenerative myelopathy (DM) is a late-onset, slowly progressive degeneration of spinal cord white matter which is reported primarily in large breed dogs. The missense mutation SOD1:c.118G>A is associated with this pathology in several dog breeds, including the German Shepherd Dog (GSD). The aims of the present study were to develop a tool for the rapid screening of the SOD1 mutation site in dogs and to evaluate the association of the polymorphism with DM in the German Shepherd breed. Two different techniques were compared: a minisequencing test and a real-time pcr allelic discrimination assay. Both approaches resulted effective and efficient. A sample of 47 dogs were examined. Ten subjects presented the symptoms of the illness; for one of them the diagnosis was confirmed by postmortem investigations and it resulted to be an A/A homozygote. In another clinically suspected dog, heterozygote A/G, the histopathological examination of the medulla showed moderate axon and myelin degenerative changes. GSD shows a frequency of the mutant allele equal to 0.17, quite high being a high-risk allele. Because canine DM has a late onset in adulthood and homozygous mutant dogs are likely as fertile as other genotypes, the natural selection is mild and the mutant allele may reach high frequencies. A diagnostic test, easy to implement, may contribute to control the gene diffusion in populations. The SOD1:c.118G>A mutation could be a useful marker for breeding strategies intending to reduce the incidence of DM.

Novel missense mutations in MYO7A underlying postlingual high- or low-frequency non-syndromic hearing impairment in two large families from China.

PubMed

Sun, Yi; Chen, Jing; Sun, Hanjun; Cheng, Jing; Li, Jianzhong; Lu, Yu; Lu, Yanping; Jin, Zhanguo; Zhu, Yuhua; Ouyang, Xiaomei; Yan, Denise; Dai, Pu; Han, Dongyi; Yang, Weiyan; Wang, Rongguang; Liu, Xuezhong; Yuan, Huijun

2011-01-01

The myosin VIIA (MYO7A) gene encodes a protein classified as an unconventional myosin. Mutations within MYO7A can lead to both syndromic and non-syndromic hearing impairment in humans. Among different mutations reported in MYO7A, only five led to non-syndromic sensorineural deafness autosomal dominant type 11 (DFNA11). Here, we present the clinical, genetic and molecular characteristics of two large Chinese DFNA11 families with either high- or low-frequency hearing loss. Affected individuals of family DX-J033 have a sloping audiogram at young ages with high frequency are most affected. With increasing age, all test frequencies are affected. Affected members of family HB-S037 present with an ascending audiogram affecting low frequencies at young ages, and then all frequencies are involved with increasing age. Genome-wide linkage analysis mapped the disease loci within the DFNA11 interval in both families. DNA sequencing of MYO7A revealed two novel nucleotide variations, c.652G > A (p.D218N) and c.2011G > A (p.G671S), in the two families. It is for the first time that the mutations identified in MYO7A in the present study are being implicated in DFNA11 in a Chinese population. For the first time, we tested electrocochleography (ECochG) in a DFNA11 family with low-frequency hearing loss. We speculate that the low-frequency sensorineural hearing loss in this DFNA11 family was not associated with endolymphatic hydrops.

Hybridization-Induced Aggregation Technology for Practical Clinical Testing: KRAS Mutation Detection in Lung and Colorectal Tumors.

PubMed

Sloane, Hillary S; Landers, James P; Kelly, Kimberly A

2016-07-01

KRAS mutations have emerged as powerful predictors of response to targeted therapies in the treatment of lung and colorectal cancers; thus, prospective KRAS genotyping is essential for appropriate treatment stratification. Conventional mutation testing technologies are not ideal for routine clinical screening, as they often involve complex, time-consuming processes and/or costly instrumentation. In response, we recently introduced a unique analytical strategy for revealing KRAS mutations, based on the allele-specific hybridization-induced aggregation (HIA) of oligonucleotide probe-conjugated microbeads. Using simple, inexpensive instrumentation, this approach allows for the detection of any common KRAS mutation in <10 minutes after PCR. Here, we evaluate the clinical utility of the HIA method for mutation detection (HIAMD). In the analysis of 20 lung and colon tumor pathology specimens, we observed a 100% correlation between the KRAS mutation statuses determined by HIAMD and sequencing. In addition, we were able to detect KRAS mutations in a background of 75% wild-type DNA-a finding consistent with that reported for sequencing. With this, we show that HIAMD allows for the rapid and cost-effective detection of KRAS mutations, without compromising analytical performance. These results indicate the validity of HIAMD as a mutation-testing technology suitable for practical clinical testing. Further expansion of this platform may involve the detection of mutations in other key oncogenic pathways. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

A novel twelve class fluctuation test reveals higher than expected mutation rates for influenza A viruses

PubMed Central

Pauly, Matthew D; Procario, Megan C; Lauring, Adam S

2017-01-01

Influenza virus’ low replicative fidelity contributes to its capacity for rapid evolution. Clonal sequencing and fluctuation tests have suggested that the influenza virus mutation rate is 2.7 × 10–6 - 3.0 × 10–5 substitutions per nucleotide per strand copied (s/n/r). However, sequencing assays are biased toward mutations with minimal fitness impacts and fluctuation tests typically investigate only a subset of all possible single nucleotide mutations. We developed a fluctuation test based on reversion to fluorescence in a set of virally encoded mutant green fluorescent proteins, which allowed us to measure the rates of selectively neutral mutations representative of the twelve different mutation types. We measured an overall mutation rate of 1.8 × 10–4 s/n/r for PR8 (H1N1) and 2.5 × 10–4 s/n/r for Hong Kong 2014 (H3N2) and a transitional bias of 2.7–3.6. Our data suggest that each replicated genome will have an average of 2–3 mutations and highlight the importance of mutational load in influenza virus evolution. DOI: http://dx.doi.org/10.7554/eLife.26437.001 PMID:28598328

Pitfalls in genetic testing: the story of missed SCN1A mutations.

PubMed

Djémié, Tania; Weckhuysen, Sarah; von Spiczak, Sarah; Carvill, Gemma L; Jaehn, Johanna; Anttonen, Anna-Kaisa; Brilstra, Eva; Caglayan, Hande S; de Kovel, Carolien G; Depienne, Christel; Gaily, Eija; Gennaro, Elena; Giraldez, Beatriz G; Gormley, Padhraig; Guerrero-López, Rosa; Guerrini, Renzo; Hämäläinen, Eija; Hartmann, Corinna; Hernandez-Hernandez, Laura; Hjalgrim, Helle; Koeleman, Bobby P C; Leguern, Eric; Lehesjoki, Anna-Elina; Lemke, Johannes R; Leu, Costin; Marini, Carla; McMahon, Jacinta M; Mei, Davide; Møller, Rikke S; Muhle, Hiltrud; Myers, Candace T; Nava, Caroline; Serratosa, Jose M; Sisodiya, Sanjay M; Stephani, Ulrich; Striano, Pasquale; van Kempen, Marjan J A; Verbeek, Nienke E; Usluer, Sunay; Zara, Federico; Palotie, Aarno; Mefford, Heather C; Scheffer, Ingrid E; De Jonghe, Peter; Helbig, Ingo; Suls, Arvid

2016-07-01

Sanger sequencing, still the standard technique for genetic testing in most diagnostic laboratories and until recently widely used in research, is gradually being complemented by next-generation sequencing (NGS). No single mutation detection technique is however perfect in identifying all mutations. Therefore, we wondered to what extent inconsistencies between Sanger sequencing and NGS affect the molecular diagnosis of patients. Since mutations in SCN1A, the major gene implicated in epilepsy, are found in the majority of Dravet syndrome (DS) patients, we focused on missed SCN1A mutations. We sent out a survey to 16 genetic centers performing SCN1A testing. We collected data on 28 mutations initially missed using Sanger sequencing. All patients were falsely reported as SCN1A mutation-negative, both due to technical limitations and human errors. We illustrate the pitfalls of Sanger sequencing and most importantly provide evidence that SCN1A mutations are an even more frequent cause of DS than already anticipated.

Laboratory practice guidelines for detecting and reporting JAK2 and MPL mutations in myeloproliferative neoplasms: a report of the Association for Molecular Pathology.

PubMed

Gong, Jerald Z; Cook, James R; Greiner, Timothy C; Hedvat, Cyrus; Hill, Charles E; Lim, Megan S; Longtine, Janina A; Sabath, Daniel; Wang, Y Lynn

2013-11-01

Recurrent mutations in JAK2 and MPL genes are genetic hallmarks of BCR-ABL1-negative myeloproliferative neoplasms. Detection of JAK2 and MPL mutations has been incorporated into routine diagnostic algorithms for these diseases. This Special Article summarizes results from a nationwide laboratory survey of JAK2 and MPL mutation analysis. Based on the current practice pattern and the literature, this Special Article provides recommendations and guidelines for laboratory practice for detection of mutations in the JAK2 and MPL genes, including clinical manifestations for prompting the mutation analysis, current and recommended methodologies for testing the mutations, and standardization for reporting the test results. This Special Article also points to future directions for genomic testing in BCR-ABL1-negative myeloproliferative neoplasms. Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Participation of Korean families at high risk for hereditary breast and ovarian cancer in BRCA1/2 genetic testing.

PubMed

Sun, Young; Kang, Eunyoung; Baek, Hyunnam; Jung, Jaehag; Hwang, Euijun; Koo, Jauk; Kim, Eun-Kyu; Kim, Sung-Won

2015-06-01

The aim of our study was to determine the rate of participation in genetic testing, to determine the reasons for non-participation and to identify the factors affecting participation in BRCA genetic testing for high-risk patients. This study was performed through a retrospective review of 804 individuals who underwent genetic counseling for BRCA1/2 gene mutations at Seoul National University Bundang Hospital between July 2003 and September 2012. In total, 728 (90.5%) individuals underwent BRCA1/2 mutation screening after the initial genetic counseling; 88.2% of 647 probands and 100% of 157 family members were screened. In multivariate analysis, family history of breast cancer and younger age were independent variables affecting participation in genetic testing. Of the 132 people who initially declined genetic testing, 58 (43.9%) postponed the decision, 30 (22.7%) needed time to discuss the issue with family members, 22 (16.7%) did not want to know if they had a BRCA1/2 mutation, and 22 (16.7%) declined the test because of financial problems. When analyzing refusal of testing according to the time period before and after the implementation of national health insurance coverage for BRCA1/2 genetic testing, the critical reason given for refusal was different. After insurance coverage, refusal for financial reason was decreased from 61.1 to 9.6%. A family history of breast cancer and a younger age were important factors associated with participation in genetic testing. National health insurance decreased the proportion of individuals who did not participate in testing owing to a financial reason. In genetic counseling, we have to understand these issues and consider several factors that may influence an individual's decision to be tested. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Distributions of Mutational Effects and the Estimation of Directional Selection in Divergent Lineages of Arabidopsis thaliana.

PubMed

Park, Briton; Rutter, Matthew T; Fenster, Charles B; Symonds, V Vaughan; Ungerer, Mark C; Townsend, Jeffrey P

2017-08-01

Mutations are crucial to evolution, providing the ultimate source of variation on which natural selection acts. Due to their key role, the distribution of mutational effects on quantitative traits is a key component to any inference regarding historical selection on phenotypic traits. In this paper, we expand on a previously developed test for selection that could be conducted assuming a Gaussian mutation effect distribution by developing approaches to also incorporate any of a family of heavy-tailed Laplace distributions of mutational effects. We apply the test to detect directional natural selection on five traits along the divergence of Columbia and Landsberg lineages of Arabidopsis thaliana , constituting the first test for natural selection in any organism using quantitative trait locus and mutation accumulation data to quantify the intensity of directional selection on a phenotypic trait. We demonstrate that the results of the test for selection can depend on the mutation effect distribution specified. Using the distributions exhibiting the best fit to mutation accumulation data, we infer that natural directional selection caused divergence in the rosette diameter and trichome density traits of the Columbia and Landsberg lineages. Copyright © 2017 by the Genetics Society of America.

Estimation of the frequency of occult mutations for an autosomal recessive disease in the presence of genetic heterogeneity: application to genetic hearing loss disorders.

PubMed

Kimberling, William J

2005-11-01

The routine testing for pathologic mutation(s) in a patient's DNA has become the foundation of modern molecular genetic diagnosis. It is especially valuable when the phenotype shows genetic heterogeneity, and its importance will grow as treatments become genotype specific. However, the technology of mutation detection is imperfect and mutations are often missed. This can be especially troublesome when dealing with a recessive disorder where the combination of genetic heterogeneity and missed mutation creates an imprecision in the genotypic assessment of individuals who do not appear to have the expected complement of two pathologic mutations. This article describes a statistical approach to the estimation of the likelihood of a genetic diagnosis under these conditions. In addition to providing a means of testing for missed mutations, it also provides a method of estimating and testing for the presence of genetic heterogeneity in the absence of linkage data. Gene frequencies as well as estimates of sensitivity and specificity can be obtained as well. The test is applied to GJB2 recessive nonsyndromic deafness, Usher syndrome types Ib and IIa, and Pendred-enlarged vestibular aqueduct syndrome. Copyright 2005 Wiley-Liss, Inc.

Setup of a Protocol of Molecular Diagnosis of β-Thalassemia Mutations in Tunisia using Denaturing High-Performance Liquid Chromatography (DHPLC).

PubMed

Sahli, Chaima Abdelhafidh; Ben Salem, Ikbel; Jouini, Latifa; Laouini, Naouel; Dabboubi, Rym; Hadj Fredj, Sondes; Siala, Hajer; Othmeni, Rym; Dakhlaoui, Boutheina; Fattoum, Slaheddine; Bibi, Amina; Messaoud, Taieb

2016-09-01

β-Thalassemia is one of the most prevalent worldwide autosomal recessive disorders. It presents a great molecular heterogeneity resulting from more than 200 causative mutations in the β-globin gene. In Tunisia, β-thalassemia represents the most prevalent monogenic hemoglobin disorder with 2.21% of carriers. Efficient and reliable mutation-screening methods are essential in order to establish appropriate prevention programs for at risk couples. The aim of the present study is to develop an efficient method based on the denaturing high-performance liquid chromatography (DHPLC) in which the whole β-globin gene (HBB) is screened for mutations covering about 90% of the spectrum. We have performed the validation of a DHPLC assay for direct genotyping of 11 known β-thalassemia mutations in the Tunisian population. DHPLC assay was established based on the analysis of 62 archival β-thalassemia samples previously genotyped then validated with full concordance on 50 tests with blind randomized samples previously genotyped with DNA sequencing and with 96% of consistency on 40 samples as a prospective study. Compared to other genotyping techniques, the DHPLC method can meet the requirements of direct genotyping of known β-thalassemia mutations in Tunisia and to be applied as a powerful tool for the genetic screening of prenatal and postnatal individuals. © 2016 Wiley Periodicals, Inc.

Mitochondrial recessive ataxia syndrome mimicking dominant spinocerebellar ataxia.

PubMed

Palin, Eino J H; Hakonen, Anna H; Korpela, Mari; Paetau, Anders; Suomalainen, Anu

2012-04-15

We studied the genetic background of a family with SCA, showing dominant inheritance and anticipation. Muscle histology, POLG1 gene sequence, neuropathology and mitochondrial DNA analyses in a mother and a son showed typical findings for a mitochondrial disorder, and both were shown to be homozygous for a recessive POLG1 mutation, underlying mitochondrial recessive ataxia syndrome, MIRAS. The healthy father was a heterozygous carrier for the same mutation. Recessively inherited MIRAS mutations should be tested in dominantly inherited SCAs cases of unknown cause, as the high carrier frequency of MIRAS may result in two independent introductions of the mutant allele in the family and thereby mimic dominant inheritance. Copyright © 2011 Elsevier B.V. All rights reserved.

Computational design of thermostabilizing point mutations for G protein-coupled receptors

PubMed Central

Popov, Petr; Peng, Yao; Shen, Ling; Stevens, Raymond C; Cherezov, Vadim; Liu, Zhi-Jie

2018-01-01

Engineering of GPCR constructs with improved thermostability is a key for successful structural and biochemical studies of this transmembrane protein family, targeted by 40% of all therapeutic drugs. Here we introduce a comprehensive computational approach to effective prediction of stabilizing mutations in GPCRs, named CompoMug, which employs sequence-based analysis, structural information, and a derived machine learning predictor. Tested experimentally on the serotonin 5-HT2C receptor target, CompoMug predictions resulted in 10 new stabilizing mutations, with an apparent thermostability gain ~8.8°C for the best single mutation and ~13°C for a triple mutant. Binding of antagonists confers further stabilization for the triple mutant receptor, with total gains of ~21°C as compared to wild type apo 5-HT2C. The predicted mutations enabled crystallization and structure determination for the 5-HT2C receptor complexes in inactive and active-like states. While CompoMug already shows high 25% hit rate and utility in GPCR structural studies, further improvements are expected with accumulation of structural and mutation data. PMID:29927385

Specific and straightforward molecular investigation of β-thalassemia mutations in the Malaysian Malays and Chinese using direct TaqMan genotyping assays.

PubMed

Kho, S L; Chua, K H; George, E; Tan, J A M A

2013-07-15

Beta-thalassemia is a life-threatening inherited blood disorder. Rapid characterization of β-globin gene mutations is necessary because of the high frequency of Malaysian β-thalassemia carriers. A combination real-time polymerase chain reaction genotyping assay using TaqMan probes was developed to confirm β-globin gene mutations. In this study, primers and probes were designed to specifically identify 8 common β-thalassemia mutations in the Malaysian Malay and Chinese ethnic groups using the Primer Express software. "Blind tests" using DNA samples from healthy individuals and β-thalassemia patients with different genotypes were performed to determine the specificity and sensitivity of this newly designed assay. Our results showed 100% sensitivity and specificity for this novel assay. In conclusion, the TaqMan genotyping assay is a straightforward assay that allows detection of β-globin gene mutations in less than 40 min. The simplicity and reproducibility of the TaqMan genotyping assay permit its use in laboratories as a rapid and cost-effective diagnostic tool for confirmation of common β-thalassemia mutations in Malaysia.

Translational Control in Bone Marrow Failure

DTIC Science & Technology

2014-04-01

expressing the ATG2 ORF and the ATG3 ORF, we also evaluated the ATG> GTG mutation, which produces all 3 internal ORFs. (We did not test ORF4...expressing shortened forms of NE reduced clonogenic capacity (Fig. 4 of Tidwell et al.). When comparing the shorter isoforms of NE to each other, the GTG ...mutated from ATG to either ATA or GTG , shortened forms of NE were evident (Figure 2, c.3G.A, c.1A.G). When the highly conserved nucleotide in the Kozak

Accurate computation of survival statistics in genome-wide studies.

PubMed

Vandin, Fabio; Papoutsaki, Alexandra; Raphael, Benjamin J; Upfal, Eli

2015-05-01

A key challenge in genomics is to identify genetic variants that distinguish patients with different survival time following diagnosis or treatment. While the log-rank test is widely used for this purpose, nearly all implementations of the log-rank test rely on an asymptotic approximation that is not appropriate in many genomics applications. This is because: the two populations determined by a genetic variant may have very different sizes; and the evaluation of many possible variants demands highly accurate computation of very small p-values. We demonstrate this problem for cancer genomics data where the standard log-rank test leads to many false positive associations between somatic mutations and survival time. We develop and analyze a novel algorithm, Exact Log-rank Test (ExaLT), that accurately computes the p-value of the log-rank statistic under an exact distribution that is appropriate for any size populations. We demonstrate the advantages of ExaLT on data from published cancer genomics studies, finding significant differences from the reported p-values. We analyze somatic mutations in six cancer types from The Cancer Genome Atlas (TCGA), finding mutations with known association to survival as well as several novel associations. In contrast, standard implementations of the log-rank test report dozens-hundreds of likely false positive associations as more significant than these known associations.

Accurate Computation of Survival Statistics in Genome-Wide Studies

PubMed Central

Vandin, Fabio; Papoutsaki, Alexandra; Raphael, Benjamin J.; Upfal, Eli

2015-01-01

A key challenge in genomics is to identify genetic variants that distinguish patients with different survival time following diagnosis or treatment. While the log-rank test is widely used for this purpose, nearly all implementations of the log-rank test rely on an asymptotic approximation that is not appropriate in many genomics applications. This is because: the two populations determined by a genetic variant may have very different sizes; and the evaluation of many possible variants demands highly accurate computation of very small p-values. We demonstrate this problem for cancer genomics data where the standard log-rank test leads to many false positive associations between somatic mutations and survival time. We develop and analyze a novel algorithm, Exact Log-rank Test (ExaLT), that accurately computes the p-value of the log-rank statistic under an exact distribution that is appropriate for any size populations. We demonstrate the advantages of ExaLT on data from published cancer genomics studies, finding significant differences from the reported p-values. We analyze somatic mutations in six cancer types from The Cancer Genome Atlas (TCGA), finding mutations with known association to survival as well as several novel associations. In contrast, standard implementations of the log-rank test report dozens-hundreds of likely false positive associations as more significant than these known associations. PMID:25950620

Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach.

PubMed

Pender, Alexandra; Garcia-Murillas, Isaac; Rana, Sareena; Cutts, Rosalind J; Kelly, Gavin; Fenwick, Kerry; Kozarewa, Iwanka; Gonzalez de Castro, David; Bhosle, Jaishree; O'Brien, Mary; Turner, Nicholas C; Popat, Sanjay; Downward, Julian

2015-01-01

Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma.

Efficient Genotyping of KRAS Mutant Non-Small Cell Lung Cancer Using a Multiplexed Droplet Digital PCR Approach

PubMed Central

Pender, Alexandra; Garcia-Murillas, Isaac; Rana, Sareena; Cutts, Rosalind J.; Kelly, Gavin; Fenwick, Kerry; Kozarewa, Iwanka; Gonzalez de Castro, David; Bhosle, Jaishree; O’Brien, Mary; Turner, Nicholas C.; Popat, Sanjay; Downward, Julian

2015-01-01

Droplet digital PCR (ddPCR) can be used to detect low frequency mutations in oncogene-driven lung cancer. The range of KRAS point mutations observed in NSCLC necessitates a multiplex approach to efficient mutation detection in circulating DNA. Here we report the design and optimisation of three discriminatory ddPCR multiplex assays investigating nine different KRAS mutations using PrimePCR™ ddPCR™ Mutation Assays and the Bio-Rad QX100 system. Together these mutations account for 95% of the nucleotide changes found in KRAS in human cancer. Multiplex reactions were optimised on genomic DNA extracted from KRAS mutant cell lines and tested on DNA extracted from fixed tumour tissue from a cohort of lung cancer patients without prior knowledge of the specific KRAS genotype. The multiplex ddPCR assays had a limit of detection of better than 1 mutant KRAS molecule in 2,000 wild-type KRAS molecules, which compared favourably with a limit of detection of 1 in 50 for next generation sequencing and 1 in 10 for Sanger sequencing. Multiplex ddPCR assays thus provide a highly efficient methodology to identify KRAS mutations in lung adenocarcinoma. PMID:26413866

Murine models of osteosarcoma: A piece of the translational puzzle.

PubMed

Walia, Mannu K; Castillo-Tandazo, Wilson; Mutsaers, Anthony J; Martin, Thomas John; Walkley, Carl R

2018-06-01

Osteosarcoma (OS) is the most common cancer of bone in children and young adults. Despite extensive research efforts, there has been no significant improvement in patient outcome for many years. An improved understanding of the biology of this cancer and how genes frequently mutated contribute to OS may help improve outcomes for patients. While our knowledge of the mutational burden of OS is approaching saturation, our understanding of how these mutations contribute to OS initiation and maintenance is less clear. Murine models of OS have now been demonstrated to be highly valid recapitulations of human OS. These models were originally based on the frequent disruption of p53 and Rb in familial OS syndromes, which are also common mutations in sporadic OS. They have been applied to significantly improve our understanding about the functions of recurrently mutated genes in disease. The murine models can be used as a platform for preclinical testing and identifying new therapeutic targets, in addition to testing the role of additional mutations in vivo. Most recently these models have begun to be used for discovery based approaches and screens, which hold significant promise in furthering our understanding of the genetic and therapeutic sensitivities of OS. In this review, we discuss the mouse models of OS that have been reported in the last 3-5 years and newly identified pathways from these studies. Finally, we discuss the preclinical utilization of the mouse models of OS for identifying and validating actionable targets to improve patient outcome. © 2017 Wiley Periodicals, Inc.

Primary and acquired EGFR T790M-mutant NSCLC patients identified by routine mutation testing show different characteristics but may both respond to osimertinib treatment.

PubMed

Li, Weihua; Qiu, Tian; Guo, Lei; Ling, Yun; Gao, Yibo; Ying, Jianming; He, Jie

2018-06-01

Primary EGFR T790M mutation is occasionally identified by routine mutation testing in tyrosine kinase inhibitor (TKI)-naive patients with non-small cell lung cancer (NSCLC). We herein aimed to compare the characteristics of primary and acquired T790M mutations in NSCLC patients, and their response to osimertinib. Using amplification refractory mutation system (ARMS) detection, primary T790M was identified in 0.5% (46/8723) of TKI-naive patients, whereas acquired T790M was detected in 49.7% (71/143) of TKI-relapsed patients. T790M always coexisted with a sensitizing EGFR mutation. Primary T790M more commonly coexisted with L858R, whereas acquired T790M was more likely to coexist with exon 19 deletions. Moreover, next-generation sequencing (NGS) showed that concomitant sensitizing EGFR and primary T790M mutant allele frequencies (MAFs) were highly concordant, but acquired T790M MAFs were significantly lower than the sensitizing EGFR MAFs. Sixteen acquired T790M-mutant patients received osimertinib. The median progression-free survival (PFS) was 8.1 months. Four primary T790M-mutant patients received osimertinib and the median PFS was 8.0 months. Together, our study demonstrates that primary and acquired T790M-mutant patients show distinct differences in some clinical and molecular characteristics, but may both respond to osimertinib treatment. Copyright © 2018 Elsevier B.V. All rights reserved.

The prevalence of mutations in the gene encoding filaggrin in the population of Polish patients with atopic dermatitis

PubMed Central

Kaczmarek-Skamira, Elżbieta; Romańska-Gocka, Krystyna; Czajkowski, Rafał; Kałużna, Lucyna; Zegarska, Barbara

2016-01-01

Introduction The genetic background of atopic dermatitis (AD) is complex, involves many genes and their participation varies in varied populations, and depends on the intensity and course of a disease. Changes in the nucleotide sequence of the FLG gene and a reduced number or a deficit of the functional product of processed profilaggrin can be one of risk factors for atopic dermatitis. Aim To determine the prevalence of R501X and 2282del4 mutations of the FLG gene in patients with AD. Material and methods The studied group included 60 patients with clinically diagnosed AD, and the control group included 61 healthy volunteers. The study protocol included collection of biological material for tests, DNA isolation and evaluation of its quality and quantity, and PCR amplification of the isolated genetic material. Results In the studied group, both changes in the nucleotide sequence of the FLG gene were detected and in the control group no tested mutations were detected. In 18 (30%) patients with AD, 22 mutations (4 heterozygous and 1 homozygous ones of R501X and 10 heterozygous and 7 homozygous ones of 2282del4) were detected. Conclusions A high rate of mutations of the FLG gene in patients with clinically diagnosed AD and pathologically dry skin was observed in the studied population. The 2282del4 mutation occurred more often than R501X. PMID:27279822

Screening for germline BRCA1, BRCA2, TP53 and CHEK2 mutations in families at-risk for hereditary breast cancer identified in a population-based study from Southern Brazil

PubMed Central

Palmero, Edenir Inêz; Alemar, Bárbara; Schüler-Faccini, Lavínia; Hainaut, Pierre; Moreira-Filho, Carlos Alberto; Ewald, Ingrid Petroni; dos Santos, Patricia Koehler; Ribeiro, Patricia Lisbôa Izetti; de Oliveira, Cristina Brinkmann; Kelm, Florence Le Calvez; Tavtigian, Sean; Cossio, Silvia Liliana; Giugliani, Roberto; Caleffi, Maira; Ashton-Prolla, Patricia

2016-01-01

Abstract In Brazil, breast cancer is a public health care problem due to its high incidence and mortality rates. In this study, we investigated the prevalence of hereditary breast cancer syndromes (HBCS) in a population-based cohort in Brazils southernmost capital, Porto Alegre. All participants answered a questionnaire about family history (FH) of breast, ovarian and colorectal cancer and those with a positive FH were invited for genetic cancer risk assessment (GCRA). If pedigree analysis was suggestive of HBCS, genetic testing of the BRCA1, BRCA2, TP53, and CHEK2 genes was offered. Of 902 women submitted to GCRA, 214 had pedigrees suggestive of HBCS. Fifty of them underwent genetic testing: 18 and 40 for BRCA1/BRCA2 and TP53 mutation screening, respectively, and 7 for CHEK2 1100delC testing. A deleterious BRCA2 mutation was identified in one of the HBOC probands and the CHEK2 1100delC mutation occurred in one of the HBCC families. No deleterious germline alterations were identified in BRCA1 or TP53. Although strict inclusion criteria and a comprehensive testing approach were used, the suspected genetic risk in these families remains unexplained. Further studies in a larger cohort are necessary to better understand the genetic component of hereditary breast cancer in Southern Brazil. PMID:27223485

Identification of mutations in the PI3K-AKT-mTOR signalling pathway in patients with macrocephaly and developmental delay and/or autism.

PubMed

Yeung, Kit San; Tso, Winnie Wan Yee; Ip, Janice Jing Kun; Mak, Christopher Chun Yu; Leung, Gordon Ka Chun; Tsang, Mandy Ho Yin; Ying, Dingge; Pei, Steven Lim Cho; Lee, So Lun; Yang, Wanling; Chung, Brian Hon-Yin

2017-01-01

Macrocephaly, which is defined as a head circumference greater than or equal to + 2 standard deviations, is a feature commonly observed in children with developmental delay and/or autism spectrum disorder. Although PTEN is a well-known gene identified in patients with this syndromic presentation, other genes in the PI3K-AKT-mTOR signalling pathway have also recently been suggested to have important roles. The aim of this study is to characterise the mutation spectrum of this group of patients. We performed whole-exome sequencing of 21 patients with macrocephaly and developmental delay/autism spectrum disorder. Sources of genomic DNA included blood, buccal mucosa and saliva. Germline mutations were validated by Sanger sequencing, whereas somatic mutations were validated by droplet digital PCR. We identified ten pathogenic/likely pathogenic mutations in PTEN ( n  = 4), PIK3CA ( n  = 3), MTOR ( n  = 1) and PPP2R5D ( n  = 2) in ten patients. An additional PTEN mutation, which was classified as variant of unknown significance, was identified in a patient with a pathogenic PTEN mutation, making him harbour bi-allelic germline PTEN mutations. Two patients harboured somatic PIK3CA mutations, and the level of somatic mosaicism in blood DNA was low. Patients who tested positive for mutations in the PI3K-AKT-mTOR pathway had a lower developmental quotient than the rest of the cohort (DQ = 62.8 vs. 76.1, p = 0.021). Their dysmorphic features were non-specific, except for macrocephaly. Among the ten patients with identified mutations, brain magnetic resonance imaging was performed in nine, all of whom showed megalencephaly. We identified mutations in the PI3K-AKT-mTOR signalling pathway in nearly half of our patients with macrocephaly and developmental delay/autism spectrum disorder. These patients have subtle dysmorphic features and mild developmental issues. Clinically, patients with germline mutations are difficult to distinguish from patients with somatic mutations, and therefore, sequencing of buccal or saliva DNA is important to identify somatic mosaicism. Given the high diagnostic yield and the management implications, we suggest implementing comprehensive genetic testing in the PI3K-AKT-mTOR pathway in the clinical evaluation of patients with macrocephaly and developmental delay and/or autism spectrum disorder.

[Study of a family with epidermolysis bullosa simplex resulting from a novel mutation of KRT14 gene].

PubMed

Meng, Lanlan; Du, Juan; Li, Wen; Lu, Guangxiu; Tan, Yueqiu

2017-08-10

To determine the molecular etiology for a Chinese pedigree affected with epidermolysis bullosa simplex (EBS). Target region sequencing using a hereditary epidermolysis bullosa capture array combined with Sanger sequencing and bioinformatics analysis were used. Mutation taster, PolyPhen-2, Provean, and SIFT software and NCBI online were employed to assess the pathogenicity and conservation of detected mutations. One hundred healthy unrelated individuals were used as controls. Target region sequencing showed that the proband has carried a unreported heterozygous c.1234A>G (p.Ile412Val) mutation of the KRT14 gene, which was confirmed by Sanger sequencing in other 8 affected individuals but not among healthy members of the pedigree. Bioinformatics analysis indicated that the mutation is highly pathogenic. Remarkably, 3 members of the family (2 affected and 1 unaffected) have carried a heterozygous c.1237G>A (p.Ala413Thr) mutation of the KRT14 gene, which was collected in Human Gene Mutation Database (HGMD). Bioinformatics analysis indicated that the mutation may not be pathogenic. Both mutations were not detected among the 100 healthy controls. The novel c.1234A>G(p.Ile412Val) mutation of the KRT14 gene is probably responsible for the disease, while c.1237G>A (p.Ala413Thr) mutation of KRT14 gene may be a polymorphism. Compared with Sanger sequencing, target region capture sequencing is more efficient and can significantly reduce the cost of genetic testing for EBS.

First evidence of a large CHEK2 duplication involved in cancer predisposition in an Italian family with hereditary breast cancer

PubMed Central

2014-01-01

Background CHEK2 is a multi-cancer susceptibility gene whose common germline mutations are known to contribute to the risk of developing breast and prostate cancer. Case presentation Here, we describe an Italian family with a high number of cases of breast cancer and other types of tumour subjected to the MLPA test to verify the presence of BRCA1, BRCA2 and CHEK2 deletions and duplications. We identified a new 23-kb duplication in the CHEK2 gene extending from intron 5 to 13 that was associated with breast cancer in the family. The presence and localisation of the alteration was confirmed by a second analysis by Next-Generation Sequencing. Conclusions This finding suggests that CHEK2 mutations are heterogeneous and that techniques other than sequencing, such as MLPA, are needed to identify CHEK2 mutations. It also indicates that CHEK2 rare variants, such as duplications, can confer a high susceptibility to cancer development and should thus be studied in depth as most of our knowledge of CHEK2 concerns common mutations. PMID:24986639

First evidence of a large CHEK2 duplication involved in cancer predisposition in an Italian family with hereditary breast cancer.

PubMed

Tedaldi, Gianluca; Danesi, Rita; Zampiga, Valentina; Tebaldi, Michela; Bedei, Lucia; Zoli, Wainer; Amadori, Dino; Falcini, Fabio; Calistri, Daniele

2014-07-01

CHEK2 is a multi-cancer susceptibility gene whose common germline mutations are known to contribute to the risk of developing breast and prostate cancer. Here, we describe an Italian family with a high number of cases of breast cancer and other types of tumour subjected to the MLPA test to verify the presence of BRCA1, BRCA2 and CHEK2 deletions and duplications. We identified a new 23-kb duplication in the CHEK2 gene extending from intron 5 to 13 that was associated with breast cancer in the family. The presence and localisation of the alteration was confirmed by a second analysis by Next-Generation Sequencing. This finding suggests that CHEK2 mutations are heterogeneous and that techniques other than sequencing, such as MLPA, are needed to identify CHEK2 mutations. It also indicates that CHEK2 rare variants, such as duplications, can confer a high susceptibility to cancer development and should thus be studied in depth as most of our knowledge of CHEK2 concerns common mutations.

Unveiling adaptation using high-resolution lineage tracking

NASA Astrophysics Data System (ADS)

Blundell, Jamie; Levy, Sasha; Fisher, Daniel; Petrov, Dmitri; Sherlock, Gavin

2013-03-01

Human diseases such as cancer and microbial infections are adaptive processes inside the human body with enormous population sizes: between 106 -1012 cells. In spite of this our understanding of adaptation in large populations is limited. The key problem is the difficulty in identifying anything more than a handful of rare, large-effect beneficial mutations. The development and use of molecular barcodes allows us to uniquely tag hundreds of thousands of cells and enable us to track tens of thousands of adaptive mutations in large yeast populations. We use this system to test some of the key theories on which our understanding of adaptation in large populations is based. We (i) measure the fitness distribution in an evolving population at different times, (ii) identify when an appreciable fraction of clones in the population have at most a single adaptive mutation and isolate a large number of clones with independent single adaptive mutations, and (iii) use this clone collection to determine the distribution of fitness effects of single beneficial mutations.

Correlation between Genotypic and Phenotypic Testing for Resistance to Rifampin in Mycobacterium tuberculosis Clinical Isolates in Haiti: Investigation of Cases with Discrepant Susceptibility Results

PubMed Central

Ocheretina, Oksana; Escuyer, Vincent E.; Mabou, Marie-Marcelle; Royal-Mardi, Gertrude; Collins, Sean; Vilbrun, Stalz C.; Pape, Jean W.; Fitzgerald, Daniel W.

2014-01-01

The World Health Organization has recommended use of molecular-based tests MTBDRplus and GeneXpert MTB/RIF to diagnose multidrug-resistant tuberculosis in developing and high-burden countries. Both tests are based on detection of mutations in the Rifampin (RIF) Resistance-Determining Region of DNA-dependent RNA Polymerase gene (rpoB). Such mutations are found in 95–98% of Mycobacterium tuberculosis strains determined to be RIF-resistant by the “gold standard” culture-based drug susceptibility testing (DST). We report the phenotypic and genotypic characterization of 153 consecutive clinical Mycobacterium tuberculosis strains diagnosed as RIF-resistant by molecular tests in our laboratory in Port-au-Prince, Haiti. 133 isolates (86.9%) were resistant to both RIF and Isoniazid and 4 isolates (2.6%) were RIF mono-resistant in MGIT SIRE liquid culture-based DST. However the remaining 16 isolates (10.5%) tested RIF-sensitive by the assay. Five strains with discordant genotypic and phenotypic susceptibility results had RIF minimal inhibitory concentration (MIC) close to the cut-off value of 1 µg/ml used in phenotypic susceptibility assays and were confirmed as resistant by DST on solid media. Nine strains had sub-critical RIF MICs ranging from 0.063 to 0.5 µg/ml. Finally two strains were pan-susceptible and harbored a silent rpoB mutation. Our data indicate that not only detection of the presence but also identification of the nature of rpoB mutation is needed to accurately diagnose resistance to RIF in Mycobacterium tuberculosis. Observed clinical significance of low-level resistance to RIF supports the re-evaluation of the present critical concentration of the drug used in culture-based DST assays. PMID:24599230

JAG1 mutations are found in approximately one third of patients presenting with only one or two clinical features of Alagille syndrome.

PubMed

Guegan, K; Stals, K; Day, M; Turnpenny, P; Ellard, S

2012-07-01

Alagille syndrome is a multisystem disorder characterized by highly variable expressivity, most frequently caused by heterozygous JAG1 gene mutations. Classic diagnostic criteria combine the presence of bile duct paucity on liver biopsy with three of five systems affected; liver, heart, skeleton, eye and dysmorphic facies. The aim of this study was to determine the prevalence and distribution of JAG1 mutations in patients referred for routine clinical diagnostic testing. Clinical data were available for 241 patients from 135 families. The index cases were grouped according to the number of systems affected (heart, liver, skeletal, eye and facies) and the mutation frequency calculated for each group. JAG1 mutations were identified in 59/135 (44%) probands. The highest mutation detection rates were observed in patients with the most frequent presenting features of Alagille syndrome; ranging from 20% (one system) to 86% (five systems). The overall mutation pick-up rate in a clinical diagnostic setting was lower than in previous research studies. Identification of a JAG1 gene mutation is particularly useful for those patients with atypical or mild Alagille syndrome who do not meet classic diagnostic criteria as it provides a definite molecular diagnosis and allows accurate genetic counselling for the family. © 2011 John Wiley & Sons A/S.

Optimized p53 immunohistochemistry is an accurate predictor of TP53 mutation in ovarian carcinoma

PubMed Central

Köbel, Martin; Piskorz, Anna M; Lee, Sandra; Lui, Shuhong; LePage, Cecile; Marass, Francesco; Rosenfeld, Nitzan; Mes Masson, Anne‐Marie

2016-01-01

Abstract TP53 mutations are ubiquitous in high‐grade serous ovarian carcinomas (HGSOC), and the presence of TP53 mutation discriminates between high and low‐grade serous carcinomas and is now an important biomarker for clinical trials targeting mutant p53. p53 immunohistochemistry (IHC) is widely used as a surrogate for TP53 mutation but its accuracy has not been established. The objective of this study was to test whether improved methods for p53 IHC could reliably predict TP53 mutations independently identified by next generation sequencing (NGS). Four clinical p53 IHC assays and tagged‐amplicon NGS for TP53 were performed on 171 HGSOC and 80 endometrioid carcinomas (EC). p53 expression was scored as overexpression (OE), complete absence (CA), cytoplasmic (CY) or wild type (WT). p53 IHC was evaluated as a binary classifier where any abnormal staining predicted deleterious TP53 mutation and as a ternary classifier where OE, CA or WT staining predicted gain‐of‐function (GOF or nonsynonymous), loss‐of‐function (LOF including stopgain, indel, splicing) or no detectable TP53 mutations (NDM), respectively. Deleterious TP53 mutations were detected in 169/171 (99%) HGSOC and 7/80 (8.8%) EC. The overall accuracy for the best performing IHC assay for binary and ternary prediction was 0.94 and 0.91 respectively, which improved to 0.97 (sensitivity 0.96, specificity 1.00) and 0.95 after secondary analysis of discordant cases. The sensitivity for predicting LOF mutations was lower at 0.76 because p53 IHC detected mutant p53 protein in 13 HGSOC with LOF mutations. CY staining associated with LOF was seen in 4 (2.3%) of HGSOC. Optimized p53 IHC can approach 100% specificity for the presence of TP53 mutation and its high negative predictive value is clinically useful as it can exclude the possibility of a low‐grade serous tumour. 4.1% of HGSOC cases have detectable WT staining while harboring a TP53 LOF mutation, which limits sensitivity for binary prediction of mutation to 96%. PMID:27840695

Uncommon EGFR mutations in cytological specimens of 1,874 newly diagnosed Indonesian lung cancer patients.

PubMed

Syahruddin, Elisna; Wulandari, Laksmi; Sri Muktiati, Nunuk; Rima, Ana; Soeroso, Noni; Ermayanti, Sabrina; Levi, Michael; Hidajat, Heriawaty; Widjajahakim, Grace; Utomo, Ahmad Rusdan Handoyo

2018-01-01

We aimed to evaluate the distribution of individual epidermal growth factor receptor ( EGFR ) mutation subtypes found in routine cytological specimens. A retrospective audit was performed on EGFR testing results of 1,874 consecutive cytological samples of newly diagnosed or treatment-naïve Indonesian lung cancer patients (years 2015-2016). Testing was performed by ISO15189 accredited central laboratory. Overall test failure rate was 5.1%, with the highest failure (7.1%) observed in pleural effusion and lowest (1.6%) in needle aspiration samples. EGFR mutation frequency was 44.4%. Tyrosine kinase inhibitor (TKI)-sensitive common EGFR mutations (ins/dels exon 19, L858R) and uncommon mutations (G719X, T790M, L861Q) contributed 57.1% and 29%, respectively. Approximately 13.9% of mutation-positive patients carried a mixture of common and uncommon mutations. Women had higher EGFR mutation rate (52.9%) vs men (39.1%; p <0.05). In contrast, uncommon mutations conferring either TKI responsive (G719X, L861Q) or TKI resistance (T790M, exon 20 insertions) were consistently more frequent in men than in women (67.3% vs 32.7% or 69.4% vs 30.6%; p <0.05). Up to 10% EGFR mutation-positive patients had baseline single mutation T790M, exon 20 insertion, or in coexistence with TKI-sensitive mutations. Up to 9% patients had complex or multiple EGFR mutations, whereby 48.7% patients harbored TKI-resistant mutations. One patient presented third-generation TKI-resistant mutation L792F simultaneously with T790M. Routine diagnostic cytological techniques yielded similar success rate to detect EGFR mutations. Uncommon EGFR mutations were frequent events in Indonesian lung cancer patients.

Should EGFR mutations be tested in advanced lung squamous cell carcinomas to guide frontline treatment?

PubMed

Chiu, Chao-Hua; Chou, Teh-Ying; Chiang, Chi-Lu; Tsai, Chun-Ming

2014-10-01

There is no argument over using epidermal growth factor receptor (EGFR) mutation status to guide the frontline treatment for advanced lung adenocarcinoma (LADC); however, the role of the testing in lung squamous cell carcinoma (LSQC) remains controversial. Currently, the guidelines/consensus statements regarding EGFR mutation testing in LSQC are not consistent among different oncology societies. American Society of Clinical Oncology recommends performing EGFR mutation testing in all patients; European Society for Medical Oncology, College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology, and National Comprehensive Cancer Network suggest for some selected group. EGFR mutation is rarely found in LSQC; however, more importantly, it is not a valid predictive biomarker for EGFR tyrosine kinase inhibitors (EGFR-TKI) in LSQC as it has been shown in LADC. Available data showed that the response rate and progression-free survival in EGFR mutant LSQC patients treated with EGFR-TKI are not better than that observed in patients treated with platinum-doublet chemotherapy in the first-line setting. Therefore, in contrast to advanced LADC, EGFR mutation testing may not be necessarily performed upfront in advanced LSQC because not only the mutation rate is low, but also the predictive value is insufficient. For LSQC patients with known sensitizing-EGFR mutations, both conventional chemotherapy and EGFR-TKI are acceptable frontline treatment options.

High Satisfaction and Low Distress in Breast Cancer Patients One Year after BRCA-Mutation Testing without Prior Face-to-Face Genetic Counseling.

PubMed

Sie, Aisha S; Spruijt, Liesbeth; van Zelst-Stams, Wendy A G; Mensenkamp, Arjen R; Ligtenberg, Marjolijn J L; Brunner, Han G; Prins, Judith B; Hoogerbrugge, Nicoline

2016-06-01

According to standard practice following referral to clinical genetics, most high risk breast cancer (BC) patients in many countries receive face-to-face genetic counseling prior to BRCA-mutation testing (DNA-intake). We evaluated a novel format by prospective study: replacing the intake consultation with telephone, written and digital information sent home. Face-to-face counseling then followed BRCA-mutation testing (DNA-direct). One year after BRCA-result disclosure, 108 participants returned long-term follow-up questionnaires, of whom 59 (55 %) had previously chosen DNA-direct (intervention) versus DNA-intake (standard practice i.e., control: 45 %). Questionnaires assessed satisfaction and psychological distress. All participants were satisfied and 85 % of DNA-direct participants would choose this procedure again; 10 % would prefer DNA-intake and 5 % were undecided. In repeated measurements ANOVA, general distress (GHQ-12, p = 0.01) and BC-specific distress (IES-bc, p = 0.03) were lower in DNA-direct than DNA-intake at all time measurements. Heredity-specific distress (IES-her) did not differ significantly between groups. Multivariate regression analyses showed that choice of procedure did not significantly contribute to either general or heredity-specific distress. BC-specific distress (after BC diagnosis) did contribute to both general and heredity-specific distress. This suggests that higher distress scores reflected BC experience, rather than the type of genetic diagnostic procedure. In conclusion, the large majority of BC patients that used DNA-direct reported high satisfaction without increased distress both in the short term, and 1 year after conclusion of genetic testing.

EZH2 and CD79B mutational status over time in B-cell non-Hodgkin lymphomas detected by high-throughput sequencing using minimal samples

PubMed Central

Saieg, Mauro Ajaj; Geddie, William R; Boerner, Scott L; Bailey, Denis; Crump, Michael; da Cunha Santos, Gilda

2013-01-01

BACKGROUND: Numerous genomic abnormalities in B-cell non-Hodgkin lymphomas (NHLs) have been revealed by novel high-throughput technologies, including recurrent mutations in EZH2 (enhancer of zeste homolog 2) and CD79B (B cell antigen receptor complex-associated protein beta chain) genes. This study sought to determine the evolution of the mutational status of EZH2 and CD79B over time in different samples from the same patient in a cohort of B-cell NHLs, through use of a customized multiplex mutation assay. METHODS: DNA that was extracted from cytological material stored on FTA cards as well as from additional specimens, including archived frozen and formalin-fixed histological specimens, archived stained smears, and cytospin preparations, were submitted to a multiplex mutation assay specifically designed for the detection of point mutations involving EZH2 and CD79B, using MassARRAY spectrometry followed by Sanger sequencing. RESULTS: All 121 samples from 80 B-cell NHL cases were successfully analyzed. Mutations in EZH2 (Y646) and CD79B (Y196) were detected in 13.2% and 8% of the samples, respectively, almost exclusively in follicular lymphomas and diffuse large B-cell lymphomas. In one-third of the positive cases, a wild type was detected in a different sample from the same patient during follow-up. CONCLUSIONS: Testing multiple minimal tissue samples using a high-throughput multiplex platform exponentially increases tissue availability for molecular analysis and might facilitate future studies of tumor progression and the related molecular events. Mutational status of EZH2 and CD79B may vary in B-cell NHL samples over time and support the concept that individualized therapy should be based on molecular findings at the time of treatment, rather than on results obtained from previous specimens. Cancer (Cancer Cytopathol) 2013;121:377–386. © 2013 American Cancer Society. PMID:23361872

Ultra-deep sequencing confirms immunohistochemistry as a highly sensitive and specific method for detecting BRAF V600E mutations in colorectal carcinoma.

PubMed

Rössle, Matthias; Sigg, Michèle; Rüschoff, Jan H; Wild, Peter J; Moch, Holger; Weber, Achim; Rechsteiner, Markus P

2013-11-01

The activating BRAF (V600) mutation is a well-established negative prognostic biomarker in metastatic colorectal carcinoma (CRC). A recently developed monoclonal mouse antibody (clone VE1) has been shown to detect reliably BRAF (V600E) mutated protein by immunohistochemistry (IHC). In this study, we aimed to compare the detection of BRAF (V600E) mutations by IHC, Sanger sequencing (SaS), and ultra-deep sequencing (UDS) in CRC. VE1-IHC was established in a cohort of 68 KRAS wild-type CRCs. The VE1-IHC was only positive in the three patients with a known BRAF (V600E) mutation as assessed by SaS and UDS. The test cohort consisted of 265 non-selected, consecutive CRC samples. Thirty-nine out of 265 cases (14.7%) were positive by VE1-IHC. SaS of 20 randomly selected IHC negative tumors showed BRAF wild-type (20/20). Twenty-four IHC-positive cases were confirmed by SaS (24/39; 61.5%) and 15 IHC-positive cases (15/39; 38.5%) showed a BRAF wild-type by SaS. UDS detected a BRAF (V600E) mutation in 13 of these 15 discordant cases. In one tumor, the mutation frequency was below our threshold for UDS positivity, while in another case, UDS could not be performed due to low DNA amount. Statistical analysis showed sensitivities of 100% and 63% and specificities of 95 and 100% for VE1-IHC and SaS, respectively, compared to combined results of SaS and UDS. Our data suggests that there is high concordance between UDS and IHC using the anti-BRAF(V600E) (VE1) antibody. Thus, VE1 immunohistochemistry is a highly sensitive and specific method in detecting BRAF (V600E) mutations in colorectal carcinoma.

Teaching the Fluctuation Test "In Silico" by Using Mutate: A Program to Distinguish between the Adaptive and Spontaneous Mutation Hypotheses

ERIC Educational Resources Information Center

Carvajal-Rodriguez, Antonio

2012-01-01

Mutate is a program developed for teaching purposes to impart a virtual laboratory class for undergraduate students of Genetics in Biology. The program emulates the so-called fluctuation test whose aim is to distinguish between spontaneous and adaptive mutation hypotheses in bacteria. The plan is to train students in certain key multidisciplinary…

Epidermal growth factor receptor and anaplastic lymphoma kinase testing and mutation prevalence in patients with advanced non-small cell lung cancer in Switzerland: A comprehensive evaluation of real world practices.

PubMed

Ess, S M; Herrmann, C; Frick, H; Krapf, M; Cerny, T; Jochum, W; Früh, M

2017-11-01

In order to improve outcomes, identification of the epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) genes has become crucial in advanced non-small-cell lung cancer (NSCLC). The aim of the present study is to analyse time trends and frequency of testing, factors affecting testing as well as prevalence of mutations in the Swiss population. We analysed EGFR and ALK testing in a cohort of patients with newly diagnosed metastasised non-squamous NSCLC in the catchment area of the cancer registry Eastern Switzerland in the years 2008-2014. We analysed prevalence of mutations and studied clinicopathological characteristics and survival of tested and non-tested patients and of patients with and without mutations. Among 718 patients identified, 11% (51/447) harboured an EGFR mutation in the exons 18, 19 or 21 and further 12% (31/265) showed a positive test result for ALK rearrangements. In non-smokers the proportions of mutations were 31% and 23% respectively. Testing rates increased over time and reached 79% in 2014. We observed significantly lower testing rates and poorer survival in elderly, patients with limited life expectancy and patients treated at hospitals not involved in clinical research. Outcomes can be further improved in a considerable proportion of patients with advanced non-squamous NSCLC. © 2017 John Wiley & Sons Ltd.

Prediction of BRCA Mutations Using the BRCAPRO Model in Clinic-Based African American, Hispanic, and Other Minority Families in the United States

PubMed Central

Huo, Dezheng; Senie, Ruby T.; Daly, Mary; Buys, Saundra S.; Cummings, Shelly; Ogutha, Jacqueline; Hope, Kisha; Olopade, Olufunmilayo I.

2009-01-01

Purpose BRCAPRO, a BRCA mutation carrier prediction model, was developed on the basis of studies in individuals of Ashkenazi Jewish and European ancestry. We evaluated the performance of the BRCAPRO model among clinic-based minority families. We also assessed the clinical utility of mutation status of probands (the first individual tested in a family) in the recommendation of BRCA mutation testing for other at-risk family members. Patients and Methods A total of 292 minority families with at least one member who was tested for BRCA mutations were identified through the Breast Cancer Family Registry and the University of Chicago. Using the BRCAPRO model, the predicted likelihood of carrying BRCA mutations was generated. Area under the receiver operating characteristic curves (AUCs) were calculated. Results There were 104 African American, 130 Hispanic, 37 Asian-American, and 21 other minority families. The AUC was 0.748 (95% CI, 0.672 to 0.823) for all minorities combined. There was a statistically nonsignificant trend for BRCAPRO to perform better in Hispanic families than in other minority families. After taking into account the mutation status of probands, BRCAPRO performance in additional tested family members was improved: the AUC increased from 0.760 to 0.902. Conclusion The findings support the use of BRCAPRO in pretest BRCA mutation prediction among minority families in clinical settings, but there is room for improvement in ethnic groups other than Hispanics. Knowledge of the mutation status of the proband provides additional predictive value, which may guide genetic counselors in recommending BRCA testing of additional relatives when a proband has tested negative. PMID:19188678

Genetics of triple-negative breast cancer: Implications for patient care.

PubMed

Afghahi, Anosheh; Telli, Melinda L; Kurian, Allison W

Patients with triple-negative breast cancer (TNBC), defined as lacking expression of the estrogen and progesterone receptors (ER/PR) and amplification of the HER2 oncogene, often have a more aggressive disease course than do patients with hormone receptor-positive breast cancer, including higher rates of visceral and central nervous system metastases, early cancer recurrences and deaths. Triple-negative breast cancer is associated with a young age at diagnosis and both African and Ashkenazi Jewish ancestry, the latter due to three common founder mutations in the highly penetrant cancer susceptibility genes BRCA1 and BRCA2 (BRCA1/2). In the past decade, there has been a surge both in genetic testing technology and in patient access to such testing. Advances in genetic testing have enabled more rapid and less expensive commercial sequencing than could be imagined only a few years ago. Massively parallel, next-generation sequencing allows the simultaneous analysis of many different genes. Studies of TNBC patients in the current era have revealed associations of TNBC with mutations in several moderate penetrance breast cancer susceptibility genes, including PALB2, BARD1, BRIP1, RAD51C and RAD51D. Interestingly, many of these genes, like BRCA1/2, are involved in homologous recombination DNA double-stranded repair. In this review, we summarize the current understanding of pathogenic germline gene mutations associated with TNBC and the early detection and prevention strategies for women at risk of developing this high-risk breast cancer subtype. Furthermore, we discuss recent the advances in targeted therapies for TNBC patients with a hereditary predisposition, including the role of poly (ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2 mutation-associated breast cancers. Copyright © 2016 Elsevier Inc. All rights reserved.

Detection of BRAF-V600E and V600K in melanoma circulating tumour cells by droplet digital PCR.

PubMed

Reid, Anna L; Freeman, James B; Millward, Michael; Ziman, Melanie; Gray, Elin S

2015-10-01

Defining the BRAF mutation status in metastatic melanoma patients is critical to selecting patients for therapeutic treatment with targeted therapies. Circulating tumour cells (CTCs) can provide an alternative source of contemporaneous tumour genetic material. However methodologies to analyse the presence of rare mutations in a background of wild-type DNA requires a detailed assessment. Here we evaluate the sensitivity of two technologies for cancer mutation detection and the suitability of whole genome amplified DNA as a template for the detection of BRAF-V600 mutations. Serial dilutions of mutant BRAF-V600E DNA in wild-type DNA were tested using both competitive allele-specific PCR (castPCR) and droplet digital PCR (ddPCR), with and without previous whole genome amplification (WGA). Using immunomagnetic beads, we partially enriched CTCs from blood obtained from metastatic melanoma patients with confirmed BRAF mutation positive tumours and extracted RNA and DNA from the CTCs. We used RT-PCR of RNA to confirm the presence of melanoma cells in the CTC fraction then the DNAs of CTC positive fractions were WGA and tested for BRAF V600E or V600K mutations by ddPCRs. WGA DNA produced lower than expected fractional abundances by castPCR analysis but not by ddPCR. Moreover, ddPCR was found to be 200 times more sensitive than castPCR and in combination with WGA produced the most concordant results, with a limit of detection of 0.0005%. BRAF-V600E or V600K mutated DNA was detected in 77% and 44%, respectively, of enriched CTC fractions from metastatic melanoma patients carrying the corresponding mutations. Our results demonstrate that using ddPCR in combination with WGA DNA allows the detection with high sensitivity of cancer mutations in partially enriched CTC fractions. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Whole exome sequencing reveals concomitant mutations of multiple FA genes in individual Fanconi anemia patients

PubMed Central

2014-01-01

Background Fanconi anemia (FA) is a rare inherited genetic syndrome with highly variable clinical manifestations. Fifteen genetic subtypes of FA have been identified. Traditional complementation tests for grouping studies have been used generally in FA patients and in stepwise methods to identify the FA type, which can result in incomplete genetic information from FA patients. Methods We diagnosed five pediatric patients with FA based on clinical manifestations, and we performed exome sequencing of peripheral blood specimens from these patients and their family members. The related sequencing data were then analyzed by bioinformatics, and the FANC gene mutations identified by exome sequencing were confirmed by PCR re-sequencing. Results Homozygous and compound heterozygous mutations of FANC genes were identified in all of the patients. The FA subtypes of the patients included FANCA, FANCM and FANCD2. Interestingly, four FA patients harbored multiple mutations in at least two FA genes, and some of these mutations have not been previously reported. These patients’ clinical manifestations were vastly different from each other, as were their treatment responses to androstanazol and prednisone. This finding suggests that heterozygous mutation(s) in FA genes could also have diverse biological and/or pathophysiological effects on FA patients or FA gene carriers. Interestingly, we were not able to identify de novo mutations in the genes implicated in DNA repair pathways when the sequencing data of patients were compared with those of their parents. Conclusions Our results indicate that Chinese FA patients and carriers might have higher and more complex mutation rates in FANC genes than have been conventionally recognized. Testing of the fifteen FANC genes in FA patients and their family members should be a regular clinical practice to determine the optimal care for the individual patient, to counsel the family and to obtain a better understanding of FA pathophysiology. PMID:24885126

Whole exome sequencing reveals concomitant mutations of multiple FA genes in individual Fanconi anemia patients.

PubMed

Chang, Lixian; Yuan, Weiping; Zeng, Huimin; Zhou, Quanquan; Wei, Wei; Zhou, Jianfeng; Li, Miaomiao; Wang, Xiaomin; Xu, Mingjiang; Yang, Fengchun; Yang, Yungui; Cheng, Tao; Zhu, Xiaofan

2014-05-15

Fanconi anemia (FA) is a rare inherited genetic syndrome with highly variable clinical manifestations. Fifteen genetic subtypes of FA have been identified. Traditional complementation tests for grouping studies have been used generally in FA patients and in stepwise methods to identify the FA type, which can result in incomplete genetic information from FA patients. We diagnosed five pediatric patients with FA based on clinical manifestations, and we performed exome sequencing of peripheral blood specimens from these patients and their family members. The related sequencing data were then analyzed by bioinformatics, and the FANC gene mutations identified by exome sequencing were confirmed by PCR re-sequencing. Homozygous and compound heterozygous mutations of FANC genes were identified in all of the patients. The FA subtypes of the patients included FANCA, FANCM and FANCD2. Interestingly, four FA patients harbored multiple mutations in at least two FA genes, and some of these mutations have not been previously reported. These patients' clinical manifestations were vastly different from each other, as were their treatment responses to androstanazol and prednisone. This finding suggests that heterozygous mutation(s) in FA genes could also have diverse biological and/or pathophysiological effects on FA patients or FA gene carriers. Interestingly, we were not able to identify de novo mutations in the genes implicated in DNA repair pathways when the sequencing data of patients were compared with those of their parents. Our results indicate that Chinese FA patients and carriers might have higher and more complex mutation rates in FANC genes than have been conventionally recognized. Testing of the fifteen FANC genes in FA patients and their family members should be a regular clinical practice to determine the optimal care for the individual patient, to counsel the family and to obtain a better understanding of FA pathophysiology.

Analysis of genotoxic activity of ketamine and rocuronium bromide using the somatic mutation and recombination test in Drosophila melanogaster.

PubMed

Koksal, Pakize Muge; Gürbüzel, Mehmet

2015-03-01

The present study evaluated the mutagenic and recombinogenic effects of two commonly used anesthetic agents, ketamine and rocuronium bromide, in medicine using the wing somatic mutation and recombination test (SMART) in Drosophila. The standard (ST) cross and the high-bioactivation (HB) cross with high sensitivity to procarcinogens and promutagens were used. The SMART test is based on the loss of heterozygosity, which occurs via various mechanisms, such as chromosome loss and deletion, half-translocation, mitotic recombination, mutation, and non-disjunction. Genetic alterations occurring in the somatic cells of the wing's imaginal discs result in mutant clones in the wing blade. Three-day-old trans-heterozygous larvae with two recessive markers, multiple wing hairs (mwh) and flare (flr(3)), were treated with ketamine and rocuronium bromide. Analysis of the ST cross indicated that ketamine exhibited genotoxicity activity and that this activity was particularly dependent on homologous mitotic recombination at concentrations of 250 μg/ml and above. Rocuronium bromide did not exert mutagenic and/or recombinogenic effects. In the HB cross, ketamine at a concentration of 1000 μg/ml and rocuronium bromide at all concentrations, with the exception of 250 μg/ml (inconclusive), exerted genotoxic effects, which could also be associated with the increase in mitotic recombination. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecular characterization of mutations associated with resistance to second-line tuberculosis drug among multidrug-resistant tuberculosis patients from high prevalence tuberculosis city in Morocco.

PubMed

Oudghiri, Amal; Karimi, Hind; Chetioui, Fouad; Zakham, Fathiah; Bourkadi, Jamal Eddine; Elmessaoudi, My Driss; Laglaoui, Amin; Chaoui, Imane; El Mzibri, Mohammed

2018-02-27

The emergence of extensively drug-resistant tuberculosis (XDR-TB) has raised public health concern for global TB control. Although multi drug-resistant tuberculosis (MDR- TB) prevalence and associated genetic mutations in Morocco are well documented, scarce information on XDR TB is available. Hence, the evaluation of pre-XDR and XDR prevalence, as well as the mutation status of gyrA, gyrB, rrs, tlyA genes and eis promoter region, associated with resistance to second line drugs, is of great value for better management of M/XDR TB in Morocco. To evaluate pre-XDR and XDR prevalence, as well as the mutation status of gyrA, gyrB, rrs, tlyA genes and eis promoter region, associated with resistance to second line drug resistance, in 703 clinical isolates from TB patients recruited in Casablanca, and to assess the usefulness of molecular tools in clinical laboratories for better management of M/XDR TB in Morocco. Drug susceptibility testing (DST) was performed by the proportional method for first line drugs, and then the selected MDR isolates were tested for second line drugs (Ofloxacin, Kanamycin, Amikacin and Capreomycin). Along with DST, all samples were subjected to rpoB, katG and p-inhA mutation analysis by PCR and DNA sequencing. MDR isolates as well as 30 pan-susceptible strains were subjected to PCR and DNA sequencing of gyrA, gyrB, rrs, tlyA genes and eis promoter, associated with resistance to fluoroquinolones and injectable drugs. Among the 703 analysed strains, 12.8% were MDR; Ser531Leu and Ser315Thr being the most common recorded mutations within rpoB and katG genes associated with RIF and INH resistance respectively. Drug susceptibility testing for second line drugs showed that among the 90 MDR strains, 22.2% (20/90) were resistant to OFX, 2.22% (2/90) to KAN, 3.33% (3/90) to AMK and 1.11% (1/90) to CAP. Genotypic analysis revealed that 19 MDR strains harbored mutations in the gyrA gene; the most recorded mutation being Asp91Ala accounting for 47.6% (10/21), and 2 isolates harbored mutations in the promoter region of eis gene. No mutation was found in gyrB, rrs and tlyA genes. Moreover, none of the pan-susceptible isolates displayed mutations in targeted genes. Most of mutations associated with SLD resistance occurred in gyrA gene (codons 90-94) and eis promoter region. These findings highlight the impact of mutations in gyrA on the development of fluroquinolones resistance and provide the first estimates of the proportion of pre-XDR-TB among MDR-TB cases in Morocco.

Identification of Germline Genetic Mutations in Pancreatic Cancer Patients

PubMed Central

Salo-Mullen, Erin E.; O’Reilly, Eileen; Kelsen, David; Ashraf, Asad M.; Lowery, Maeve; Yu, Kenneth; Reidy, Diane; Epstein, Andrew S.; Lincoln, Anne; Saldia, Amethyst; Jacobs, Lauren M.; Rau-Murthy, Rohini; Zhang, Liying; Kurtz, Robert; Saltz, Leonard; Offit, Kenneth; Robson, Mark; Stadler, Zsofia K.

2016-01-01

Background Pancreatic adenocarcinoma (PAC) is part of several cancer predisposition syndromes; however, indications for genetic counseling/testing are not well-defined. We sought to determine mutation prevalence and characteristics that predict for inherited predisposition to PAC. Methods We identified 175 consecutive PAC patients who underwent clinical genetics assessment at Memorial Sloan Kettering between 2011–2014. Clinical data, family history, and germline results were evaluated. Results Among 159 PAC patients who pursued genetic testing, 24 pathogenic mutations were identified (15.1%; 95%CI, 9.5%–20.7%), including BRCA2(n=13), BRCA1(n=4), p16(n=2), PALB2(n=1), and Lynch syndrome(n=4). BRCA1/BRCA2 prevalence was 13.7% in Ashkenazi Jewish(AJ) (n=95) and 7.1% in non-AJ(n=56) patients. In AJ patients with strong, weak, or absent family history of BRCA-associated cancers, mutation prevalence was 16.7%, 15.8%, and 7.4%, respectively. Mean age at diagnosis in all mutation carriers was 58.5y(range 45–75y) compared to 64y(range 27–87y) in non-mutation carriers(P=0.02). Although BRCA2 was the most common mutation identified, no patients with early-onset PAC(≤50y) harbored a BRCA2 mutation and the mean age at diagnosis in BRCA2 carriers was equivalent to non-mutation carriers(P=0.34). Mutation prevalence in early-onset patients(n=21) was 28.6%, including BRCA1(n=2), p16(n=2), MSH2(n=1) and MLH1(n=1). Conclusion Mutations in BRCA2 account for over 50% of PAC patients with an identified susceptibility syndrome. AJ patients had high BRCA1/BRCA2 prevalence regardless of personal/family history, suggesting that ancestry alone indicates a need for genetic evaluation. With the exception of BRCA2-associated PAC, inherited predisposition to PAC is associated with earlier age at PAC diagnosis suggesting that this subset of patients may also represent a population warranting further evaluation. PMID:26440929

Endometrial tumour BRAF mutations and MLH1 promoter methylation as predictors of germline mismatch repair gene mutation status: a literature review.

PubMed

Metcalf, Alexander M; Spurdle, Amanda B

2014-03-01

Colorectal cancer (CRC) that displays high microsatellite instability (MSI-H) can be caused by either germline mutations in mismatch repair (MMR) genes, or non-inherited transcriptional silencing of the MLH1 promoter. A correlation between MLH1 promoter methylation, specifically the 'C' region, and BRAF V600E status has been reported in CRC studies. Germline MMR mutations also greatly increase risk of endometrial cancer (EC), but no systematic review has been undertaken to determine if these tumour markers may be useful predictors of MMR mutation status in EC patients. Endometrial cancer cohorts meeting review inclusion criteria encompassed 2675 tumours from 20 studies for BRAF V600E, and 447 tumours from 11 studies for MLH1 methylation testing. BRAF V600E mutations were reported in 4/2675 (0.1%) endometrial tumours of unknown MMR mutation status, and there were 7/823 (0.9%) total sequence variants in exon 11 and 27/1012 (2.7%) in exon 15. Promoter MLH1 methylation was not observed in tumours from 32 MLH1 mutation carriers, or for 13 MSH2 or MSH6 mutation carriers. MMR mutation-negative individuals with tumour MLH1 and PMS2 IHC loss displayed MLH1 methylation in 48/51 (94%) of tumours. We have also detailed specific examples that show the importance of MLH1 promoter region, assay design, and quantification of methylation. This review shows that BRAF mutations occurs so infrequently in endometrial tumours they can be discounted as a useful marker for predicting MMR-negative mutation status, and further studies of endometrial cohorts with known MMR mutation status are necessary to quantify the utility of tumour MLH1 promoter methylation as a marker of negative germline MMR mutation status in EC patients.

Epidermolysis bullosa with congenital pyloric atresia: novel mutations in the beta 4 integrin gene (ITGB4) and genotype/phenotype correlations.

PubMed

Nakano, A; Pulkkinen, L; Murrell, D; Rico, J; Lucky, A W; Garzon, M; Stevens, C A; Robertson, S; Pfendner, E; Uitto, J

2001-05-01

Epidermolysis bullosa with pyloric atresia (EB-PA: OMIM 226730), also known as Carmi syndrome, is a rare autosomal recessive genodermatosis that manifests with neonatal mucocutaneous fragility associated with congenital pyloric atresia. The disease is frequently lethal within the first year, but nonlethal cases have been reported. Mutations in the genes encoding subunit polypeptides of the alpha 6 beta 4 integrin (ITGA6 and ITGB4) have been demonstrated in EB-PA patients. To extend the repertoire of mutations and to identify genotype-phenotype correlations, we examined seven new EB-PA families, four with lethal and three with nonlethal disease variants. DNA from patients was screened for mutations using heteroduplex analysis followed by nucleotide sequencing of PCR products spanning all beta 4 integrin-coding sequences. Mutation analysis disclosed 12 distinct mutations, 11 of them novel. Four mutations predicted a premature termination codon as a result of nonsense mutations or small out-of-frame insertions or deletions, whereas seven were missense mutations. This brings the total number of distinct ITGB4 mutations to 33. The mutation database indicates that premature termination codons are associated predominantly with the lethal EB-PA variants, whereas missense mutations are more prevalent in nonlethal forms. However, the consequences of the missense mutations are position dependent, and substitutions of highly conserved amino acids may have lethal consequences. In general, indirect immunofluorescence studies of affected skin revealed negative staining for beta 4 integrin in lethal cases and positive, but attenuated, staining in nonlethal cases and correlated with clinical phenotype. The data on specific mutations in EB-PA patients allows prenatal testing and preimplantation genetic diagnosis in families at risk.

Protein and genetic diagnosis of limb girdle muscular dystrophy type 2A: The yield and the pitfalls.

PubMed

Fanin, Marina; Angelini, Corrado

2015-08-01

Limb girdle muscular dystrophy type 2A (LGMD2A) is the most frequent form of LGMD worldwide. Comprehensive clinical assessment and laboratory testing is essential for diagnosis of LGMD2A. Muscle immunoblot analysis of calpain-3 is the most useful tool to direct genetic testing, as detection of calpain-3 deficiency has high diagnostic value. However, calpain-3 immunoblot testing lacks sensitivity in about 30% of cases due to gene mutations that inactivate the enzyme. The best diagnostic strategy should be determined on a case-by-case basis, depending on which tissues are available, and which molecular and/or genetic methods are adopted. In this work we survey the current knowledge, advantages, limitations, and pitfalls of protein testing and mutation detection in LGMD2A and provide an update of genetic epidemiology. © 2015 Wiley Periodicals, Inc.

Risk assessment, genetic counseling, and genetic testing for BRCA-related cancer in women: a systematic review to update the U.S. Preventive Services Task Force recommendation.

PubMed

Nelson, Heidi D; Pappas, Miranda; Zakher, Bernadette; Mitchell, Jennifer Priest; Okinaka-Hu, Leila; Fu, Rongwei

2014-02-18

Mutations in breast cancer susceptibility genes (BRCA1 and BRCA2) are associated with increased risks for breast, ovarian, and other types of cancer. To review new evidence on the benefits and harms of risk assessment, genetic counseling, and genetic testing for BRCA-related cancer in women. MEDLINE and PsycINFO between 2004 and 30 July 2013, the Cochrane Central Register of Controlled Trials and Cochrane Database of Systematic Reviews from 2004 through the second quarter of 2013, Health Technology Assessment during the fourth quarter of 2012, Scopus, and reference lists. English-language studies about accuracy of risk assessment and benefits and harms of genetic counseling, genetic testing, and interventions to reduce cancer incidence and mortality. Individual investigators extracted data on participants, study design, analysis, follow-up, and results, and a second investigator confirmed key data. Investigators independently dual-rated study quality and applicability by using established criteria. Five referral models accurately estimated individual risk for BRCA mutations. Genetic counseling increased the accuracy of risk perception and decreases the intention for genetic testing among unlikely carriers and cancer-related worry, anxiety, and depression. No trials evaluated the effectiveness of intensive screening or risk-reducing medications in mutation carriers, although false-positive rates, unneeded imaging, and unneeded surgeries were higher with screening. Among high-risk women and mutation carriers, risk-reducing mastectomy decreased breast cancer by 85% to 100% and breast cancer mortality by 81% to 100% compared with women without surgery; risk-reducing salpingo-oophorectomy decreased breast cancer incidence by 37% to 100%, ovarian cancer by 69% to 100%, and all-cause mortality by 55% to 100%. The analysis included only English-language articles;efficacy trials in mutation carriers were lacking. Studies of risk assessment, genetic counseling, genetic testing, and interventions to reduce cancer and mortality indicate potential benefits and harms that vary according to risk.

Molecular characterization, fitness and mycotoxin production of Fusarium graminearum laboratory strains resistant to benzimidazoles.

PubMed

Sevastos, A; Markoglou, A; Labrou, N E; Flouri, F; Malandrakis, A

2016-03-01

Six benzimidazole (BMZ)-resistant Fusarium graminearum strains were obtained after UV mutagenesis and selection on carbendazim (MBC)-amended medium. In vitro bioassays resulted in the identification of two resistant phenotypes that were highly HR (Rf: 40-170, based on EC50) and moderately MR (Rf: 10-20) resistant to carbendazim. Cross resistance studies with other fungicides showed that all mutant strains tested were also resistant to other BMZs, such as benomyl and thiabendazole, but retained their parental sensitivity to fungicides belonging to other chemical groups. A point mutation at codon 6 (His6Asn) was found in the β2-tubulin gene of MR isolates while another mutation at codon 200 (Phe200Tyr) was present in one MR and one HR isolates. Interestingly, low temperatures suppressed MBC-resistance in all isolates bearing the H6N mutation. The three-dimensional homology model of the wild-type and mutants of β-tubulins were constructed, and the possible carbendazim binding site was analyzed. Studies on fitness parameters showed that the mutation(s) for resistance to BMZs did not affect the mycelial growth rate whereas adverse effects were found in sporulation and conidial germination in most of the resistant mutants. Pathogenicity tests on corn cobs revealed that mutants were less or equally aggressive to the wild-type strain but expressed their BMZ-resistance after inoculation on maize cobs treated with MBC. Analysis of mycotoxin production by high performance liquid chromatography revealed that only two HR strains produced zearalenone (ZEA) at concentrations similar to that of the wild-type strain, while no ZEA levels were detected in the rest of the mutants. Copyright © 2015 Elsevier Inc. All rights reserved.

TumorNext: A comprehensive tumor profiling assay that incorporates high resolution copy number analysis and germline status to improve testing accuracy

PubMed Central

Gray, Phillip N.; Vuong, Huy; Tsai, Pei; Lu, Hsaio-Mei; Mu, Wenbo; Hsuan, Vickie; Hoo, Jayne; Shah, Swati; Uyeda, Lisa; Fox, Susanne; Patel, Harshil; Janicek, Mike; Brown, Sandra; Dobrea, Lavinia; Wagman, Lawrence; Plimack, Elizabeth; Mehra, Ranee; Golemis, Erica A.; Bilusic, Marijo; Zibelman, Matthew; Elliott, Aaron

2016-01-01

The development of targeted therapies for both germline and somatic DNA mutations has increased the need for molecular profiling assays to determine the mutational status of specific genes. Moreover, the potential of off-label prescription of targeted therapies favors classifying tumors based on DNA alterations rather than traditional tissue pathology. Here we describe the analytical validation of a custom probe-based NGS tumor panel, TumorNext, which can detect single nucleotide variants, small insertions and deletions in 142 genes that are frequently mutated in somatic and/or germline cancers. TumorNext also detects gene fusions and structural variants, such as tandem duplications and inversions, in 15 frequently disrupted oncogenes and tumor suppressors. The assay uses a matched control and custom bioinformatics pipeline to differentiate between somatic and germline mutations, allowing precise variant classification. We tested 170 previously characterized samples, of which > 95% were formalin-fixed paraffin embedded tissue from 8 different cancer types, and highlight examples where lack of germline status may have led to the inappropriate prescription of therapy. We also describe the validation of the Affymetrix OncoScan platform, an array technology for high resolution copy number variant detection for use in parallel with the NGS panel that can detect single copy amplifications and hemizygous deletions. We analyzed 80 previously characterized formalin-fixed paraffin-embedded specimens and provide examples of hemizygous deletion detection in samples with known pathogenic germline mutations. Thus, the TumorNext combined approach of NGS and OncoScan potentially allows for the identification of the “second hit” in hereditary cancer patients. PMID:27626691

High Frequency of Copy-Neutral Loss of Heterozygosity in Patients with Myelofibrosis.

PubMed

Rego de Paula Junior, Milton; Nonino, Alexandre; Minuncio Nascimento, Juliana; Bonadio, Raphael S; Pic-Taylor, Aline; de Oliveira, Silviene F; Wellerson Pereira, Rinaldo; do Couto Mascarenhas, Cintia; Forte Mazzeu, Juliana

2018-01-01

Myelofibrosis is the rarest and most severe type of Philadelphia-negative classical myeloproliferative neoplasms. Although mutually exclusive driver mutations in JAK2, MPL, or CALR that activate JAK-STAT pathway have been related to the pathogenesis of the disease, chromosome abnormalities have also been associated with the phenotype and prognosis of the disease. Here, we report the use of a chromosomal microarray platform consisting of both oligo and SNP probes to improve the detection of chromosome abnormalities in patients with myelofibrosis. Sixteen patients with myelofibrosis were tested, and the results were compared to karyotype analysis. Driver mutations in JAK2, MPL, or CALR were investigated by PCR and MLPA. Conventional cytogenetics revealed chromosome abnormalities in 3 out of 16 cases (18.7%), while chromosomal microarray analysis detected copy-number variations (CNV) or copy-neutral loss of heterozygosity (CN-LOH) alterations in 11 out of 16 (68.7%) patients. These included 43 CN-LOH, 14 deletions, 1 trisomy, and 1 duplication. Ten patients showed multiple chromosomal abnormalities, varying from 2 to 13 CNVs or CN-LOHs. Mutational status for JAK2, CALR, and MPL by MLPA revealed a total of 3/16 (18.7%) patients positive for the JAK2 V617F mutation, 9 with CALR deletion or insertion and 1 positive for MPL mutation. Considering that most of the CNVs identified were smaller than the karyotype resolution and the high frequency of CN-LOHs in our study, we propose that chromosomal microarray platforms that combine oligos and SNP should be used as a first-tier genetic test in patients with myelofibrosis. © 2018 S. Karger AG, Basel.

Clinical and genetic characteristics of chinese patients with Birt-Hogg-Dubé syndrome.

PubMed

Liu, Yaping; Xu, Zhiyan; Feng, Ruie; Zhan, Yongzhong; Wang, Jun; Li, Guozhen; Li, Xue; Zhang, Weihong; Hu, Xiaowen; Tian, Xinlun; Xu, Kai-Feng; Zhang, Xue

2017-05-30

Birt-Hogg-Dubé syndrome (BHD) is an autosomal dominant disorder, the main manifestations of which are fibrofolliculomas, renal tumors, pulmonary cysts and recurrent pneumothorax. The known causative gene for BHD syndrome is the folliculin (FLCN) gene on chromosome 17p11.2. Studies of the FLCN mutation for BHD syndrome are less prevalent in Chinese populations than in Caucasian populations. Our study aims to investigate the genotype spectrum in a group of Chinese patients with BHD. We enrolled 51 patients with symptoms highly suggestive of BHD from January 2014 to February 2017. The FLCN gene was examined using PCR and Sanger sequencing in every patient, for those whose Sanger sequencing showed negative mutation results, multiplex ligation-dependent probe amplification (MLPA) testing was conducted to detect any losses of large segments. Among the 51 patients, 27 had FLCN germline mutations. In total, 20 mutations were identified: 14 were novel mutations, including 3 splice acceptor site mutations, 2 different deletions, 6 nonsense mutations, 1 missense mutation, 1 small insertion, and 1 deletion of the whole exon 8. We found a similar genotype spectrum but different mutant loci in Chinese patients with BHD compared with European and American patients, thus providing stronger evidence for the clinical molecular diagnosis of BHD in China. It suggests that mutation analysis of the FLCN gene should be systematically conducted in patients with cystic lung diseases.

Contribution of mammography to MRI screening in BRCA mutation carriers by BRCA status and age: individual patient data meta-analysis

PubMed Central

Phi, Xuan-Anh; Saadatmand, Sepideh; De Bock, Geertruida H; Warner, Ellen; Sardanelli, Francesco; Leach, Martin O; Riedl, Christopher C; Trop, Isabelle; Hooning, Maartje J; Mandel, Rodica; Santoro, Filippo; Kwan-Lim, Gek; Helbich, Thomas H; Tilanus-Linthorst, Madeleine MA; van den Heuvel, Edwin R; Houssami, Nehmat

2016-01-01

Background: We investigated the additional contribution of mammography to screening accuracy in BRCA1/2 mutation carriers screened with MRI at different ages using individual patient data from six high-risk screening trials. Methods: Sensitivity and specificity of MRI, mammography and the combination of these tests were compared stratified for BRCA mutation and age using generalised linear mixed models with random effect for studies. Number of screens needed (NSN) for additional mammography-only detected cancer was estimated. Results: In BRCA1/2 mutation carriers of all ages (BRCA1=1219 and BRCA2=732), adding mammography to MRI did not significantly increase screening sensitivity (increased by 3.9% in BRCA1 and 12.6% in BRCA2 mutation carriers, P>0.05). However, in women with BRCA2 mutation younger than 40 years, one-third of breast cancers were detected by mammography only. Number of screens needed for mammography to detect one breast cancer not detected by MRI was much higher for BRCA1 compared with BRCA2 mutation carriers at initial and repeat screening. Conclusions: Additional screening sensitivity from mammography above that from MRI is limited in BRCA1 mutation carriers, whereas mammography contributes to screening sensitivity in BRCA2 mutation carriers, especially those ⩽40 years. The evidence from our work highlights that a differential screening schedule by BRCA status is worth considering. PMID:26908327

Extensive molecular analysis suggested the strong genetic heterogeneity of idiopathic chronic pancreatitis.

PubMed

Sofia, Valentina Maria; Da Sacco, Letizia; Surace, Cecilia; Tomaiuolo, Anna Cristina; Genovese, Silvia; Grotta, Simona; Gnazzo, Maria; Petrocchi, Stefano; Ciocca, Laura; Alghisi, Federico; Montemitro, Enza; Martemucci, Luigi; Elce, Ausilia; Lucidi, Vincenzina; Castaldo, Giuseppe; Angioni, Adriano

2016-05-26

Genetic features of Chronic Pancreatitis (CP) have been extensively investigated mainly testing genes associated to the trypsinogen activation pathway. However, different molecular pathways involving other genes may be implicated in CP pathogenesis. 80 patients with Idiopathic CP were investigated using Next Generation Sequencing approach with a panel of 70 genes related to six different pancreatic pathways: premature activation of trypsinogen; modifier genes of Cystic Fibrosis phenotype; pancreatic secretion and ion homeostasis; Calcium signalling and zymogen granules exocytosis; autophagy; autoimmune pancreatitis related genes. We detected mutations in 34 out of 70 genes examined; 64/80 patients (80.0%) were positive for mutations in one or more genes, 16/80 patients (20.0%) had no mutations. Mutations in CFTR were detected in 32/80 patients (40.0%) and 22 of them exhibited at least one mutation in genes of other pancreatic pathways. Of the remaining 48 patients, 13/80 (16.3%) had mutations in genes involved in premature activation of trypsinogen and 19/80 (23.8%) had mutations only in genes of the other pathways: 38/64 patients positive for mutations showed variants in two or more genes (59.3%). Our data, although to be extended with functional analysis of novel mutations, suggest a high rate of genetic heterogeneity in chronic pancreatitis and that trans-heterozygosity may predispose to the idiopathic CP phenotype.

Contribution of mammography to MRI screening in BRCA mutation carriers by BRCA status and age: individual patient data meta-analysis.

PubMed

Phi, Xuan-Anh; Saadatmand, Sepideh; De Bock, Geertruida H; Warner, Ellen; Sardanelli, Francesco; Leach, Martin O; Riedl, Christopher C; Trop, Isabelle; Hooning, Maartje J; Mandel, Rodica; Santoro, Filippo; Kwan-Lim, Gek; Helbich, Thomas H; Tilanus-Linthorst, Madeleine M A; van den Heuvel, Edwin R; Houssami, Nehmat

2016-03-15

We investigated the additional contribution of mammography to screening accuracy in BRCA1/2 mutation carriers screened with MRI at different ages using individual patient data from six high-risk screening trials. Sensitivity and specificity of MRI, mammography and the combination of these tests were compared stratified for BRCA mutation and age using generalised linear mixed models with random effect for studies. Number of screens needed (NSN) for additional mammography-only detected cancer was estimated. In BRCA1/2 mutation carriers of all ages (BRCA1 = 1,219 and BRCA2 = 732), adding mammography to MRI did not significantly increase screening sensitivity (increased by 3.9% in BRCA1 and 12.6% in BRCA2 mutation carriers, P > 0.05). However, in women with BRCA2 mutation younger than 40 years, one-third of breast cancers were detected by mammography only. Number of screens needed for mammography to detect one breast cancer not detected by MRI was much higher for BRCA1 compared with BRCA2 mutation carriers at initial and repeat screening. Additional screening sensitivity from mammography above that from MRI is limited in BRCA1 mutation carriers, whereas mammography contributes to screening sensitivity in BRCA2 mutation carriers, especially those ⩽ 40 years. The evidence from our work highlights that a differential screening schedule by BRCA status is worth considering.

The genomic basis of adaptation to the fitness cost of rifampicin resistance in Pseudomonas aeruginosa

PubMed Central

Toll-Riera, Macarena; Heilbron, Karl

2016-01-01

Antibiotic resistance carries a fitness cost that must be overcome in order for resistance to persist over the long term. Compensatory mutations that recover the functional defects associated with resistance mutations have been argued to play a key role in overcoming the cost of resistance, but compensatory mutations are expected to be rare relative to generally beneficial mutations that increase fitness, irrespective of antibiotic resistance. Given this asymmetry, population genetics theory predicts that populations should adapt by compensatory mutations when the cost of resistance is large, whereas generally beneficial mutations should drive adaptation when the cost of resistance is small. We tested this prediction by determining the genomic mechanisms underpinning adaptation to antibiotic-free conditions in populations of the pathogenic bacterium Pseudomonas aeruginosa that carry costly antibiotic resistance mutations. Whole-genome sequencing revealed that populations founded by high-cost rifampicin-resistant mutants adapted via compensatory mutations in three genes of the RNA polymerase core enzyme, whereas populations founded by low-cost mutants adapted by generally beneficial mutations, predominantly in the quorum-sensing transcriptional regulator gene lasR. Even though the importance of compensatory evolution in maintaining resistance has been widely recognized, our study shows that the roles of general adaptation in maintaining resistance should not be underestimated and highlights the need to understand how selection at other sites in the genome influences the dynamics of resistance alleles in clinical settings. PMID:26763710

Genome-Wide Mutation Rate Response to pH Change in the Coral Reef Pathogen Vibrio shilonii AK1.

PubMed

Strauss, Chloe; Long, Hongan; Patterson, Caitlyn E; Te, Ronald; Lynch, Michael

2017-08-22

Recent application of mutation accumulation techniques combined with whole-genome sequencing (MA/WGS) has greatly promoted studies of spontaneous mutation. However, such explorations have rarely been conducted on marine organisms, and it is unclear how marine habitats have influenced genome stability. This report resolves the mutation rate and spectrum of the coral reef pathogen Vibrio shilonii , which causes coral bleaching and endangers the biodiversity maintained by coral reefs. We found that its mutation rate and spectrum are highly similar to those of other studied bacteria from various habitats, despite the saline environment. The mutational properties of this marine bacterium are thus controlled by other general evolutionary forces such as natural selection and genetic drift. We also found that as pH drops, the mutation rate decreases and the mutation spectrum is biased in the direction of generating G/C nucleotides. This implies that evolutionary features of this organism and perhaps other marine microbes might be altered by the increasingly acidic ocean water caused by excess CO 2 emission. Nonetheless, further exploration is needed as the pH range tested in this study was rather narrow and many other possible mutation determinants, such as carbonate increase, are associated with ocean acidification. IMPORTANCE This study explored the pH dependence of a bacterial genome-wide mutation rate. We discovered that the genome-wide rates of appearance of most mutation types decrease linearly and that the mutation spectrum is biased in generating more G/C nucleotides with pH drop in the coral reef pathogen V. shilonii . Copyright © 2017 Strauss et al.

Uncommon EGFR mutations in cytological specimens of 1,874 newly diagnosed Indonesian lung cancer patients

PubMed Central

Syahruddin, Elisna; Wulandari, Laksmi; Sri Muktiati, Nunuk; Rima, Ana; Soeroso, Noni; Ermayanti, Sabrina; Levi, Michael; Hidajat, Heriawaty; Widjajahakim, Grace; Utomo, Ahmad Rusdan Handoyo

2018-01-01

Purpose We aimed to evaluate the distribution of individual epidermal growth factor receptor (EGFR) mutation subtypes found in routine cytological specimens. Patients and methods A retrospective audit was performed on EGFR testing results of 1,874 consecutive cytological samples of newly diagnosed or treatment-naïve Indonesian lung cancer patients (years 2015–2016). Testing was performed by ISO15189 accredited central laboratory. Results Overall test failure rate was 5.1%, with the highest failure (7.1%) observed in pleural effusion and lowest (1.6%) in needle aspiration samples. EGFR mutation frequency was 44.4%. Tyrosine kinase inhibitor (TKI)-sensitive common EGFR mutations (ins/dels exon 19, L858R) and uncommon mutations (G719X, T790M, L861Q) contributed 57.1% and 29%, respectively. Approximately 13.9% of mutation-positive patients carried a mixture of common and uncommon mutations. Women had higher EGFR mutation rate (52.9%) vs men (39.1%; p<0.05). In contrast, uncommon mutations conferring either TKI responsive (G719X, L861Q) or TKI resistance (T790M, exon 20 insertions) were consistently more frequent in men than in women (67.3% vs 32.7% or 69.4% vs 30.6%; p<0.05). Up to 10% EGFR mutation–positive patients had baseline single mutation T790M, exon 20 insertion, or in coexistence with TKI-sensitive mutations. Up to 9% patients had complex or multiple EGFR mutations, whereby 48.7% patients harbored TKI-resistant mutations. One patient presented third-generation TKI-resistant mutation L792F simultaneously with T790M. Conclusion Routine diagnostic cytological techniques yielded similar success rate to detect EGFR mutations. Uncommon EGFR mutations were frequent events in Indonesian lung cancer patients. PMID:29615847

[Mutation Analysis of 19 STR Loci in 20 723 Cases of Paternity Testing].

PubMed

Bi, J; Chang, J J; Li, M X; Yu, C Y

2017-06-01

To observe and analyze the confirmed cases of paternity testing, and to explore the mutation rules of STR loci. The mutant STR loci were screened from 20 723 confirmed cases of paternity testing by Goldeneye 20A system．The mutation rates, and the sources, fragment length, steps and increased or decreased repeat sequences of mutant alleles were counted for the analysis of the characteristics of mutation-related factors. A total of 548 mutations were found on 19 STR loci, and 557 mutation events were observed. The loci mutation rate was 0.07‰-2.23‰. The ratio of paternal to maternal mutant events was 3.06:1. One step mutation was the main mutation, and the number of the increased repeat sequences was almost the same as the decreased repeat sequences. The repeat sequences were more likely to decrease in two steps mutation and above. Mutation mainly occurred in the medium allele, and the number of the increased repeat sequences was almost the same as the decreased repeat sequences. In long allele mutations, the decreased repeat sequences were significantly more than the increased repeat sequences. The number of the increased repeat sequences was almost the same as the decreased repeat sequences in paternal mutation, while the decreased repeat sequences were more than the increased in maternal mutation. There are significant differences in the mutation rate of each locus. When one or two loci do not conform to the genetic law, other detection system should be added, and PI value should be calculated combined with the information of the mutate STR loci in order to further clarify the identification opinions. Copyright© by the Editorial Department of Journal of Forensic Medicine

A Korean patient with glutaric aciduria type 1 with a novel mutation in the glutaryl CoA dehydrogenase gene.

PubMed

Kim, Hee Su; Yu, Hee Joon; Lee, Jeehun; Park, Hyung-Doo; Kim, Ji Hye; Shin, Hyung-Jin; Jin, Dong Kyu; Lee, Munhyang

2014-01-01

Mutations in the glutaryl-CoA dehydrogenase gene can result in Glutaric aciduria type 1(GA 1) by accumulation of glutaric acid, 3-hydroxyglutaric acid (3-OH-GA), and glutarylcarnitine (C5DC). GA 1 is characterized by macrocephaly, subdural hemorrhage (SDH), and dystonic movement disorder after acute encephalopathic crisis. We report a Korean patient with GA1 and a novel mutation. A 16-month-old boy presented with SDH, macrocephaly, and developmental delay. In the neurologic examination, the patient had mild axial hypotonia, but otherwise normal neurologic functions. The brain MRI showed large amounts of bilateral SDH and high signal intensity in both basal ganglia and thalamus. Metabolic screening tests detected highly elevated urinary GA levels but 3-OH-glutaric acid was normal. C5DC was 0.94 μM/L (reference range < 0.3 μM/L). The patient had compound heterozygous mutations of the GCDH gene: p.Arg257Gln (c.770G>A) and p.Cys308Arg (c.922T>C). p.Cys308Arg is a novel mutation; reports of p.Arg257Gln were also rare both in Caucasians and Asian populations. In summary, we hereby report one Korean patient with GA1 with clinical, biochemical, and radiologic characteristics confirmed by genetic analysis.

Whole-exome sequencing and targeted gene sequencing provide insights into the role of PALB2 as a male breast cancer susceptibility gene.

PubMed

Silvestri, Valentina; Zelli, Veronica; Valentini, Virginia; Rizzolo, Piera; Navazio, Anna Sara; Coppa, Anna; Agata, Simona; Oliani, Cristina; Barana, Daniela; Castrignanò, Tiziana; Viel, Alessandra; Russo, Antonio; Tibiletti, Maria Grazia; Zanna, Ines; Masala, Giovanna; Cortesi, Laura; Manoukian, Siranoush; Azzollini, Jacopo; Peissel, Bernard; Bonanni, Bernardo; Peterlongo, Paolo; Radice, Paolo; Palli, Domenico; Giannini, Giuseppe; Chillemi, Giovanni; Montagna, Marco; Ottini, Laura

2017-01-01

Male breast cancer (MBC) is a rare disease whose etiology appears to be largely associated with genetic factors. BRCA1 and BRCA2 mutations account for about 10% of all MBC cases. Thus, a fraction of MBC cases are expected to be due to genetic factors not yet identified. To further explain the genetic susceptibility for MBC, whole-exome sequencing (WES) and targeted gene sequencing were applied to high-risk, BRCA1/2 mutation-negative MBC cases. Germ-line DNA of 1 male and 2 female BRCA1/2 mutation-negative breast cancer (BC) cases from a pedigree showing a first-degree family history of MBC was analyzed with WES. Targeted gene sequencing for the validation of WES results was performed for 48 high-risk, BRCA1/2 mutation-negative MBC cases from an Italian multicenter study of MBC. A case-control series of 433 BRCA1/2 mutation-negative MBC and female breast cancer (FBC) cases and 849 male and female controls was included in the study. WES in the family identified the partner and localizer of BRCA2 (PALB2) c.419delA truncating mutation carried by the proband, her father, and her paternal uncle (all affected with BC) and the N-acetyltransferase 1 (NAT1) c.97C>T nonsense mutation carried by the proband's maternal aunt. Targeted PALB2 sequencing detected the c.1984A>T nonsense mutation in 1 of the 48 BRCA1/2 mutation-negative MBC cases. NAT1 c.97C>T was not found in the case-control series. These results add strength to the evidence showing that PALB2 is involved in BC risk for both sexes and indicate that consideration should be given to clinical testing of PALB2 for BRCA1/2 mutation-negative families with multiple MBC and FBC cases. Cancer 2017;123:210-218. © 2016 American Cancer Society. © 2016 American Cancer Society.

Combined mutation and copy-number variation detection by targeted next-generation sequencing in uveal melanoma.

PubMed

Smit, Kyra N; van Poppelen, Natasha M; Vaarwater, Jolanda; Verdijk, Robert; van Marion, Ronald; Kalirai, Helen; Coupland, Sarah E; Thornton, Sophie; Farquhar, Neil; Dubbink, Hendrikus-Jan; Paridaens, Dion; de Klein, Annelies; Kiliç, Emine

2018-05-01

Uveal melanoma is a highly aggressive cancer of the eye, in which nearly 50% of the patients die from metastasis. It is the most common type of primary eye cancer in adults. Chromosome and mutation status have been shown to correlate with the disease-free survival. Loss of chromosome 3 and inactivating mutations in BAP1, which is located on chromosome 3, are strongly associated with 'high-risk' tumors that metastasize early. Other genes often involved in uveal melanoma are SF3B1 and EIF1AX, which are found to be mutated in intermediate- and low-risk tumors, respectively. To obtain genetic information of all genes in one test, we developed a targeted sequencing method that can detect mutations in uveal melanoma genes and chromosomal anomalies in chromosome 1, 3, and 8. With as little as 10 ng DNA, we obtained enough coverage on all genes to detect mutations, such as substitutions, deletions, and insertions. These results were validated with Sanger sequencing in 28 samples. In >90% of the cases, the BAP1 mutation status corresponded to the BAP1 immunohistochemistry. The results obtained in the Ion Torrent single-nucleotide polymorphism assay were confirmed with several other techniques, such as fluorescence in situ hybridization, multiplex ligation-dependent probe amplification, and Illumina SNP array. By validating our assay in 27 formalin-fixed paraffin-embedded and 43 fresh uveal melanomas, we show that mutations and chromosome status can reliably be obtained using targeted next-generation sequencing. Implementing this technique as a diagnostic pathology application for uveal melanoma will allow prediction of the patients' metastatic risk and potentially assess eligibility for new therapies.

Tumor Mutational Burden Guides Therapy in a Treatment Refractory POLE-Mutant Uterine Carcinosarcoma.

PubMed

Bhangoo, Munveer S; Boasberg, Peter; Mehta, Pareen; Elvin, Julia A; Ali, Siraj M; Wu, Winnie; Klempner, Samuel J

2018-05-01

Gynecologic carcinosarcomas, previously known as malignant mixed Müllerian tumors, are uncommon malignancies that demonstrate an aggressive biology and lack a standard therapeutic approach. Molecular analyses have revealed recurrent alterations in chromatin remodeling genes, but clinical support for therapeutic significance is lacking. We prospectively identified a patient with refractory uterine carcinosarcoma whose tumor was subject to molecular profiling at diagnosis and again at radiographic progression. Initial molecular testing did not assess tumor mutational burden, DNA polymerase ɛ ( POLE ), or microsatellite status. After the failure of several lines of chemotherapy, comprehensive genomic profiling of a repeat biopsy identified two missense mutations of the exonuclease domain of POLE (P286R and T323A). Tumor mutational burden was elevated (169 mutations per DNA megabase), consistent with an ultramutator phenotype. As seen in previously reported POLE -endometrioid cases, our patient harbored alterations in PIK3CA , ARID1A , and PTEN and was microsatellite stable, with appreciable tumor-infiltrating lymphocytes. She achieved an ongoing durable response with pembrolizumab. This is the first report of programmed cell death protein 1 response in uterine carcinosarcoma. Uterine carcinosarcoma is an uncommon and aggressive histologic variant of endometrial carcinoma with a poor prognosis.Inactivating DNA polymerase ɛ ( POLE ) mutations have been associated with high tumor mutational burden (TMB) and response to immune checkpoint inhibition.To the authors' knowledge, this is the first report of response to immune checkpoint inhibitor therapy in a patient with uterine carcinosarcoma.This case further supports expanding genomic profiling to include assessment of tumor mutational burden across tumor types, given the potential for immune checkpoint inhibitor therapy in TMB-high tumors. © AlphaMed Press 2018.

Aspect ratio has no effect on genotoxicity of multi-wall carbon nanotubes.

PubMed

Kim, Jin Sik; Lee, Kyu; Lee, Young Hee; Cho, Hyun Sun; Kim, Ki Heon; Choi, Kyung Hee; Lee, Sang Hee; Song, Kyung Seuk; Kang, Chang Soo; Yu, Il Je

2011-07-01

Carbon nanotubes (CNTs) have specific physico-chemical and electrical properties that are useful for telecommunications, medicine, materials, manufacturing processes and the environmental and energy sectors. Yet, despite their many advantages, it is also important to determine whether CNTs may represent a hazard to the environment and human health. Like asbestos, the aspect ratio (length:diameter) and metal components of CNTs are known to have an effect on the toxicity of carbon nanotubes. Thus, to evaluate the toxic potential of CNTs in relation to their aspect ratio and metal contamination, in vivo and in vitro genotoxicity tests were conducted using high-aspect-ratio (diameter: 10-15 nm, length: ~10 μm) and low-aspect-ratio multi-wall carbon nanotubes (MWCNTs, diameter: 10-15 nm, length: ~150 nm) according to OECD test guidelines 471 (bacterial reverse mutation test), 473 (in vitro chromosome aberration test), and 474 (in vivo micronuclei test) with a good laboratory practice system. To determine the treatment concentration for all the tests, a solubility and dispersive test was performed, and a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) solution found to be more suitable than distilled water. Neither the high- nor the low-aspect-ratio MWCNTs induced any genotoxicity in a bacterial reverse mutation test (~1,000 μg/plate), in vitro chromosome aberration test (without S9: ~6.25 μg/ml, with S9: ~50 μg/ml), or in vivo micronuclei test (~50 mg/kg). However, the high-aspect-ratio MWCNTs were found to be more toxic than the low-aspect-ratio MWCNTs. Thus, while high-aspect-ratio MWCNTs do not induce direct genotoxicity or metabolic activation-mediated genotoxicity, genotoxicity could still be induced indirectly through oxidative stress or inflammation.

Targeted Prostate Cancer Screening in BRCA1 and BRCA2 Mutation Carriers: Results from the Initial Screening Round of the IMPACT Study

PubMed Central

Bancroft, Elizabeth K.; Page, Elizabeth C.; Castro, Elena; Lilja, Hans; Vickers, Andrew; Sjoberg, Daniel; Assel, Melissa; Foster, Christopher S.; Mitchell, Gillian; Drew, Kate; Mæhle, Lovise; Axcrona, Karol; Evans, D. Gareth; Bulman, Barbara; Eccles, Diana; McBride, Donna; van Asperen, Christi; Vasen, Hans; Kiemeney, Lambertus A.; Ringelberg, Janneke; Cybulski, Cezary; Wokolorczyk, Dominika; Selkirk, Christina; Hulick, Peter J.; Bojesen, Anders; Skytte, Anne-Bine; Lam, Jimmy; Taylor, Louise; Oldenburg, Rogier; Cremers, Ruben; Verhaegh, Gerald; van Zelst-Stams, Wendy A.; Oosterwijk, Jan C.; Blanco, Ignacio; Salinas, Monica; Cook, Jackie; Rosario, Derek J.; Buys, Saundra; Conner, Tom; Ausems, Margreet G.; Ong, Kai-ren; Hoffman, Jonathan; Domchek, Susan; Powers, Jacquelyn; Teixeira, Manuel R.; Maia, Sofia; Foulkes, William D.; Taherian, Nassim; Ruijs, Marielle; den Enden, Apollonia T. Helderman-van; Izatt, Louise; Davidson, Rosemarie; Adank, Muriel A.; Walker, Lisa; Schmutzler, Rita; Tucker, Kathy; Kirk, Judy; Hodgson, Shirley; Harris, Marion; Douglas, Fiona; Lindeman, Geoffrey J.; Zgajnar, Janez; Tischkowitz, Marc; Clowes, Virginia E.; Susman, Rachel; Ramón y Cajal, Teresa; Patcher, Nicholas; Gadea, Neus; Spigelman, Allan; van Os, Theo; Liljegren, Annelie; Side, Lucy; Brewer, Carole; Brady, Angela F.; Donaldson, Alan; Stefansdottir, Vigdis; Friedman, Eitan; Chen-Shtoyerman, Rakefet; Amor, David J.; Copakova, Lucia; Barwell, Julian; Giri, Veda N.; Murthy, Vedang; Nicolai, Nicola; Teo, Soo-Hwang; Greenhalgh, Lynn; Strom, Sara; Henderson, Alex; McGrath, John; Gallagher, David; Aaronson, Neil; Ardern-Jones, Audrey; Bangma, Chris; Dearnaley, David; Costello, Philandra; Eyfjord, Jorunn; Rothwell, Jeanette; Falconer, Alison; Gronberg, Henrik; Hamdy, Freddie C.; Johannsson, Oskar; Khoo, Vincent; Kote-Jarai, Zsofia; Lubinski, Jan; Axcrona, Ulrika; Melia, Jane; McKinley, Joanne; Mitra, Anita V.; Moynihan, Clare; Rennert, Gad; Suri, Mohnish; Wilson, Penny; Killick, Emma; Moss, Sue; Eeles, Rosalind A.

2014-01-01

Background Men with germline breast cancer 1, early onset (BRCA1) or breast cancer 2, early onset (BRCA2) gene mutations have a higher risk of developing prostate cancer (PCa) than noncarriers. IMPACT (Identification of Men with a genetic predisposition to ProstAte Cancer: Targeted screening in BRCA1/2 mutation carriers and controls) is an international consortium of 62 centres in 20 countries evaluating the use of targeted PCa screening in men with BRCA1/2 mutations. Objective To report the first year's screening results for all men at enrolment in the study. Design, setting and participants We recruited men aged 40–69 yr with germline BRCA1/2 mutations and a control group of men who have tested negative for a pathogenic BRCA1 or BRCA2 mutation known to be present in their families. All men underwent prostate-specific antigen (PSA) testing at enrolment, and those men with PSA >3 ng/ml were offered prostate biopsy. Outcome measurements and statistical analysis PSA levels, PCa incidence, and tumour characteristics were evaluated. The Fisher exact test was used to compare the number of PCa cases among groups and the differences among disease types. Results and limitations We recruited 2481 men (791 BRCA1 carriers, 531 BRCA1 controls; 731 BRCA2 carriers, 428 BRCA2 controls). A total of 199 men (8%) presented with PSA >3.0 ng/ml, 162 biopsies were performed, and 59 PCas were diagnosed (18 BRCA1 carriers, 10 BRCA1 controls; 24 BRCA2 carriers, 7 BRCA2 controls); 66% of the tumours were classified as intermediate- or high-risk disease. The positive predictive value (PPV) for biopsy using a PSA threshold of 3.0 ng/ml in BRCA2 mutation carriers was 48%—double the PPV reported in population screening studies. A significant difference in detecting intermediate- or high-risk disease was observed in BRCA2 carriers. Ninety-five percent of the men were white, thus the results cannot be generalised to all ethnic groups. Conclusions The IMPACT screening network will be useful for targeted PCa screening studies in men with germline genetic risk variants as they are discovered. These preliminary results support the use of targeted PSA screening based on BRCA genotype and show that this screening yields a high proportion of aggressive disease. Patient summary In this report, we demonstrate that germline genetic markers can be used to identify men at higher risk of prostate cancer. Targeting screening at these men resulted in the identification of tumours that were more likely to require treatment. PMID:24484606

iMARS--mutation analysis reporting software: an analysis of spontaneous cII mutation spectra.

PubMed

Morgan, Claire; Lewis, Paul D

2006-01-31

The sensitivity of any mutational assay is determined by the level at which spontaneous mutations occur in the corresponding untreated controls. Establishing the type and frequency at which mutations occur naturally within a test system is essential if one is to draw scientifically sound conclusions regarding chemically induced mutations. Currently, mutation-spectra analysis is laborious and time-consuming. Thus, we have developed iMARS, a comprehensive mutation-spectrum analysis package that utilises routinely used methodologies and visualisation tools. To demonstrate the use and capabilities of iMARS, we have analysed the distribution, types and sequence context of spontaneous base substitutions derived from the cII gene mutation assay in transgenic animals. Analysis of spontaneous mutation spectra revealed variation both within and between the transgenic rodent test systems Big Blue Mouse, MutaMouse and Big Blue Rat. The most common spontaneous base substitutions were G:C-->A:T transitions and G:C-->T:A transversions. All Big Blue Mouse spectra were significantly different from each other by distribution and nearly all by mutation type, whereas the converse was true for the other test systems. Twenty-eight mutation hotspots were observed across all spectra generally occurring in CG, GA/TC, GG and GC dinucleotides. A mutation hotspot at nucleotide 212 occurred at a higher frequency in MutaMouse and Big Blue Rat. In addition, CG dinucleotides were the most mutable in all spectra except two Big Blue Mouse spectra. Thus, spontaneous base-substitution spectra showed more variation in distribution, type and sequence context in Big Blue Mouse relative to spectra derived from MutaMouse and Big Blue Rat. The results of our analysis provide a baseline reference for mutation studies utilising the cII gene in transgenic rodent models. The potential differences in spontaneous base-substitution spectra should be considered when making comparisons between these test systems. The ease at which iMARS has allowed us to carry out an exhaustive investigation to assess mutation distribution, mutation type, strand bias, target sequences and motifs, as well as predict mutation hotspots provides us with a valuable tool in helping to distinguish true chemically induced hotspots from background mutations and gives a true reflection of mutation frequency.

Non-dystrophic myotonia: prospective study of objective and patient reported outcomes.

PubMed

Trivedi, Jaya R; Bundy, Brian; Statland, Jeffrey; Salajegheh, Mohammad; Rayan, Dipa Raja; Venance, Shannon L; Wang, Yunxia; Fialho, Doreen; Matthews, Emma; Cleland, James; Gorham, Nina; Herbelin, Laura; Cannon, Stephen; Amato, Anthony; Griggs, Robert C; Hanna, Michael G; Barohn, Richard J

2013-07-01

Non-dystrophic myotonias are rare diseases caused by mutations in skeletal muscle chloride and sodium ion channels with considerable phenotypic overlap between diseases. Few prospective studies have evaluated the sensitivity of symptoms and signs of myotonia in a large cohort of patients. We performed a prospective observational study of 95 participants with definite or clinically suspected non-dystrophic myotonia recruited from six sites in the USA, UK and Canada between March 2006 and March 2009. We used the common infrastructure and data elements provided by the NIH-funded Rare Disease Clinical Research Network. Outcomes included a standardized symptom interview and physical exam; the Short Form-36 and the Individualized Neuromuscular Quality of Life instruments; electrophysiological short and prolonged exercise tests; manual muscle testing; and a modified get-up-and-go test. Thirty-two participants had chloride channel mutations, 34 had sodium channel mutations, nine had myotonic dystrophy type 2, one had myotonic dystrophy type 1, and 17 had no identified mutation. Phenotype comparisons were restricted to those with sodium channel mutations, chloride channel mutations, and myotonic dystrophy type 2. Muscle stiffness was the most prominent symptom overall, seen in 66.7% to 100% of participants. In comparison with chloride channel mutations, participants with sodium mutations had an earlier age of onset of stiffness (5 years versus 10 years), frequent eye closure myotonia (73.5% versus 25%), more impairment on the Individualized Neuromuscular Quality of Life summary score (20.0 versus 9.44), and paradoxical eye closure myotonia (50% versus 0%). Handgrip myotonia was seen in three-quarters of participants, with warm up of myotonia in 75% chloride channel mutations, but also 35.3% of sodium channel mutations. The short exercise test showed ≥10% decrement in the compound muscle action potential amplitude in 59.3% of chloride channel participants compared with 27.6% of sodium channel participants, which increased post-cooling to 57.6% in sodium channel mutations. In evaluation of patients with clinical and electrical myotonia, despite considerable phenotypic overlap, the presence of eye closure myotonia, paradoxical myotonia, and an increase in short exercise test sensitivity post-cooling suggest sodium channel mutations. Outcomes designed to measure stiffness or the electrophysiological correlates of stiffness may prove useful for future clinical trials, regardless of underlying mutation, and include patient-reported stiffness, bedside manoeuvres to evaluate myotonia, muscle specific quality of life instruments and short exercise testing.

A novel FKRP-related muscular dystrophy founder mutation in South African Afrikaner patients with a phenotype suggestive of a dystrophinopathy.

PubMed

Mudau, M M; Essop, F; Krause, A

2016-12-21

Fukutin-related protein (FKRP) muscular dystrophy is an autosomal recessive disorder caused by mutations in the FKRP gene. The condition is often misdiagnosed as a dystrophinopathy. A previously unreported mutation, c.1100T>C in exon 4 of FKRP, had been identified in homozygous form in two white South African (SA) Afrikaner patients clinically diagnosed with a dystrophinopathy. To investigate whether the c.1100T>C mutation and the common European FKRP mutation c.826C>A are present in other patients of Afrikaner origin with suspected dystrophinopathy, and whether a founder haplotype exists. The c.1100T>C mutation was initially tested for using an amplification refractory mutation system technique in 45 white SA Afrikaner patients who had tested negative using multiplex ligation probe amplification screening for exonic deletions/duplications in the dystrophin gene. Sequencing analysis was used to confirm the c.1100T>C mutation and screen for the c.826C>A mutation. Two cohorts (each numbering 100) of Afrikaans and other white controls were screened for the c.1100T>C and c.826C>A mutations, respectively. Of the 45 patients, 8 patients (17.8%) were homozygous for c.1100T>C, 2 (4.4%) were compound heterozygotes for c.1100T>C and c.826C>A, and 1 (2.2%) was heterozygous for c.1100T>C with a second unidentified mutation. The c.1100T>C mutation was found in 1/100 controls, but no heterozygotes for the c.826C>A mutation were identified. Linked marker analysis for c.1100T>C showed a common haplotype, suggesting a probable founder mutation in the SA Afrikaner population. FKRP mutations may be relatively common in Afrikaners, and screening should be considered in patients who have a suggestive phenotype and test negative for a dystrophinopathy. This test will be useful for offering diagnostic, carrier and prenatal testing for affected individuals and their families. As FKRP muscular dystrophy is autosomal recessive in inheritance, the implications of a positive diagnosis in a family differ significantly from those of an X-linked dystrophinopathy.

Determination of ubiquitin fitness landscapes under different chemical stresses in a classroom setting.

PubMed

Mavor, David; Barlow, Kyle; Thompson, Samuel; Barad, Benjamin A; Bonny, Alain R; Cario, Clinton L; Gaskins, Garrett; Liu, Zairan; Deming, Laura; Axen, Seth D; Caceres, Elena; Chen, Weilin; Cuesta, Adolfo; Gate, Rachel E; Green, Evan M; Hulce, Kaitlin R; Ji, Weiyue; Kenner, Lillian R; Mensa, Bruk; Morinishi, Leanna S; Moss, Steven M; Mravic, Marco; Muir, Ryan K; Niekamp, Stefan; Nnadi, Chimno I; Palovcak, Eugene; Poss, Erin M; Ross, Tyler D; Salcedo, Eugenia C; See, Stephanie K; Subramaniam, Meena; Wong, Allison W; Li, Jennifer; Thorn, Kurt S; Conchúir, Shane Ó; Roscoe, Benjamin P; Chow, Eric D; DeRisi, Joseph L; Kortemme, Tanja; Bolon, Daniel N; Fraser, James S

2016-04-25

Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum.

Testing computational prediction of missense mutation phenotypes: Functional characterization of 204 mutations of human cystathionine beta synthase

PubMed Central

Wei, Qiong; Wang, Liqun; Wang, Qiang; Kruger, Warren D.; Dunbrack, Roland L.

2010-01-01

Predicting the phenotypes of missense mutations uncovered by large-scale sequencing projects is an important goal in computational biology. High-confidence predictions can be an aid in focusing experimental and association studies on those mutations most likely to be associated with causative relationships between mutation and disease. As an aid in developing these methods further, we have derived a set of random mutations of the enzymatic domains of human cystathionine beta synthase. This enzyme is a dimeric protein that catalyzes the condensation of serine and homocysteine to produce cystathionine. Yeast missing this enzyme cannot grow on medium lacking a source of cysteine, while transfection of functional human CBS into yeast strains missing endogenous enzyme can successfully complement for the missing gene. We used PCR mutagenesis with error-prone Taq polymerase to produce 948 colonies, and compared cell growth in the presence or absence of a cysteine source as a measure of CBS function. We were able to infer the phenotypes of 204 single-site mutants, 79 of them deleterious and 125 neutral. This set was used to test the accuracy of six publicly available prediction methods for phenotype prediction of missense mutations: SIFT, PolyPhen, PMut, SNPs3D, PhD-SNP, and nsSNPAnalyzer. The top methods are PolyPhen, SIFT, and nsSNPAnalyzer, which have similar performance. Using kernel discriminant functions, we found that the difference in position-specific scoring matrix values is more predictive than the wild-type PSSM score alone, and that the relative surface area in the biologically relevant complex is more predictive than that of the monomeric proteins. PMID:20455263

Validation of Version 3.0 of the Breast Cancer Genetics Referral Screening Tool (B-RST™).

PubMed

Bellcross, Cecelia; Hermstad, April; Tallo, Christine; Stanislaw, Christine

2018-05-08

Despite increased awareness of hereditary breast and ovarian cancer among clinicians and the public, many BRCA1/2 mutation carriers remain unaware of their risk status. The Breast Cancer Genetics Referral Screening Tool (B-RST™) was created and validated to easily identify individuals at increased risk for hereditary breast and ovarian cancer for referral to cancer genetics services. The purpose of this study was to revise B-RST™ to maximize sensitivity against BRCA1/2 mutation status. We analyzed pedigrees of 277 individuals who had undergone BRCA1/2 testing to determine modifications to the B-RST™ 2.0 algorithm that would maximize sensitivity for mutations, while maintaining simplicity. We used McNemar's chi-square test to compare validation measures between the revised version (3.0) and the 2.0 version. Algorithmic changes made to B-RST™ 2.0 increased the sensitivity against BRCA1/2 mutation analysis from 71.1 to 94.0% (P < 0.0001). While specificity decreased, all screen-positive individuals were appropriate for cancer genetics referral, the primary purpose of the tool. Despite calls for BRCA1/2 population screening, there remains a critical need to identify those most at risk who should receive cancer genetics services. B-RST™ version 3.0 demonstrates high sensitivity for BRCA1/2 mutations, yet remains a simple and quick screening tool for at-risk individuals.

Machine Learning to Improve the Effectiveness of ANRS in Predicting HIV Drug Resistance.

PubMed

Singh, Yashik

2017-10-01

Human immunodeficiency virus infection and acquired immune deficiency syndrome (HIV/AIDS) is one of the major burdens of disease in developing countries, and the standard-of-care treatment includes prescribing antiretroviral drugs. However, antiretroviral drug resistance is inevitable due to selective pressure associated with the high mutation rate of HIV. Determining antiretroviral resistance can be done by phenotypic laboratory tests or by computer-based interpretation algorithms. Computer-based algorithms have been shown to have many advantages over laboratory tests. The ANRS (Agence Nationale de Recherches sur le SIDA) is regarded as a gold standard in interpreting HIV drug resistance using mutations in genomes. The aim of this study was to improve the prediction of the ANRS gold standard in predicting HIV drug resistance. A genome sequence and HIV drug resistance measures were obtained from the Stanford HIV database (http://hivdb.stanford.edu/). Feature selection was used to determine the most important mutations associated with resistance prediction. These mutations were added to the ANRS rules, and the difference in the prediction ability was measured. This study uncovered important mutations that were not associated with the original ANRS rules. On average, the ANRS algorithm was improved by 79% ± 6.6%. The positive predictive value improved by 28%, and the negative predicative value improved by 10%. The study shows that there is a significant improvement in the prediction ability of ANRS gold standard.

Diagnosis and management of differentiated thyroid cancer using molecular biology.

PubMed

Witt, Robert L; Ferris, Robert L; Pribitkin, Edmund A; Sherman, Steven I; Steward, David L; Nikiforov, Yuri E

2013-04-01

To define molecular biology in clinical practice for diagnosis, surgical management, and prognostication of differentiated thyroid cancer. Ovid Medline 2006-2012 Manuscripts with clinical correlates. Papillary thyroid carcinomas harbor point mutations of the BRAF and RAS genes or RET/PTC rearrangements, all of which activate the mitogen-activated protein kinase pathway. These mutually exclusive mutations are found in 70% of PTC. BRAF mutation is found in 45% of papillary thyroid cancer and is highly specific. Follicular carcinomas are known to harbor RAS mutation or PAX8/PPARγ rearrangement. These mutations are also mutually exclusive and identified in 70% of follicular carcinomas. Molecular classifiers measure the expression of a large number of genes on a microarray chip providing a substantial negative predictive value pending further validation. 1) 20% to 30% of cytologically classified Follicular Neoplasms and Follicular Lesion of Undetermined Significance collectively are malignant on final pathology. Approximately 70% to 80% of thyroid lobectomies performed solely for diagnostic purposes are benign. Molecular alteration testing may reduce the number of unnecessary thyroid procedures, 2) may reduce the number of completion thyroidectomies, and 3) may lead to more individualized operative and postoperative management. Molecular testing for BRAF, RAS, RET/PTC, and PAX8/PPARγ for follicular lesion of undetermined significance and follicular neoplasm improve specificity, whereas molecular classifiers may add negative predictive value to fine needle aspiration diagnosis. Copyright © 2013 The American Laryngological, Rhinological, and Otological Society, Inc.

Murine knockin model for progranulin-deficient frontotemporal dementia with nonsense-mediated mRNA decay.

PubMed

Nguyen, Andrew D; Nguyen, Thi A; Zhang, Jiasheng; Devireddy, Swathi; Zhou, Ping; Karydas, Anna M; Xu, Xialian; Miller, Bruce L; Rigo, Frank; Ferguson, Shawn M; Huang, Eric J; Walther, Tobias C; Farese, Robert V

2018-03-20

Frontotemporal dementia (FTD) is the most common neurodegenerative disorder in individuals under age 60 and has no treatment or cure. Because many cases of FTD result from GRN nonsense mutations, an animal model for this type of mutation is highly desirable for understanding pathogenesis and testing therapies. Here, we generated and characterized Grn R493X knockin mice, which model the most common human GRN mutation, a premature stop codon at arginine 493 (R493X). Homozygous Grn R493X mice have markedly reduced Grn mRNA levels, lack detectable progranulin protein, and phenocopy Grn knockout mice, with CNS microgliosis, cytoplasmic TDP-43 accumulation, reduced synaptic density, lipofuscinosis, hyperinflammatory macrophages, excessive grooming behavior, and reduced survival. Inhibition of nonsense-mediated mRNA decay (NMD) by genetic, pharmacological, or antisense oligonucleotide-based approaches showed that NMD contributes to the reduced mRNA levels in Grn R493X mice and cell lines and in fibroblasts from patients containing the GRN R493X mutation. Moreover, the expressed truncated R493X mutant protein was functional in several assays in progranulin-deficient cells. Together, these findings establish a murine model for in vivo testing of NMD inhibition or other therapies as potential approaches for treating progranulin deficiency caused by the R493X mutation. Copyright © 2018 the Author(s). Published by PNAS.

Shared genetic predisposition in rheumatoid arthritis-interstitial lung disease and familial pulmonary fibrosis.

PubMed

Juge, Pierre-Antoine; Borie, Raphaël; Kannengiesser, Caroline; Gazal, Steven; Revy, Patrick; Wemeau-Stervinou, Lidwine; Debray, Marie-Pierre; Ottaviani, Sébastien; Marchand-Adam, Sylvain; Nathan, Nadia; Thabut, Gabriel; Richez, Christophe; Nunes, Hilario; Callebaut, Isabelle; Justet, Aurélien; Leulliot, Nicolas; Bonnefond, Amélie; Salgado, David; Richette, Pascal; Desvignes, Jean-Pierre; Lioté, Huguette; Froguel, Philippe; Allanore, Yannick; Sand, Olivier; Dromer, Claire; Flipo, René-Marc; Clément, Annick; Béroud, Christophe; Sibilia, Jean; Coustet, Baptiste; Cottin, Vincent; Boissier, Marie-Christophe; Wallaert, Benoit; Schaeverbeke, Thierry; Dastot le Moal, Florence; Frazier, Aline; Ménard, Christelle; Soubrier, Martin; Saidenberg, Nathalie; Valeyre, Dominique; Amselem, Serge; Boileau, Catherine; Crestani, Bruno; Dieudé, Philippe

2017-05-01

Despite its high prevalence and mortality, little is known about the pathogenesis of rheumatoid arthritis-associated interstitial lung disease (RA-ILD). Given that familial pulmonary fibrosis (FPF) and RA-ILD frequently share the usual pattern of interstitial pneumonia and common environmental risk factors, we hypothesised that the two diseases might share additional risk factors, including FPF-linked genes. Our aim was to identify coding mutations of FPF-risk genes associated with RA-ILD.We used whole exome sequencing (WES), followed by restricted analysis of a discrete number of FPF-linked genes and performed a burden test to assess the excess number of mutations in RA-ILD patients compared to controls.Among the 101 RA-ILD patients included, 12 (11.9%) had 13 WES-identified heterozygous mutations in the TERT , RTEL1 , PARN or SFTPC coding regions . The burden test, based on 81 RA-ILD patients and 1010 controls of European ancestry, revealed an excess of TERT , RTEL1 , PARN or SFTPC mutations in RA-ILD patients (OR 3.17, 95% CI 1.53-6.12; p=9.45×10 -4 ). Telomeres were shorter in RA-ILD patients with a TERT , RTEL1 or PARN mutation than in controls (p=2.87×10 -2 ).Our results support the contribution of FPF-linked genes to RA-ILD susceptibility. Copyright ©ERS 2017.

De Novo Paternal FBN1 Mutation Detected in Embryos Before Implantation.

PubMed

Wang, Shuling; Niu, Ziru; Wang, Hui; Ma, Minyue; Zhang, Wei; Fang Wang, Shu; Wang, Jun; Yan, Hong; Liu, Yifan; Duan, Na; Zhang, Xiandong; Yao, Yuanqing

2017-06-26

BACKGROUND Marfan syndrome (MFS) is an autosomal dominant disease caused by mutations in the Fibrillin (FBN)1 gene and characterized by disorders in the cardiovascular, skeletal, and visual systems. The diversity of mutations and phenotypic heterogeneity of MFS make prenatal molecular diagnoses difficult. In this study, we used pre-implantation genetic diagnosis (PGD) to identify the pathogenic mutation in a male patient with MFS and to determine whether his offspring would be free of the disease. MATERIAL AND METHODS The history and pedigree of the proband were analyzed. Mutation analysis was performed on the couple and immediate family members. The couple chose IVF treatment and 4 blastocysts were biopsied. PGD was carried out by targeted high-throughput sequencing of the FBN1 gene in the embryos, along with single-nucleotide polymorphism haplotyping. Sanger sequencing was used to confirm the causative mutation. RESULTS c.2647T>C (p.Trp883Arg) was identified as the de novo likely pathogenic mutation in the proband. Whole-genome amplification and sequencing of the 3 embryos revealed that they did not carry the mutation, and 1 blastocyst was transferred back to the uterus. The amniocentesis test result analyzed by Sanger sequencing confirmed the PGD. A premature but healthy infant free of heart malformations was born. CONCLUSIONS The de novo mutation c.2647T>C (p.Trp883Arg) in FBN1 was identified in a Chinese patient with MFS. Embryos without the mutation were identified by PGD and resulted in a successful pregnancy.

CFTR Mutations Spectrum and the Efficiency of Molecular Diagnostics in Polish Cystic Fibrosis Patients

PubMed Central

Ziętkiewicz, Ewa; Rutkiewicz, Ewa; Pogorzelski, Andrzej; Klimek, Barbara; Voelkel, Katarzyna; Witt, Michał

2014-01-01

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR). In light of the strong allelic heterogeneity and regional specificity of the mutation spectrum, the strategy of molecular diagnostics and counseling in CF requires genetic tests to reflect the frequency profile characteristic for a given population. The goal of the study was to provide an updated comprehensive estimation of the distribution of CFTR mutations in Polish CF patients and to assess the effectiveness of INNOLiPA_CFTR tests in Polish population. The analyzed cohort consisted of 738 patients with the clinically confirmed CF diagnosis, prescreened for molecular defects using INNOLiPA_CFTR panels from Innogenetics. A combined efficiency of INNOLiPA CFTR_19 and CFTR_17_TnUpdate tests was 75.5%; both mutations were detected in 68.2%, and one mutation in 14.8% of the affected individuals. The group composed of all the patients with only one or with no mutation detected (109 and 126 individuals, respectively) was analyzed further using a mutation screening approach, i.e. SSCP/HD (single strand conformational polymorphism/heteroduplex) analysis of PCR products followed by sequencing of the coding sequence. As a result, 53 more mutations were found in 97 patients. The overall efficiency of the CF allele detection was 82.5% (7.0% increase compared to INNOLiPA tests alone). The distribution of the most frequent mutations in Poland was assessed. Most of the mutations repetitively found in Polish patients had been previously described in other European populations. The most frequent mutated allele, F508del, represented 54.5% of Polish CF chromosomes. Another eight mutations had frequencies over 1%, 24 had frequencies between 1 and 0.1%; c.2052-2053insA and c.3468+2_3468+3insT were the most frequent non-INNOLiPA mutations. Mutation distribution described herein is also relevant to the Polish diaspora. Our study also demonstrates that the reported efficiency of mutation detection strongly depends on the diagnostic experience of referring health centers. PMID:24586523

CFTR mutations spectrum and the efficiency of molecular diagnostics in Polish cystic fibrosis patients.

PubMed

Ziętkiewicz, Ewa; Rutkiewicz, Ewa; Pogorzelski, Andrzej; Klimek, Barbara; Voelkel, Katarzyna; Witt, Michał

2014-01-01

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR). In light of the strong allelic heterogeneity and regional specificity of the mutation spectrum, the strategy of molecular diagnostics and counseling in CF requires genetic tests to reflect the frequency profile characteristic for a given population. The goal of the study was to provide an updated comprehensive estimation of the distribution of CFTR mutations in Polish CF patients and to assess the effectiveness of INNOLiPA_CFTR tests in Polish population. The analyzed cohort consisted of 738 patients with the clinically confirmed CF diagnosis, prescreened for molecular defects using INNOLiPA_CFTR panels from Innogenetics. A combined efficiency of INNOLiPA CFTR_19 and CFTR_17_TnUpdate tests was 75.5%; both mutations were detected in 68.2%, and one mutation in 14.8% of the affected individuals. The group composed of all the patients with only one or with no mutation detected (109 and 126 individuals, respectively) was analyzed further using a mutation screening approach, i.e. SSCP/HD (single strand conformational polymorphism/heteroduplex) analysis of PCR products followed by sequencing of the coding sequence. As a result, 53 more mutations were found in 97 patients. The overall efficiency of the CF allele detection was 82.5% (7.0% increase compared to INNOLiPA tests alone). The distribution of the most frequent mutations in Poland was assessed. Most of the mutations repetitively found in Polish patients had been previously described in other European populations. The most frequent mutated allele, F508del, represented 54.5% of Polish CF chromosomes. Another eight mutations had frequencies over 1%, 24 had frequencies between 1 and 0.1%; c.2052-2053insA and c.3468+2_3468+3insT were the most frequent non-INNOLiPA mutations. Mutation distribution described herein is also relevant to the Polish diaspora. Our study also demonstrates that the reported efficiency of mutation detection strongly depends on the diagnostic experience of referring health centers.

BRAF V600E mutations in papillary craniopharyngioma

PubMed Central

Brastianos, Priscilla K.; Santagata, Sandro

2016-01-01

Papillary craniopharyngioma is an intracranial tumor that results in high levels of morbidity. We recently demonstrated that the vast majority of these tumors harbor the oncogenic BRAF V600E mutation. The pathologic diagnosis of papillary craniopharyngioma can now be confirmed using mutation specific immunohistochemistry and targeted genetic testing. Treatment with targeted agents is now also a possibility in select situations. We recently reported a patient with a multiply recurrent papillary craniopharyngioma in whom targeting both BRAF and MEK resulted in a dramatic therapeutic response with a marked anti-tumor immune response. This work shows that activation of the MAPK pathway is the likely principal oncogenic driver of these tumors. We will now investigate the efficacy of this approach in a multicenter phase II clinical trial. Post-treatment resection samples will be monitored for the emergence of resistance mechanisms. Further advances in the non-invasive diagnosis of papillary craniopharyngioma by radiologic criteria and by cell-free DNA testing could someday allow neo-adjuvant therapy for this disease in select patient populations. PMID:26563980

Variations in the detection of ZAP-70 in chronic lymphocytic leukemia: Comparison with IgV(H) mutation analysis.

PubMed

Sheikholeslami, M R; Jilani, I; Keating, M; Uyeji, J; Chen, K; Kantarjian, H; O'Brien, S; Giles, F; Albitar, M

2006-07-15

Lack of immunoglobulin heavy chain genes (IgV(H)) mutation in patients with chronic lymphocytic leukemia (CLL) is associated with rapid disease progression and shorter survival. The zeta-chain (T-cell receptor) associated protein kinase 70 kDa (ZAP-70) has been reported to be a surrogate marker for IgV(H) mutation status, and its expression in leukemic cells correlates with unmutated IgV(H). However, ZAP-70 detection by flow cytometry varies significantly dependant on the antibodies used, the method of performing the assay, and the condition of the cells in the specimen. The clinical value of ZAP-70 testing when samples are shipped under poorly controlled conditions is not known. Furthermore, testing in a research environment may differ from testing in a routine clinical laboratory. We validated an assay for ZAP-70 by comparing results with clinical outcome and the mutation status of the IgV(H). Using stored samples, we show significant correlation between ZAP-70 expression and clinical outcome as well as IgV(H) mutation at a cut-off point of 15%. While positive samples (>15% positivity) remain positive when kept in the laboratory environment for 48 h after initial testing, results obtained from samples from CLL patients tested after shipping at room temperature for routine testing showed no correlation with IgV(H) mutation status when 15% cut-off was used. In these samples, cut-point of 10% correlated with the IgV(H) mutation (P = 0.0001). This data suggests that although ZAP-70 positivity correlates with IgV(H) mutation status and survival, variations in sample handling and preparation may influence results. We show that IgV(H) mutation results, unlike ZAP-70 remain correlated with CD38 expression and beta-2 microglobulin in shipped samples, and ZAP-70 testing should not be used as the sole criterion for stratifying patients for therapy. (c) 2006 International Society for Analytical Cytology.

Establishing an EGFR mutation screening service for non-small cell lung cancer - sample quality criteria and candidate histological predictors.

PubMed

Leary, Alexandra F; Castro, David Gonzalez de; Nicholson, Andrew G; Ashley, Sue; Wotherspoon, Andrew; O'Brien, Mary E R; Popat, Sanjay

2012-01-01

EGFR screening requires good quality tissue, sensitivity and turn-around time (TAT). We report our experience of routine screening, describing sample type, TAT, specimen quality (cellularity and DNA yield), histopathological description, mutation result and clinical outcome. Non-small cell lung cancer (NSCLC) sections were screened for EGFR mutations (M+) in exons 18-21. Clinical, pathological and screening outcome data were collected for year 1 of testing. Screening outcome alone was collected for year 2. In year 1, 152 samples were tested, most (72%) were diagnostic. TAT was 4.9 days (95%confidence interval (CI)=4.5-5.5). EGFR-M+ prevalence was 11% and higher (20%) among never-smoking women with adenocarcinomas (ADCs), but 30% of mutations occurred in current/ex-smoking men. EGFR-M+ tumours were non-mucinous ADCs and 100% thyroid transcription factor (TTF1+). No mutations were detected in poorly differentiated NSCLC-not otherwise specified (NOS). There was a trend for improved overall survival (OS) among EGFR-M+ versus EGFR-M- patients (median OS=78 versus 17 months). In year 1, test failure rate was 19%, and associated with scant cellularity and low DNA concentrations. However 75% of samples with poor cellularity but representative of tumour were informative and mutation prevalence was 9%. In year 2, 755 samples were tested; mutation prevalence was 13% and test failure only 5.4%. Although samples with low DNA concentration (<2 ng/μL) had more test failures (30% versus 3.9% for [DNA]>2.2 ng/μL), the mutation rate was 9.2%. Routine epidermal growth factor receptor (EGFR) screening using diagnostic samples is fast and feasible even on samples with poor cellularity and DNA content. Mutations tend to occur in better-differentiated non-mucinous TTF1+ ADCs. Whether these histological criteria may be useful to select patients for EGFR testing merits further investigation. Copyright © 2011 Elsevier Ltd. All rights reserved.

Mutations in gp41 are correlated with coreceptor tropism but do not improve prediction methods substantially.

PubMed

Thielen, Alexander; Lengauer, Thomas; Swenson, Luke C; Dong, Winnie W Y; McGovern, Rachel A; Lewis, Marilyn; James, Ian; Heera, Jayvant; Valdez, Hernan; Harrigan, P Richard

2011-01-01

The main determinants of HIV-1 coreceptor usage are located in the V3-loop of gp120, although mutations in V2 and gp41 are also known. Incorporation of V2 is known to improve prediction algorithms; however, this has not been confirmed for gp41 mutations. Samples with V3 and gp41 genotypes and Trofile assay (Monogram Biosciences, South San Francisco, CA, USA) results were taken from the HOMER cohort (n=444) and from patients screened for the MOTIVATE studies (n=1,916; 859 with maraviroc outcome data). Correlations of mutations with tropism were assessed using Fisher's exact test and prediction models trained using support vector machines. Models were validated by cross-validation, by testing models from one dataset on the other, and by analysing virological outcome. Several mutations within gp41 were highly significant for CXCR4 usage; most strikingly an insertion occurring in 7.7% of HOMER-R5 and 46.3% of HOMER-X4 samples (MOTIVATE 5.7% and 25.2%, respectively). Models trained on gp41 sequence alone achieved relatively high areas under the receiver-operating characteristic curve (AUCs; HOMER 0.713 and MOTIVATE 0.736) that were almost as good as V3 models (0.773 and 0.884, respectively). However, combining the two regions improved predictions only marginally (0.813 and 0.902, respectively). Similar results were found when models were trained on HOMER and validated on MOTIVATE or vice versa. The difference in median log viral load decrease at week 24 between patients with R5 and X4 virus was 1.65 (HOMER 2.45 and MOTIVATE 0.79) for V3 models, 1.59 for gp41-models (2.42 and 0.83, respectively) and 1.58 for the combined predictor (2.44 and 0.86, respectively). Several mutations within gp41 showed strong correlation with tropism in two independent datasets. However, incorporating gp41 mutations into prediction models is not mandatory because they do not improve substantially on models trained on V3 sequences alone.

Correction of nonsense BMPR2 and SMAD9 mutations by ataluren in pulmonary arterial hypertension.

PubMed

Drake, Kylie M; Dunmore, Benjamin J; McNelly, Lauren N; Morrell, Nicholas W; Aldred, Micheala A

2013-09-01

Heritable pulmonary arterial hypertension (HPAH) is a serious lung vascular disease caused by heterozygous mutations in the bone morphogenetic protein (BMP) pathway genes, BMPR2 and SMAD9. One noncanonical function of BMP signaling regulates biogenesis of a subset of microRNAs. We have previously shown that this function is abrogated in patients with HPAH, making it a highly sensitive readout of BMP pathway integrity. Ataluren (PTC124) is an investigational drug that permits ribosomal readthrough of premature stop codons, resulting in a full-length protein. It exhibits oral bioavailability and limited toxicity in human trials. Here, we tested ataluren in lung- or blood-derived cells from patients with HPAH with nonsense mutations in BMPR2 (n = 6) or SMAD9 (n = 1). Ataluren significantly increased BMP-mediated microRNA processing in six of the seven cases. Moreover, rescue was achieved even for mutations exhibiting significant nonsense-mediated mRNA decay. Response to ataluren was dose dependent, and complete correction was achieved at therapeutic doses currently used in clinical trials for cystic fibrosis. BMP receptor (BMPR)-II protein levels were normalized and ligand-dependent phosphorylation of downstream target Smads was increased. Furthermore, the usually hyperproliferative phenotype of pulmonary artery endothelial and smooth muscle cells was reversed by ataluren. These results indicate that ataluren can effectively suppress a high proportion of BMPR2 and SMAD9 nonsense mutations and correct BMP signaling in vitro. Approximately 29% of all HPAH mutations are nonsense point mutations. In light of this, we propose ataluren as a potential new personalized therapy for this significant subgroup of patients with PAH.

Rare Complex Mutational Profile in an ALK Inhibitor-resistant Non-small Cell Lung Cancer.

PubMed

Azzato, Elizabeth M; Deshpande, Charuhas; Aikawa, Vania; Aggarwal, Charu; Alley, Evan; Jacobs, Benjamin; Morrissette, Jennifer; Daber, Robert

2015-05-01

Testing for somatic alterations, including anaplastic lymphoma receptor tyrosine kinase gene (ALK) rearrangements and epidermal growth factor receptor gene (EGFR) mutations, is standard practice in the diagnostic evaluation and therapeutic management of non-small cell lung cancer (NSCLC), where the results of such tests can predict response to targeted-therapy. ALK rearrangements, EGFR mutations and mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) are considered mutually exclusive in NSCLC. Herein we identified a KRAS Q22K mutation and frameshift mutations in the genes encoding serine/threonine kinase 11 (STK11) and ataxia telangiectasia mutated serine/threonine kinase (ATM) by next-generation sequencing in a patient with ALK rearrangement-positive oligo-metastatic NSCLC, whose disease progressed while on two ALK-targeted therapies. Such a complex diagnostic genetic profile has not been reported in ALK fusion-positive NSCLC. This case highlights the utility of comprehensive molecular testing in the diagnosis of NSCLC. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Frequent PIK3CA Mutations in Colorectal and Endometrial Cancer with Double Somatic Mismatch Repair Mutations

PubMed Central

Cohen, Stacey A.; Turner, Emily H.; Beightol, Mallory B.; Jacobson, Angela; Gooley, Ted A.; Salipante, Stephen J.; Haraldsdottir, Sigurdis; Smith, Christina; Scroggins, Sheena; Tait, Jonathan F.; Grady, William M.; Lin, Edward H.; Cohn, David E.; Goodfellow, Paul J.; Arnold, Mark W.; de la Chapelle, Albert; Pearlman, Rachel; Hampel, Heather; Pritchard, Colin C.

2016-01-01

Background & Aims Double somatic mutations in mismatch repair (MMR) genes have recently been described in colorectal and endometrial cancers with microsatellite instability (MSI) not attributable to MLH1 hypermethylation or germline mutation. We sought to define the molecular phenotype of this newly recognized tumor subtype. Methods From two prospective Lynch syndrome screening studies, we identified patients with colorectal and endometrial tumors harboring ≥2 somatic MMR mutations, but normal germline MMR testing (“double somatic”). We determined the frequencies of tumor PIK3CA, BRAF, KRAS, NRAS, and PTEN mutations by targeted next-generation sequencing and used logistic-regression models to compare them to: Lynch syndrome, MLH1 hypermethylated, and microsatellite stable (MSS) tumors. We validated our findings using independent datasets from The Cancer Genome Atlas (TCGA). Results Among colorectal cancer cases, we found that 14/21 (67%) of double somatic cases had PIK3CA mutations vs. 4/18 (22%) Lynch syndrome, 2/10 (20%) MLH1 hypermethylated, and 12/78 (15%) MSS tumors; p<0.0001. PIK3CA mutations were detected in 100% of 13 double somatic endometrial cancers (p=0.04). BRAF mutations were absent in double somatic and Lynch syndrome colorectal tumors. We found highly similar results in a validation cohort from TCGA (113 colorectal, 178 endometrial cancer), with 100% of double somatic cases harboring a PIK3CA mutation (p<0.0001). Conclusions PIK3CA mutations are present in double somatic mutated colorectal and endometrial cancers at substantially higher frequencies than other MSI subgroups. PIK3CA mutation status may better define an emerging molecular entity in colorectal and endometrial cancers, with the potential to inform screening and therapeutic decision making. PMID:27302833

Prevalence and Type of BRCA Mutations in Hispanics Undergoing Genetic Cancer Risk Assessment in the Southwestern United States: A Report From the Clinical Cancer Genetics Community Research Network

PubMed Central

Weitzel, Jeffrey N.; Clague, Jessica; Martir-Negron, Arelis; Ogaz, Raquel; Herzog, Josef; Ricker, Charité; Jungbluth, Chelsy; Cina, Cheryl; Duncan, Paul; Unzeitig, Gary; Saldivar, J. Salvador; Beattie, Mary; Feldman, Nancy; Sand, Sharon; Port, Danielle; Barragan, Deborah I.; John, Esther M.; Neuhausen, Susan L.; Larson, Garrett P.

2013-01-01

Purpose To determine the prevalence and type of BRCA1 and BRCA2 (BRCA) mutations among Hispanics in the Southwestern United States and their potential impact on genetic cancer risk assessment (GCRA). Patients and Methods Hispanics (n = 746) with a personal or family history of breast and/or ovarian cancer were enrolled in an institutional review board–approved registry and received GCRA and BRCA testing within a consortium of 14 clinics. Population-based Hispanic breast cancer cases (n = 492) enrolled in the Northern California Breast Cancer Family Registry, negative by sequencing for BRCA mutations, were analyzed for the presence of the BRCA1 ex9-12del large rearrangement. Results Deleterious BRCA mutations were detected in 189 (25%) of 746 familial clinic patients (124 BRCA1, 65 BRCA2); 21 (11%) of 189 were large rearrangement mutations, of which 62% (13 of 21) were BRCA1 ex9-12del. Nine recurrent mutations accounted for 53% of the total. Among these, BRCA1 ex9-12del seems to be a Mexican founder mutation and represents 10% to 12% of all BRCA1 mutations in clinic- and population-based cohorts in the United States. Conclusion BRCA mutations were prevalent in the largest study of Hispanic breast and/or ovarian cancer families in the United States to date, and a significant proportion were large rearrangement mutations. The high frequency of large rearrangement mutations warrants screening in every case. We document the first Mexican founder mutation (BRCA1 ex9-12del), which, along with other recurrent mutations, suggests the potential for a cost-effective panel approach to ancestry-informed GCRA. PMID:23233716

Identification of mutations associated with pyrethroid resistance in the voltage-gated sodium channel of the tomato leaf miner (Tuta absoluta).

PubMed

Haddi, Khalid; Berger, Madeleine; Bielza, Pablo; Cifuentes, Dina; Field, Linda M; Gorman, Kevin; Rapisarda, Carmelo; Williamson, Martin S; Bass, Chris

2012-07-01

The tomato leaf miner, Tuta absoluta (Lepidoptera) is a significant pest of tomatoes that has undergone a rapid expansion in its range during the past six years and is now present across Europe, North Africa and parts of Asia. One of the main means of controlling this pest is through the use of chemical insecticides. In the current study insecticide bioassays were used to determine the susceptibility of five T. absoluta strains established from field collections from Europe and Brazil to pyrethroids. High levels of resistance to λ cyhalothrin and tau fluvalinate were observed in all five strains tested. To investigate whether pyrethroid resistance was mediated by mutation of the para-type sodium channel in T. absoluta the IIS4-IIS6 region of the para gene, which contains many of the mutation sites previously shown to confer knock down (kdr)-type resistance to pyrethroids across a range of different arthropod species, was cloned and sequenced. This revealed that three kdr/super-kdr-type mutations (M918T, T929I and L1014F), were present at high frequencies within all five resistant strains at known resistance 'hot-spots'. This is the first description of these mutations together in any insect population. High-throughput DNA-based diagnostic assays were developed and used to assess the prevalence of these mutations in 27 field strains from 12 countries. Overall mutant allele frequencies were high (L1014F 0.98, M918T 0.35, T929I 0.60) and remarkably no individual was observed that did not carry kdr in combination with either M918T or T929I. The presence of these mutations at high frequency in T. absoluta populations across much of its range suggests pyrethroids are likely to be ineffective for control and supports the idea that the rapid expansion of this species over the last six years may be in part mediated by the resistance of this pest to chemical insecticides. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

Prevalence of glucocerebrosidase mutations in the Israeli Ashkenazi Jewish population.

PubMed

Horowitz, M; Pasmanik-Chor, M; Borochowitz, Z; Falik-Zaccai, T; Heldmann, K; Carmi, R; Parvari, R; Beit-Or, H; Goldman, B; Peleg, L; Levy-Lahad, E; Renbaum, P; Legum, S; Shomrat, R; Yeger, H; Benbenisti, D; Navon, R; Dror, V; Shohat, M; Magal, N; Navot, N; Eyal, N

1998-01-01

Gaucher disease is the most prevalent inherited disease among Ashkenazi Jews. It is very heterogeneous due to a large number of mutations within the glucocerebrosidase gene, whose impaired activity is the cause for this disease. Aiming at determining Gaucher carrier frequency among the Ashkenazi Jewish population in Israel, 1,208 individuals were molecularly diagnosed for six mutations known to occur among Ashkenazi Jewish Gaucher patients, using the newly developed Pronto Gaucher kit. The following mutations were tested: N370S, 84GG, IVS2+1, D409H, L444P, and V394L. Molecular testing of these mutations also allows identification of the recTL allele. The results indicated that Gaucher carrier frequency is 1:17 within the tested population. The prevalence of N370S carriers is 1:17.5. This implies that approximately 1:1225 Ashkenazi Jews will be homozygous for the N370S mutation. Actually, in our study of 1,208 individuals one was found to be homozygous for the N370S mutation. The actual number of known Ashkenazi Jewish Gaucher patients with this genotype is much lower than that expected according to the frequency of the N370S mutation, suggesting a low penetrance of this mutation. Results of loading experiments in cells homozygous for the N370S mutation, as well as cells homozygous for the L444P and the D409H mutations, exemplified this phenomenon.

[Molecular genetic diagnostics and screening of hereditary hemochromatosis].

PubMed

Zlocha, J; Kovács, L; Pozgayová, S; Kupcová, V; Durínová, S

2006-06-01

Hereditary hemochromatosis is considered one of the most common hereditary diseases in population of Caucasian origin. In recent years, a candidate gene for HLA-linked hemochromatosis, HFE, has been cloned, and a single G-to-A mutation resulting in a cysteine-to-tyrosine substitution (C282Y) has been identified in up to 80% of study patients with type 1 hereditary hemochromatosis. The purpose of the paper was to confirm the importance of genetic testing for HFE mutations in making the diagnosis of hemochromatosis and find out a suitable diagnostic algorithm for the indication of this form of diagnostics in patients suspected of hereditary hemochromatosis. The examination of C282Y mutation was conducted in 500 subjects. The most frequent indications for DNA analysis were hepatopathy of unknown ethiology, liver cirrhosis, diabetes mellitus, bronze skin pigmentation in connection with high serum iron concentration, elevated transferrin saturation and elevated serum ferritin levels. In our group of patients, 29 homozygotes and 75 heterozygotes for C282Y mutation were identified, 10 patients carried both C282Y and H63D mutations of HFE gene (compound heterozygotes), whereas in 386 subjects the mutation was not found. The genotype-phenotype correlation showed that 22 homozygotes had liver affection proved by imaging and/or histologic methods. Except the liver disorders, the most common symptoms of these patients were type 2 diabetes mellitus or glucose tolerance disorder (10 patients), arthritis or joint pain (9 patients) and cardiovascular disorders, such as cardiomyopathy (2 patients). Bronze skin pigmentation was present in 9 homozygotes. Transferin saturation values were significantly higher in homozygotes for C282Y mutation as compared to C282Y heterozygotes (p < 0.001), C282Y/H63D compound heterozygotes (p < 0.05) or wild type subjects (p < 0.001) respectively. Also serum ferritin levels were significantly higher in homozygotes for C282Y mutation as compared to C282Y heterozygotes (p < 0.001), C282Y/H63D compound heterozygotes (p < 0.001) and wild type subjects (p < 0.001) respectively. Our observations confirm that DNA analysis significantly contributes to differential diagnostics of this severe, but in early recognition curable disease. Early detection and phlebotomy treatment prior to the onset of cirrhosis can reduce morbidity and normalize life expectancy. It is readily identified through biochemical testing for iron overload using serum transferrin saturation and genetic testing for C282Y homozygosity. DNA analysis is recommended in patients whose transferrin saturation is 45% or more on a repeated test. General population screening has been waived in preference to targeting high-risk groups such as first-degree relatives of affected individuals and those with secondary iron overload, especially patients with chronic liver disorders and chronic anemia. This screening strategy is likely to continue until uncertainties regarding the natural history of the disease, age-related penetrance, and management of asymptomatic individuals are clarified.

High anaplastic lymphoma kinase immunohistochemical staining in neuroblastoma and ganglioneuroblastoma is an independent predictor of poor outcome.

PubMed

Duijkers, Floor A M; Gaal, José; Meijerink, Jules P P; Admiraal, Pieter; Pieters, Rob; de Krijger, Ronald R; van Noesel, Max M

2012-03-01

Anaplastic lymphoma kinase (ALK) mutations occur in 3% to 11% of neuroblastoma (NBL) cases and are associated with high ALK levels. However, high ALK levels appear to be a mutation-independent hallmark of NBL. Evidence about the prognostic relevance of ALK mutations and ALK tumor positivity in patients with NBL has been inconclusive. In this study, we investigated the prognostic relevance of ALK positivity by IHC and ALK mutation status by PCR sequencing in 71 NBL, 12 ganglioneuroblastoma (GNBL), and 20 ganglioneuroma samples in a multivariate model. ALK mutations were present in 2 of 72 NBL and 2 of 12 GNBL samples, which all contained many ALK-positive cells (>50%). In addition, half of all NBL samples showed ALK positivity in most (>50%) of tumor cells, whereas half of the GNBL showed staining in <20% of the tumor cells. In most ganglioneuroma samples, a low percentage of tumor cells stained positive for ALK, which mainly involved ganglion cells. Higher percentages of ALK-positive cells in NBL and GNBL patient samples correlated with inferior survival in univariate and multivariate analyses with established prognostic factors, such as stage, age, and MYCN status. In conclusion, ALK positivity by IHC is an independent, poor prognostic factor in patients with GNBL and NBL. ALK IHC is an easy test suitable for future risk stratification in patients with NBL and GNBL. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Leigh syndrome T8993C mitochondrial DNA mutation: Heteroplasmy and the first clinical presentation in a Vietnamese family.

PubMed

Weerasinghe, Chamara Arachchighe Lahiru; Bui, Bich-Hong Thi; Vu, Thu Thi; Nguyen, Hong-Loan Thi; Phung, Bao-Khanh; Nguyen, Van-Minh; Pham, Van-Anh; Cao, Vu-Hung; Phan, Tuan-Nghia

2018-05-01

Leigh syndrome is a rare inherited, heterogeneous and progressive neurometabolic disorder that is mainly caused by specific mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). The present study reported a case of childhood Leigh syndrome with a point mutation at bp 8,993 in the mitochondrial ATPase6 gene. A 21‑month‑old male child had developed epilepsy, muscular weakness and vomiting, which was accompanied by high fever. Magnetic resonance imaging indicated typical characteristics of Leigh syndrome, including a symmetric abnormal signal in the dorsal medulla oblongata and Sylvian fissure enlargement in association with an abnormal signal in the periventricular white matter and in the putamina and caudate heads. The diagnosis was further supported with genetic tests including polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), sequencing, and quantitative PCR. The patient was found to carry a mitochondrial T8993C (m.T8993C) mutation in peripheral blood with 94.00±1.34% heteroplasmy. Eight of his relatives were also subjected to quantification of the m.T8993C mutation. The percentages of heteroplasmy in samples taken from the grandmother, mother, aunt, cousin 1, and cousin 2 were 16.33±1.67, 66.81±0.85, 71.66±3.22, 87.00±1.79, and 91.24±2.50%, respectively. The mutation was not found in samples taken from the father, the husband of the aunt, or the grandfather of the patient. The obtained data showed that the mutation was maternally inherited and accumulated through generations. Even though the heteroplasmy levels of his mother, aunt, cousin 1, and cousin 2 were relatively high (66.81‑91.24%), they remained asymptomatic, indicating that the threshold at which this mutation shows effects is high. To the best of our knowledge, this is the first report of a case of Leigh syndrome in a Vietnamese individual harboring a mtDNA mutation at the 8,993 bp site, and showing a correlation between the heteroplasmy and clinical phenotype. These findings may be useful in helping to improve the clinical diagnosis and treatment of Leigh syndrome.

Combinations of chlorocatechols and heavy metals cause DNA degradation in vitro but must not result in increased mutation rates in vivo.

PubMed

Schweigert, N; Belkin, S; Leong-Morgenthaler, P; Zehnder, A J; Eggen, R I

1999-01-01

Chlorocatechols introduced into the environment directly or as a result of degradation processes are highly toxic, particularly when combined with heavy metals. With in vitro DNA degradation assays, the high reactivity of chlorocatechols combined with heavy metals could be shown, whereby copper was shown to be more active than iron. Structure-activity analysis showed that the degradation potential of the chlorocatechols decreased with an increasing number of chloratoms. The addition of reactive oxygen species scavengers allowed the identification of hydrogen peroxide as an important agent leading to DNA damage in this reaction. The potential of other reactive compounds, however, can neither be determined nor excluded with this approach. Exposure of Escherichia coli and Salmonella typhimurium cultures to the same mixtures of chlorocatechols and copper surprisingly did not lead to an enhanced mutation rate. This phenomenon was explained by doing marker gene expression measurements and toxicity tests with E. coli mutants deficient in oxidative stress defense or DNA repair. In catechol-copper-exposed cultures an increased peroxide level could indeed be demonstrated, but the highly efficient defense and repair systems of E. coli avoid the phenotypical establishment of mutations. Increased mutation rates under chronic exposure, however, cannot be excluded.

Efficient molecular screening of Lynch syndrome by specific 3' promoter methylation of the MLH1 or BRAF mutation in colorectal cancer with high-frequency microsatellite instability.

PubMed

Nakagawa, Hitoshi; Nagasaka, Takeshi; Cullings, Harry M; Notohara, Kenji; Hoshijima, Naoko; Young, Joanne; Lynch, Henry T; Tanaka, Noriaki; Matsubara, Nagahide

2009-06-01

It is sometimes difficult to diagnose Lynch syndrome by the simple but strict clinical criteria, or even by the definitive genetic testing for causative germline mutation of mismatch repair genes. Thus, some practical and efficient screening strategy to select highly possible Lynch syndrome patients is exceedingly desirable. We performed a comprehensive study to evaluate the methylation status of whole MLH1 promoter region by direct bisulfite sequencing of the entire MLH1 promoter regions on Lynch and non-Lynch colorectal cancers (CRCs). Then, we established a convenient assay to detect methylation in key CpG islands responsible for the silencing of MLH1 expression. We studied the methylation status of MLH1 as well as the CpG island methylator phenotype (CIMP) and immunohistochemical analysis of mismatch repair proteins on 16 cases of Lynch CRC and 19 cases of sporadic CRCs with high-frequency microsatellite instability (MSI-H). Sensitivity to detect Lynch syndrome by MLH1 (CCAAT) methylation was 88% and the specificity was 84%. Positive likelihood ratio (PLR) was 5.5 and negative likelihood ratio (NLR) was 0.15. Sensitivity by mutational analysis of BRAF was 100%, specificity was 84%, PLR was 6.3 and NLR was zero. By CIMP analysis; sensitivity was 88%, specificity was 79%, PLR was 4.2, and NLR was 0.16. BRAF mutation or MLH1 methylation analysis combined with MSI testing could be a good alternative to screen Lynch syndrome patients in a cost effective manner. Although the assay for CIMP status also showed acceptable sensitivity and specificity, it may not be practical because of its rather complicated assay.

Channel architecture in maltoporin: dominance studies with lamB mutations influencing maltodextrin binding provide evidence for independent selectivity filters in each subunit.

PubMed Central

Ferenci, T; Lee, K S

1989-01-01

Maltoporin trimers constitute maltodextrin-selective channels in the outer membrane of Escherichia coli. To study the organization of the maltodextrin-binding site within trimers, dominance studies were undertaken with maltoporin variants of altered binding affinity. It has been established that amino acid substitutions at three dispersed regions of the maltoporin sequence (at residues 8, 82, and 360) resulted specifically in maltodextrin-binding defects and loss of maltodextrin channel selectivity; a substitution at residue 118 increased both binding affinity and maltodextrin transport. Strains heterodiploid for lamB were constructed in which these substitutions were encoded by chromosomal and plasmid-borne genes, and the relative level of maltoporin expression from these genes was estimated. Binding assays with bacteria forming maltoporin heterotrimers were performed in order to test for complementation between binding-negative alleles, negative dominance of negative over wild-type alleles, and possible dominance of negatives over the high-affinity allele. Double mutants with mutations affecting residues 8 and 118, 82 and 118, and 118 and 360 were constructed in vitro, and the dominance properties of the mutations in cis were also tested. There was no complementation between negatives and no negative dominance in heterotrimers. The high-affinity mutation was dominant over negatives in trans but not in cis. The affinity of binding sites in heterotrimer populations was characteristic of the high-affinity allele present and uninfluenced by the negative allele. These results are consistent with the presence of three discrete binding sites in a maltoporin trimer and suggest that the selectivity filter for maltodextrins is not at the interface between the three subunits. PMID:2521623

Do craniopharyngioma molecular signatures correlate with clinical characteristics?

PubMed

Omay, Sacit Bulent; Chen, Yu-Ning; Almeida, Joao Paulo; Ruiz-Treviño, Armando Saul; Boockvar, John A; Stieg, Philip E; Greenfield, Jeffrey P; Souweidane, Mark M; Kacker, Ashutosh; Pisapia, David J; Anand, Vijay K; Schwartz, Theodore H

2018-05-01

OBJECTIVE Exome sequencing studies have recently demonstrated that papillary craniopharyngiomas (PCPs) and adamantinomatous craniopharyngiomas (ACPs) have distinct genetic origins, each primarily driven by mutually exclusive alterations: either BRAF ( V600E), observed in 95% of PCPs, or CTNNB1, observed in 75%-96% of ACPs. How the presence of these molecular signatures, or their absence, correlates with clinical, radiographic, and outcome variables is unknown. METHODS The pathology records for patients who underwent surgery for craniopharyngiomas between May 2000 and March 2015 at Weill Cornell Medical College were reviewed. Craniopharyngiomas were identified and classified as PCP or ACP. Patients were placed into 1 of 3 groups based on their genomic mutations: BRAF mutation only, CTNNB1 mutation only, and tumors with neither of these mutations detected (not detected [ND]). Demographic, radiological, and clinical variables were collected, and their correlation with each genomic group was tested. RESULTS Histology correlated strongly with mutation group. All BRAF tumors with mutations were PCPs, and all CTNNB1 with mutations and ND tumors were ACPs. Preoperative and postoperative clinical symptoms and radiographic features did not correlate with any mutation group. There was a statistically significant relationship (p = 0.0323) between the age group (pediatric vs adult) and the mutation groups. The ND group tumors were more likely to involve the sella (p = 0.0065). CONCLUSIONS The mutation signature in craniopharyngioma is highly predictive of histology. The subgroup of tumors in which these 2 mutations are not detected is more likely to occur in children, be located in the sella, and be of ACP histology.

Frequency of 8 CFTR gene mutations in cystic fibrosis patients in Minas Gerais, Brazil, diagnosed by neonatal screening.

PubMed

Perone, C; Medeiros, G S; del Castillo, D M; de Aguiar, M J B; Januário, J N

2010-02-01

The nature and frequency of cystic fibrosis mutations in Brazil is not uniform due to the highly varied ethnic composition of the population. The average frequency of the F508del mutation has been reported to be 48.6%. Other common mutations in Brazil are G542X, R1162X, and N1303K. The aim of this study was to analyze the frequency of 8 mutations (F508del, G542X, R1162X, N1303K, W1282X, G85E, 3120+1G>A, and 711+1G>T) in a sample of 111 newborn patients with cystic fibrosis diagnosed by the Cystic Fibrosis Neonatal Screening Program of Minas Gerais State. The mutations were tested by allele-specific oligonucleotide PCR with specially designed primers. An allele frequency of 48.2% was observed for the F508del mutation, and allele frequencies of 5.41, 4.50, 4.05, and 3.60% were found for the R1162X, G542X, 3120+1G>A, and G85E mutations, respectively. The genotypes obtained were in Hardy-Weinberg equilibrium. These data demonstrate that the 8-mutation panel studied here has extensive coverage (68%) for the cystic fibrosis mutations in Minas Gerais. These data improve our knowledge of cystic fibrosis in Brazil, particularly in this region. In addition, this investigation contributed to the establishment of a sensitive and population-specific mutation panel, which can be helpful for molecular diagnosis of cystic fibrosis.

High-Throughput In Vivo Genotoxicity Testing: An Automated Readout System for the Somatic Mutation and Recombination Test (SMART)

PubMed Central

Kwak, Jihoon; Genovesio, Auguste; Kang, Myungjoo; Hansen, Michael Adsett Edberg; Han, Sung-Jun

2015-01-01

Genotoxicity testing is an important component of toxicity assessment. As illustrated by the European registration, evaluation, authorization, and restriction of chemicals (REACH) directive, it concerns all the chemicals used in industry. The commonly used in vivo mammalian tests appear to be ill adapted to tackle the large compound sets involved, due to throughput, cost, and ethical issues. The somatic mutation and recombination test (SMART) represents a more scalable alternative, since it uses Drosophila, which develops faster and requires less infrastructure. Despite these advantages, the manual scoring of the hairs on Drosophila wings required for the SMART limits its usage. To overcome this limitation, we have developed an automated SMART readout. It consists of automated imaging, followed by an image analysis pipeline that measures individual wing genotoxicity scores. Finally, we have developed a wing score-based dose-dependency approach that can provide genotoxicity profiles. We have validated our method using 6 compounds, obtaining profiles almost identical to those obtained from manual measures, even for low-genotoxicity compounds such as urethane. The automated SMART, with its faster and more reliable readout, fulfills the need for a high-throughput in vivo test. The flexible imaging strategy we describe and the analysis tools we provide should facilitate the optimization and dissemination of our methods. PMID:25830368

Selfish Spermatogonial Selection: Evidence from an Immunohistochemical Screen in Testes of Elderly Men

PubMed Central

Turner, Gareth D. H.; Dudka-Ruszkowska, Wioleta; Taylor, Stephen; Meyts, Ewa Rajpert-De; Goriely, Anne; Wilkie, Andrew O. M.

2012-01-01

The dominant congenital disorders Apert syndrome, achondroplasia and multiple endocrine neoplasia–caused by specific missense mutations in the FGFR2, FGFR3 and RET proteins respectively–represent classical examples of paternal age-effect mutation, a class that arises at particularly high frequencies in the sperm of older men. Previous analyses of DNA from randomly selected cadaveric testes showed that the levels of the corresponding FGFR2, FGFR3 and RET mutations exhibit very uneven spatial distributions, with localised hotspots surrounded by large mutation-negative areas. These studies imply that normal testes are mosaic for clusters of mutant cells: these clusters are predicted to have altered growth and signalling properties leading to their clonal expansion (selfish spermatogonial selection), but DNA extraction eliminates the possibility to study such processes at a tissue level. Using a panel of antibodies optimised for the detection of spermatocytic seminoma, a rare tumour of spermatogonial origin, we demonstrate that putative clonal events are frequent within normal testes of elderly men (mean age: 73.3 yrs) and can be classed into two broad categories. We found numerous small (less than 200 cells) cellular aggregations with distinct immunohistochemical characteristics, localised to a portion of the seminiferous tubule, which are of uncertain significance. However more infrequently we identified additional regions where entire seminiferous tubules had a circumferentially altered immunohistochemical appearance that extended through multiple serial sections that were physically contiguous (up to 1 mm in length), and exhibited enhanced staining for antibodies both to FGFR3 and a marker of downstream signal activation, pAKT. These findings support the concept that populations of spermatogonia in individual seminiferous tubules in the testes of older men are clonal mosaics with regard to their signalling properties and activation, thus fulfilling one of the specific predictions of selfish spermatogonial selection. PMID:22879958

The role of BRAF V600 mutation in melanoma

PubMed Central

2012-01-01

BRAF is a serine/threonine protein kinase activating the MAP kinase/ERK-signaling pathway. About 50 % of melanomas harbors activating BRAF mutations (over 90 % V600E). BRAFV600E has been implicated in different mechanisms underlying melanomagenesis, most of which due to the deregulated activation of the downstream MEK/ERK effectors. The first selective inhibitor of mutant BRAF, vemurafenib, after highly encouraging results of the phase I and II trial, was compared to dacarbazine in a phase III trial in treatment-naïve patients (BRIM-3). The study results showed a relative reduction of 63 % in risk of death and 74 % in risk of tumor progression. Considering all trials so far completed, median overall survival reached approximately 16 months for vemurafenib compared to less than 10 months for dacarbazine treatment. Vemurafenib has been extensively tested on melanoma patients expressing the BRAFV600E mutated form; it has been demonstrated to be also effective in inhibiting melanomas carrying the V600K mutation. In 2011, both FDA and EMA therefore approved vemurafenib for metastatic melanoma carrying BRAFV600 mutations. Some findings suggest that continuation of vemurafenib treatment is potentially beneficial after local therapy in a subset of patients with disease progression (PD). Among who continued vemurafenib >30 days after local therapy of PD lesion(s), a median overall survival was not reached, with a median follow-up of 15.5 months from initiation of BRAF inhibitor therapy. For patients who did not continue treatment, median overall survival from the time of disease progression was 1.4 months. A clinical phase I/II trial is evaluating the safety, tolerability and efficacy of vemurafenib in combination with the CTLA-4 inhibitor mAb ipilimumab. In the BRIM-7 trial vemurafenib is tested in association with GDC-0973, a potent and highly selective inhibitor of MEK1/2. Preliminary data seem to indicate that an additional inhibitor of mutated BRAF, GSK2118436, might be also active on a wider range of BRAF mutations (V600E-K-D-R); actually, treatment with such a compound is under evaluation in a phase III study among stage III-IV melanoma patients positive for BRAF mutations. Overall, BRAF inhibitors were well tolerated; common adverse events are arthralgia, rash, fatigue, alopecia, keratoacanthoma or cutaneous squamous-cell carcinoma, photosensitivity, nausea, and diarrhea, with some variants between different inhibitors. PMID:22554099

DeepGene: an advanced cancer type classifier based on deep learning and somatic point mutations.

PubMed

Yuan, Yuchen; Shi, Yi; Li, Changyang; Kim, Jinman; Cai, Weidong; Han, Zeguang; Feng, David Dagan

2016-12-23

With the developments of DNA sequencing technology, large amounts of sequencing data have become available in recent years and provide unprecedented opportunities for advanced association studies between somatic point mutations and cancer types/subtypes, which may contribute to more accurate somatic point mutation based cancer classification (SMCC). However in existing SMCC methods, issues like high data sparsity, small volume of sample size, and the application of simple linear classifiers, are major obstacles in improving the classification performance. To address the obstacles in existing SMCC studies, we propose DeepGene, an advanced deep neural network (DNN) based classifier, that consists of three steps: firstly, the clustered gene filtering (CGF) concentrates the gene data by mutation occurrence frequency, filtering out the majority of irrelevant genes; secondly, the indexed sparsity reduction (ISR) converts the gene data into indexes of its non-zero elements, thereby significantly suppressing the impact of data sparsity; finally, the data after CGF and ISR is fed into a DNN classifier, which extracts high-level features for accurate classification. Experimental results on our curated TCGA-DeepGene dataset, which is a reformulated subset of the TCGA dataset containing 12 selected types of cancer, show that CGF, ISR and DNN all contribute in improving the overall classification performance. We further compare DeepGene with three widely adopted classifiers and demonstrate that DeepGene has at least 24% performance improvement in terms of testing accuracy. Based on deep learning and somatic point mutation data, we devise DeepGene, an advanced cancer type classifier, which addresses the obstacles in existing SMCC studies. Experiments indicate that DeepGene outperforms three widely adopted existing classifiers, which is mainly attributed to its deep learning module that is able to extract the high level features between combinatorial somatic point mutations and cancer types.

Development of a High-Throughput Resequencing Array for the Detection of Pathogenic Mutations in Osteogenesis Imperfecta

PubMed Central

Wang, Yao; Cui, Yazhou; Zhou, Xiaoyan; Han, Jinxiang

2015-01-01

Objective Osteogenesis imperfecta (OI) is a rare inherited skeletal disease, characterized by bone fragility and low bone density. The mutations in this disorder have been widely reported to be on various exonal hotspots of the candidate genes, including COL1A1, COL1A2, CRTAP, LEPRE1, and FKBP10, thus creating a great demand for precise genetic tests. However, large genome sizes make the process daunting and the analyses, inefficient and expensive. Therefore, we aimed at developing a fast, accurate, efficient, and cheaper sequencing platform for OI diagnosis; and to this end, use of an advanced array-based technique was proposed. Method A CustomSeq Affymetrix Resequencing Array was established for high-throughput sequencing of five genes simultaneously. Genomic DNA extraction from 13 OI patients and 85 normal controls and amplification using long-range PCR (LR-PCR) were followed by DNA fragmentation and chip hybridization, according to standard Affymetrix protocols. Hybridization signals were determined using GeneChip Sequence Analysis Software (GSEQ). To examine the feasibility, the outcome from new resequencing approach was validated by conventional capillary sequencing method. Result Overall call rates using resequencing array was 96–98% and the agreement between microarray and capillary sequencing was 99.99%. 11 out of 13 OI patients with pathogenic mutations were successfully detected by the chip analysis without adjustment, and one mutation could also be identified using manual visual inspection. Conclusion A high-throughput resequencing array was developed that detects the disease-associated mutations in OI, providing a potential tool to facilitate large-scale genetic screening for OI patients. Through this method, a novel mutation was also found. PMID:25742658

Molecular Evolution of the Tissue-nonspecific Alkaline Phosphatase Allows Prediction and Validation of Missense Mutations Responsible for Hypophosphatasia*

PubMed Central

Silvent, Jérémie; Gasse, Barbara; Mornet, Etienne; Sire, Jean-Yves

2014-01-01

ALPL encodes the tissue nonspecific alkaline phosphatase (TNSALP), which removes phosphate groups from various substrates. Its function is essential for bone and tooth mineralization. In humans, ALPL mutations lead to hypophosphatasia, a genetic disorder characterized by defective bone and/or tooth mineralization. To date, 275 ALPL mutations have been reported to cause hypophosphatasia, of which 204 were simple missense mutations. Molecular evolutionary analysis has proved to be an efficient method to highlight residues important for the protein function and to predict or validate sensitive positions for genetic disease. Here we analyzed 58 mammalian TNSALP to identify amino acids unchanged, or only substituted by residues sharing similar properties, through 220 millions years of mammalian evolution. We found 469 sensitive positions of the 524 residues of human TNSALP, which indicates a highly constrained protein. Any substitution occurring at one of these positions is predicted to lead to hypophosphatasia. We tested the 204 missense mutations resulting in hypophosphatasia against our predictive chart, and validated 99% of them. Most sensitive positions were located in functionally important regions of TNSALP (active site, homodimeric interface, crown domain, calcium site, …). However, some important positions are located in regions, the structure and/or biological function of which are still unknown. Our chart of sensitive positions in human TNSALP (i) enables to validate or invalidate at low cost any ALPL mutation, which would be suspected to be responsible for hypophosphatasia, by contrast with time consuming and expensive functional tests, and (ii) displays higher predictive power than in silico models of prediction. PMID:25023282

Statewide Retrospective Review of Familial Pancreatic Cancer in Delaware, and Frequency of Genetic Mutations in Pancreatic Cancer Kindreds.

PubMed

Catts, Zohra Ali-Khan; Baig, Muhammad Khurram; Milewski, Becky; Keywan, Christine; Guarino, Michael; Petrelli, Nicholas

2016-05-01

Considering the typical rapid progression and high mortality of pancreatic cancer (PC), early detection may lead to an improved outcome. To date, there is no safe, sensitive, and cost-effective screening strategy to detect PC. Currently, screening is focused on individuals at the highest risk of developing PC based on family history. A high-risk individual is defined as having two or more first-degree relatives with PC, or one first- or second-degree relative with PC with a confirmed mutation in a gene associated with PC. The BRCA2 gene is one of the most common genes linked to pancreatic-only cancer families; however, other hereditary cancer syndromes have also been associated with an increased risk for PC. We conducted a retrospective review of pedigrees of families with a pancreatic adenocarcinoma cancer diagnosis held in the statewide Ruth Ann Minner High Risk Family Cancer Registry at the Helen F. Graham Cancer Center and Research Institute, Christiana Care Health System, Newark, DE, USA, from 2002 to 2013. The registry was queried based on how many first-, second-, or third-degree relatives of the proband were affected with PC, genetic testing status, and (if applicable) the results. These data were then categorized into families that meet familial PC (FPC) criteria, defined as two first-degree relatives with PC (FPC families), families that did not meet the FPC definition but had one first-degree relative affected with PC (first-degree families), and probands with PC (probands). Each family was counted only once in the analysis, even if multiple family members were tested. Our analysis revealed that 175 of 597 families fitting any of the above criteria completed genetic testing. Of this cohort, 52 had pathogenic alterations with nine different genes implicated. Overall, 164 of the 175 families that fitted into any of the three categories previously identified had BRCA1 or BRCA2 testing, either by DNA sequencing or next-generation sequencing via a panel test that included BRCA1/2. BRCA1 pathogenic alterations were noted in 17/164 (10.4 %) and BRCA2 pathogenic alterations were noted in 23/164 (14.0 %). FPC families (n = 46) 42/46 of the FPC families underwent BRCA1/2 testing, and 11/42 (26 % [95 % CI 12.89-39.49]) had pathogenic alterations. Specifically, 4/42 = BRCA1 (9.5 %) and 7/42 = BRCA2 (16.7 %). Additionally, 16/46 of the FPC families underwent exclusively Lynch syndrome (LS) testing, and pathogenic mutations in a mismatch repair protein were identified in 2/16. Specifically, 1/16 = MLH1 (6.3 %) and 1/16 = MSH2 (3.6 %). Overall, a genetic mutation within any gene associated with an increased PC risk was found in 28 % of FPC families. First-degree families (n = 106) 99/106 of the families with one first-degree relative underwent BRCA1/2 testing, and 21/99 (21.2 % [95 % CI 13.16-29.27]) had pathogenic alterations. Specifically, 11/99 = BRCA1 (11.1 %) and 10/99 = BRCA2 (10.1 %). 32/99 first-degree families underwent exclusively LS testing, and pathogenic mutations were identified in 4/32. Specifically, 3/32 = MLH1 (9 %) and 1/32 = MSH6 (3 %). 25/99 of the families pursued panel testing, and pathogenic alterations in any gene were identified in 3/25. Specifically, the mutations were found in 1/25 = ATM (4 %), 1/25 = CHEK2 (4 %), and 1/25 = RAD51D (4 %). Affected probands (n = 23) Lastly, all 23 probands affected with PC pursued genetic testing. Of these, 11/23 were found to have pathogenic alterations. All 23 underwent BRCA1/2 testing, and pathogenic alterations were identified in 8/23 (35 % [95 % CI 15.32-54.25]), specifically 2/23 = BRCA1 (9 %), and 6/23 = BRCA2 (26 %). 10/23 patients underwent panel testing and pathogenic alterations were found in 3/10 (30 %) patients, of whom 1/10 = MSH6 (10 %), 1/10 = ATM (10 %), and 1/10 = TP53 (10 %). This study demonstrates that a statewide high-risk family cancer registry is an important instrument in studying the risk of PC in families. Our analysis revealed 14 mutations associated with FPC, among which hereditary breast and ovarian cancer and LS were most prevalent. BRCA1 was found to have the same association with PC as BRCA2, which appears unique to our population. We plan to use our knowledge of these mutations in developing a PC screening program.

Molecular genetic analysis of the F11 gene in 14 Turkish patients with factor XI deficiency: identification of novel and recurrent mutations and their inheritance within families.

PubMed

Colakoglu, Seyma; Bayhan, Turan; Tavil, Betül; Keskin, Ebru Yılmaz; Cakir, Volkan; Gümrük, Fatma; Çetin, Mualla; Aytaç, Selin; Berber, Ergul

2018-01-01

Factor XI (FXI) deficiency is an autosomal bleeding disease associated with genetic defects in the F11 gene which cause decreased FXI levels or impaired FXI function. An increasing number of mutations has been reported in the FXI mutation database, most of which affect the serine protease domain of the protein. FXI is a heterogeneous disorder associated with a variable bleeding tendency and a variety of causative F11 gene mutations. The molecular basis of FXI deficiency in 14 patients from ten unrelated families in Turkey was analysed to establish genotype-phenotype correlations and inheritance of the mutations in the patients' families. Fourteen index cases with a diagnosis of FXI deficiency and family members of these patients were enrolled into the study. The patients' F11 genes were amplified by polymerase chain reaction and subjected to direct DNA sequencing analysis. The findings were analysed statistically using bivariate correlations, Pearson's correlation coefficient and the nonparametric Mann-Whitney test. Direct DNA sequencing analysis of the F11 genes revealed that all of the 14 patients had a F11 gene mutation. Eight different mutations were identified in the apple 1, apple 2 or serine protease domains, except one which was a splice site mutation. Six of the mutations were recurrent. Two of the mutations were novel missense mutations, p.Val522Gly and p.Cys581Arg, within the catalytic domain. The p.Trp519Stop mutation was observed in two families whereas all the other mutations were specific to a single family. Identification of mutations confirmed the genetic heterogeneity of FXI deficiency. Most of the patients with mutations did not have any bleeding complications, whereas some had severe bleeding symptoms. Genetic screening for F11 gene mutations is important to decrease the mortality and morbidity rate associated with FXI deficiency, which can be life-threatening if bleeding occurs in tissues with high fibrinolytic activity.

Mutation spectrum of RB1 mutations in retinoblastoma cases from Singapore with implications for genetic management and counselling.

PubMed

Tomar, Swati; Sethi, Raman; Sundar, Gangadhara; Quah, Thuan Chong; Quah, Boon Long; Lai, Poh San

2017-01-01

Retinoblastoma (RB) is a rare childhood malignant disorder caused by the biallelic inactivation of RB1 gene. Early diagnosis and identification of carriers of heritable RB1 mutations can improve disease outcome and management. In this study, mutational analysis was conducted on fifty-nine matched tumor and peripheral blood samples from 18 bilateral and 41 unilateral unrelated RB cases by a combinatorial approach of Multiplex Ligation-dependent Probe Amplification (MLPA) assay, deletion screening, direct sequencing, copy number gene dosage analysis and methylation assays. Screening of both blood and tumor samples yielded a mutation detection rate of 94.9% (56/59) while only 42.4% (25/59) of mutations were detected if blood samples alone were analyzed. Biallelic mutations were observed in 43/59 (72.9%) of tumors screened. There were 3 cases (5.1%) in which no mutations could be detected and germline mutations were detected in 19.5% (8/41) of unilateral cases. A total of 61 point mutations were identified, of which 10 were novel. There was a high incidence of previously reported recurrent mutations, occurring at 38.98% (23/59) of all cases. Of interest were three cases of mosaic RB1 mutations detected in the blood from patients with unilateral retinoblastoma. Additionally, two germline mutations previously reported to be associated with low-penetrance phenotypes: missense-c.1981C>T and splice variant-c.607+1G>T, were observed in a bilateral and a unilateral proband, respectively. These findings have implications for genetic counselling and risk prediction for the affected families. This is the first published report on the spectrum of mutations in RB patients from Singapore and shows that further improved mutation screening strategies are required in order to provide a definitive molecular diagnosis for every case of RB. Our findings also underscore the importance of genetic testing in supporting individualized disease management plans for patients and asymptomatic family members carrying low-penetrance, germline mosaicism or heritable unilateral mutational phenotypes.

[Views of Icelandic women towards genetic counseling - and testing of BRCA2 mutations].

PubMed

Jonsdottir, Thordis; Valdimarsdottir, Heiddis; Tryggvadottir, Laufey; Lund, Sigrun Helga; Thordardottir, Marianna; Magnusson, Magnus Karl; Valdimarsdottir, Unnur

2018-01-01

Introduction The aim of this study was to explore the attitudes of Icelandic women towards existing genetic information, genetic counseling and genetic testing for BRCA mutations which dramatically increase risk for aggressive cancers. Materials and methods Women attending the cancer prevention clinic in Reykjavik, capital of Iceland, from October 12th until November 20th 2015 received an invitation to participate. Participation involved answering a short online questionnaire about background, family history of cancer as well as attitudes towards genetic counseling, BRCA testing and preventive use of such information. Descriptive statistics and chi-square tests were used to describe differences in attitudes towards those questions between subgroups of women. Results 1129 women (69% response rate) answered the questionnaire. Mean age was 47 years (span 21-76 years). Around half (47%) had heard fairly much about the mutations. Independent of family history of cancer, the majority of women were positive towards receiving genetic counseling (79%) and to undergo genetic testing (83%) for BRCA mutation with younger women being more interested than older women. On the other hand, only 4% of the women had already received genetic counseling and 7% undergone genetic testing. Women with family history of cancer were more knowledgeable about BRCA mutations (p<0.0001) and were less afraid of the consequence of being a mutation carrier (p<0.0001) compared to those with little or no family history. Regardless of family history, half (49%) worried that results from genetic testing could influence their health insurance. Almost all, or 97% of the women, were positive or very positive toward using existing genetic information obtained through scientific work, to inform affected indi-viduals of their mutation status. Conclusion Icelandic women are positive towards genetic counseling and testing for BRCA mutations although half of them worry that a positive result might affect their health insurance. Nevertheless, almost all women believe that existing genetic information should be used to inform carriers for preventive purposes.

Monogenic diabetes in children and young adults: Challenges for researcher, clinician and patient

PubMed Central

2006-01-01

Monogenic diabetes results from one or more mutations in a single gene which might hence be rare but has great impact leading to diabetes at a very young age. It has resulted in great challenges for researchers elucidating the aetiology of diabetes and related features in other organ systems, for clinicians specifying a diagnosis that leads to improved genetic counselling, predicting of clinical course and changes in treatment, and for patients to altered treatment that has lead to coming off insulin and injections with no alternative (Glucokinase mutations), insulin injections being replaced by tablets (e.g. low dose in HNFα or high dose in potassium channel defects -Kir6.2 and SUR1) or with tablets in addition to insulin (e.g. metformin in insulin resistant syndromes). Genetic testing requires guidance to test for what gene especially given limited resources. Monogenic diabetes should be considered in any diabetic patient who has features inconsistent with their current diagnosis (unspecified neonatal diabetes, type 1 or type 2 diabetes) and clinical features of a specific subtype of monogenic diabetes (neonatal diabetes, familial diabetes, mild hyperglycaemia, syndromes). Guidance is given by clinical and physiological features in patient and family and the likelihood of the proposed mutation altering clinical care. In this article, I aimed to provide insight in the genes and mutations involved in insulin synthesis, secretion, and resistance, and to provide guidance for genetic testing by showing the clinical and physiological features and tests for each specified diagnosis as well as the opportunities for treatment. PMID:17186387

Clinical application of antenatal genetic diagnosis of osteogenesis imperfecta type IV.

PubMed

Yuan, Jing; Li, Song; Xu, YeYe; Cong, Lin

2015-04-02

Clinical analysis and genetic testing of a family with osteogenesis imperfecta type IV were conducted, aiming to discuss antenatal genetic diagnosis of osteogenesis imperfecta type IV. Preliminary genotyping was performed based on clinical characteristics of the family members and then high-throughput sequencing was applied to rapidly and accurately detect the changes in candidate genes. Genetic testing of the III5 fetus and other family members revealed missense mutation in c.2746G>A, pGly916Arg in COL1A2 gene coding region and missense and synonymous mutation in COL1A1 gene coding region. Application of antenatal genetic diagnosis provides fast and accurate genetic counseling and eugenics suggestions for patients with osteogenesis imperfecta type IV and their families.

Lessons learnt from implementation of a Lynch syndrome screening program for patients with gynaecological malignancy.

PubMed

Najdawi, Fedaa; Crook, Ashley; Maidens, Jayne; McEvoy, Christopher; Fellowes, Andrew; Pickett, Justine; Ho, Musei; Nevell, David; McIlroy, Kirsten; Sheen, Amy; Sioson, Loretta; Ahadi, Mahsa; Turchini, John; Clarkson, Adele; Hogg, Russell; Valmadre, Sue; Gard, Greg; Dooley, Susan J; Scott, Rodney J; Fox, Stephen B; Field, Michael; Gill, Anthony J

2017-08-01

Despite a trend towards universal testing, best practice to screen patients presenting with gynaecological malignancy for Lynch syndrome (LS) is uncertain. We report our institutional experience of a co-ordinated gynaecological LS screening program. All patients with endometrial carcinoma or carcinosarcoma, or gynaecological endometrioid or clear cell carcinomas undergo reflex four panel immunohistochemistry (IHC) for MLH1, PMS2, MSH2 and MSH6 followed by cascade somatic hypermethylation analysis of the MLH1 promoter locus for dual MLH1/PMS2 negative tumours. On the basis of these results, genetic counselling and targeted germline mutation testing is then offered to patients considered at high risk of LS. From 1 August 2013 to 31 December 2015, 124 patients were screened (mean age 64.6 years). Thirty-six (29.0%) demonstrated abnormal MMR IHC: 26 (72.2%) showed dual loss of MLH1/PMS2, five (13.9%) dual loss of MSH2/MSH6, three (8.3%) isolated loss of MSH6, and two (5.6%) isolated loss of PMS2. Twenty-five of 26 (96.1%) patients with dual MLH1/PMS2 loss demonstrated MLH1 promoter methylation. Therefore, 11 (8.9%) patients screened were classified as high risk for LS, of whom nine (81.8%) accepted germline mutation testing. Three (2.4% of total screened) were confirmed to have LS, two with germline PMS2 and one with germline MSH2 mutation. Massive parallel sequencing of tumour tissue demonstrated somatic mutations which were concordant with the IHC results in the remainder. Interestingly, the one MLH1/PMS2 IHC negative but not hypermethylated tumour harboured only somatic MLH1 mutations, indicating that universal cascade methylation testing in MLH1/PMS2 IHC negative tumours is very low yield and could be reconsidered in a resource-poor setting. In conclusion, universal screening for LS in patients presenting with gynaecological malignancy using the algorithm described above identified LS in three of 124 (2.4%) of our population. Only three of nine (33.3%) patients considered at high risk for LS by combined IHC and hypermethylation analysis were proven to have LS. Only one of the LS patients was less than 50 years of age and none of these patients would have been identified had more restrictive Amsterdam or Bethesda criteria been applied. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

Tumor testing to identify lynch syndrome in two Australian colorectal cancer cohorts.

PubMed

Buchanan, Daniel D; Clendenning, Mark; Rosty, Christophe; Eriksen, Stine V; Walsh, Michael D; Walters, Rhiannon J; Thibodeau, Stephen N; Stewart, Jenna; Preston, Susan; Win, Aung Ko; Flander, Louisa; Ouakrim, Driss Ait; Macrae, Finlay A; Boussioutas, Alex; Winship, Ingrid M; Giles, Graham G; Hopper, John L; Southey, Melissa C; English, Dallas; Jenkins, Mark A

2017-02-01

Tumor testing of colorectal cancers (CRC) for mismatch repair (MMR) deficiency is an effective approach to identify carriers of germline MMR gene mutation (Lynch syndrome). The aim of this study was to identify MMR gene mutation carriers in two cohorts of population-based CRC utilizing a combination of tumor and germline testing approaches. Colorectal cancers from 813 patients diagnosed with CRC < 60 years of age from the Australasian Colorectal Cancer Family Registry (ACCFR) and from 826 patients from the Melbourne Collaborative Cohort Study (MCCS) were tested for MMR protein expression using immunohistochemistry, microsatellite instability (MSI), BRAF V600E somatic mutation, and for MLH1 methylation. MMR gene mutation testing (Sanger sequencing and Multiplex Ligation Dependent Probe Amplification) was performed on germline DNA of patients with MMR-deficient tumors and a subset of MMR-proficient CRCs. Of the 813 ACCFR probands, 90 probands demonstrated tumor MMR deficiency (11.1%), and 42 had a MMR gene germline mutation (5.2%). For the MCCS, MMR deficiency was identified in the tumors of 103 probands (12.5%) and seven had a germline mutation (0.8%). All the mutation carriers were diagnosed prior to 70 years of age. Probands with a MMR-deficient CRC without MLH1 methylation and a gene mutation were considered Lynch-like and comprised 41.1% and 25.2% of the MMR-deficient CRCs for the ACCFR and MCCS, respectively. Identification of MMR gene mutation carriers in Australian CRC-affected patients is optimized by immunohistochemistry screening of CRC diagnosed before 70 years of age. A significant proportion of MMR-deficient CRCs will have unknown etiology (Lynch-like) proving problematic for clinical management. © 2016 Journal of Gastroenterology and Hepatology Foundation and John Wiley & Sons Australia, Ltd.

Tumour testing to identify Lynch syndrome in two Australian colorectal cancer cohorts

PubMed Central

Eriksen, Stine V.; Walsh, Michael D.; Walters, Rhiannon J.; Thibodeau, Stephen N.; Stewart, Jenna; Preston, Susan; Win, Aung Ko; Flander, Louisa; Ouakrim, Driss Ait; Macrae, Finlay A.; Boussioutas, Alex; Winship, Ingrid M.; Giles, Graham G.; Hopper, John L.; Southey, Melissa C.

2016-01-01

Background and Aim Tumour testing of colorectal cancers (CRC) for mismatch repair (MMR) deficiency is an effective approach to identify carriers of germline MMR gene mutation (Lynch syndrome). The aim of this study was to identify MMR gene mutation carriers in two cohorts of population-based CRC utilising a combination of tumour and germline testing approaches. Methods CRCs from 813 patients diagnosed with CRC <60 years of age from the Australasian Colorectal Cancer Family Registry (ACCFR) and from 826 patients from the Melbourne Collaborative Cohort Study (MCCS) were tested for MMR protein expression using immunohistochemistry (IHC), microsatellite instability (MSI), BRAFV600E somatic mutation and for MLH1 methylation. MMR gene mutation testing (Sanger sequencing and MLPA) was performed on germline DNA of patients with MMR-deficient tumours and a subset of MMR-proficient CRCs. Results Of the 813 ACCFR probands, 90 probands demonstrated tumour MMR-deficiency (11.1%) and 42 had a MMR gene germline mutation (5.2%). For the MCCS, MMR-deficiency was identified in the tumours of 103 probands (12.5%) and 7 had a germline mutation (0.8%). All the mutation carriers were diagnosed prior to 70 years of age. Probands with a MMR-deficient CRC without MLH1 methylation and a gene mutation were considered Lynch-like and comprised 41.1% and 22.3% of the MMR-deficient CRCs for the ACCFR and MCCS, respectively. Conclusions Identification of MMR gene mutation carriers in Australian CRC-affected patients is optimised by IHC screening of CRC diagnosed before 70 years. A significant proportion of MMR-deficient CRCs will have unknown aetiology (Lynch-like) proving problematic for clinical management. PMID:27273229

Mutational Effects and Population Dynamics During Viral Adaptation Challenge Current Models

PubMed Central

Miller, Craig R.; Joyce, Paul; Wichman, Holly A.

2011-01-01

Adaptation in haploid organisms has been extensively modeled but little tested. Using a microvirid bacteriophage (ID11), we conducted serial passage adaptations at two bottleneck sizes (104 and 106), followed by fitness assays and whole-genome sequencing of 631 individual isolates. Extensive genetic variation was observed including 22 beneficial, several nearly neutral, and several deleterious mutations. In the three large bottleneck lines, up to eight different haplotypes were observed in samples of 23 genomes from the final time point. The small bottleneck lines were less diverse. The small bottleneck lines appeared to operate near the transition between isolated selective sweeps and conditions of complex dynamics (e.g., clonal interference). The large bottleneck lines exhibited extensive interference and less stochasticity, with multiple beneficial mutations establishing on a variety of backgrounds. Several leapfrog events occurred. The distribution of first-step adaptive mutations differed significantly from the distribution of second-steps, and a surprisingly large number of second-step beneficial mutations were observed on a highly fit first-step background. Furthermore, few first-step mutations appeared as second-steps and second-steps had substantially smaller selection coefficients. Collectively, the results indicate that the fitness landscape falls between the extremes of smooth and fully uncorrelated, violating the assumptions of many current mutational landscape models. PMID:21041559

Screening for circulating RAS/RAF mutations by multiplex digital PCR.

PubMed

Andersen, Rikke Fredslund; Jakobsen, Anders

2016-07-01

Recent years have shown a large interest in the application of liquid biopsies in cancer management. Circulating tumor DNA (ctDNA) has been investigated for potential use in treatment selection, monitoring of treatment response, and early detection of recurrence. Advances have been hampered by technical challenges primarily due to the low levels of ctDNA in patients with localized disease and in patients responding to therapy. The approach presented here is a multiplex digital PCR method of screening for 31 mutations in the KRAS, NRAS, BRAF, and PIK3CA genes in the plasma. The upper level of the limit of blank, which defines the specificity of the multiplexes, was 0.006%-0.06%. Mutations found by multiplex analyses were identified and quantified by duplex analyses. The method was tested on samples from cholangiocarcinoma patients with known tumor mutational status. Mutations found in the tumor were also found in plasma samples in all cases with analyses for all other mutations being negative. There was a perfect agreement as to wild type status in tumor and plasma. The method combines a high sensitivity with the ability to analyze for several mutations at a time and could be a step towards routine clinical application of liquid biopsies. Copyright © 2016 Elsevier B.V. All rights reserved.

No correlation between germline mutation at repeat DNA and meiotic crossover in male mice exposed to X-rays or cisplatin.

PubMed

Barber, R; Plumb, M; Smith, A G; Cesar, C E; Boulton, E; Jeffreys, A J; Dubrova, Y E

2000-12-20

To test the hypothesis that mouse germline expanded simple tandem repeat (ESTR) mutations are associated with recombination events during spermatogenesis, crossover frequencies were compared with germline mutation rates at ESTR loci in male mice acutely exposed to 1Gy of X-rays or to 10mg/kg of the anticancer drug cisplatin. Ionising radiation resulted in a highly significant 2.7-3.6-fold increase in ESTR mutation rate in males mated 4, 5 and 6 weeks after exposure, but not 3 weeks after exposure. In contrast, irradiation had no effect on meiotic crossover frequencies assayed on six chromosomes using 25 polymorphic microsatellite loci spaced at approximately 20cM intervals and covering 421cM of the mouse genome. Paternal exposure to cisplatin did not affect either ESTR mutation rates or crossover frequencies, despite a report that cisplatin can increase crossover frequency in mice. Correlation analysis did not reveal any associations between the paternal ESTR mutation rate and crossover frequency in unexposed males and in those exposed to X-rays or cisplatin. This study does not, therefore, support the hypothesis that mutation induction at mouse ESTR loci results from a general genome-wide increase in meiotic recombination rate.

Estimate of within population incremental selection through branch imbalance in lineage trees

PubMed Central

Liberman, Gilad; Benichou, Jennifer I.C.; Maman, Yaakov; Glanville, Jacob; Alter, Idan; Louzoun, Yoram

2016-01-01

Incremental selection within a population, defined as limited fitness changes following mutation, is an important aspect of many evolutionary processes. Strongly advantageous or deleterious mutations are detected using the synonymous to non-synonymous mutations ratio. However, there are currently no precise methods to estimate incremental selection. We here provide for the first time such a detailed method and show its precision in multiple cases of micro-evolution. The proposed method is a novel mixed lineage tree/sequence based method to detect within population selection as defined by the effect of mutations on the average number of offspring. Specifically, we propose to measure the log of the ratio between the number of leaves in lineage trees branches following synonymous and non-synonymous mutations. The method requires a high enough number of sequences, and a large enough number of independent mutations. It assumes that all mutations are independent events. It does not require of a baseline model and is practically not affected by sampling biases. We show the method's wide applicability by testing it on multiple cases of micro-evolution. We show that it can detect genes and inter-genic regions using the selection rate and detect selection pressures in viral proteins and in the immune response to pathogens. PMID:26586802

Risk of colorectal cancer for people with a mutation in both a MUTYH and a DNA mismatch repair gene

PubMed Central

Win, Aung Ko; Reece, Jeanette C.; Buchanan, Daniel D.; Clendenning, Mark; Young, Joanne P.; Cleary, Sean P.; Kim, Hyeja; Cotterchio, Michelle; Dowty, James G.; MacInnis, Robert J.; Tucker, Katherine M.; Winship, Ingrid M.; Macrae, Finlay A.; Burnett, Terrilea; Le Marchand, Loïc; Casey, Graham; Haile, Robert W.; Newcomb, Polly A.; Thibodeau, Stephen N.; Lindor, Noralane M.; Hopper, John L.; Gallinger, Steven; Jenkins, Mark A.

2015-01-01

The base excision repair protein, MUTYH, functionally interacts with the DNA mismatch repair (MMR) system. As genetic testing moves from testing one gene at a time, to gene panel and whole exome next generation sequencing approaches, understanding the risk associated with co-existence of germline mutations in these genes will be important for clinical interpretation and management. From the Colon Cancer Family Registry, we identified 10 carriers who had both a MUTYH mutation (6 with c.1187G>A p.(Gly396Asp), 3 with c.821G>A p.(Arg274Gln), and 1 with c.536A>G p.(Tyr179Cys)) and a MMR gene mutation (3 in MLH1, 6 in MSH2, and 1 in PMS2), 375 carriers of a single (monoallelic) MUTYH mutation alone, and 469 carriers of a MMR gene mutation alone. Of the 10 carriers of both gene mutations, 8 were diagnosed with colorectal cancer. Using a weighted cohort analysis, we estimated that risk of colorectal cancer for carriers of both a MUTYH and a MMR gene mutation was substantially higher than that for carriers of a MUTYH mutation alone [hazard ratio (HR) 21.5, 95 % confidence interval (CI) 9.19–50.1; p < 0.001], but not different from that for carriers of a MMR gene mutation alone (HR 1.94, 95 % CI 0.63–5.99; p = 0.25). Within the limited power of this study, there was no evidence that a monoallelic MUTYH gene mutation confers additional risk of colorectal cancer for carriers of a MMR gene mutation alone. Our finding suggests MUTYH mutation testing in MMR gene mutation carriers is not clinically informative. PMID:26202870

Risk of colorectal cancer for people with a mutation in both a MUTYH and a DNA mismatch repair gene.

PubMed

Win, Aung Ko; Reece, Jeanette C; Buchanan, Daniel D; Clendenning, Mark; Young, Joanne P; Cleary, Sean P; Kim, Hyeja; Cotterchio, Michelle; Dowty, James G; MacInnis, Robert J; Tucker, Katherine M; Winship, Ingrid M; Macrae, Finlay A; Burnett, Terrilea; Le Marchand, Loïc; Casey, Graham; Haile, Robert W; Newcomb, Polly A; Thibodeau, Stephen N; Lindor, Noralane M; Hopper, John L; Gallinger, Steven; Jenkins, Mark A

2015-12-01

The base excision repair protein, MUTYH, functionally interacts with the DNA mismatch repair (MMR) system. As genetic testing moves from testing one gene at a time, to gene panel and whole exome next generation sequencing approaches, understandin g the risk associated with co-existence of germline mutations in these genes will be important for clinical interpretation and management. From the Colon Cancer Family Registry, we identified 10 carriers who had both a MUTYH mutation (6 with c.1187G>A p.(Gly396Asp), 3 with c.821G>A p.(Arg274Gln), and 1 with c.536A>G p.(Tyr179Cys)) and a MMR gene mutation (3 in MLH1, 6 in MSH2, and 1 in PMS2), 375 carriers of a single (monoallelic) MUTYH mutation alone, and 469 carriers of a MMR gene mutation alone. Of the 10 carriers of both gene mutations, 8 were diagnosed with colorectal cancer. Using a weighted cohort analysis, we estimated that risk of colorectal cancer for carriers of both a MUTYH and a MMR gene mutation was substantially higher than that for carriers of a MUTYH mutation alone [hazard ratio (HR) 21.5, 95% confidence interval (CI) 9.19-50.1; p < 0.001], but not different from that for carriers of a MMR gene mutation alone (HR 1.94, 95% CI 0.63-5.99; p = 0.25). Within the limited power of this study, there was no evidence that a monoallelic MUTYH gene mutation confers additional risk of colorectal cancer for carriers of a MMR gene mutation alone. Our finding suggests MUTYH mutation testing in MMR gene mutation carriers is not clinically informative.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Hong; Zeng, Hong; Lam, Robert

Mismatch repair prevents the accumulation of erroneous insertions/deletions and non-Watson–Crick base pairs in the genome. Pathogenic mutations in theMLH1gene are associated with a predisposition to Lynch and Turcot's syndromes. Although genetic testing for these mutations is available, robust classification of variants requires strong clinical and functional support. Here, the first structure of the N-terminus of human MLH1, determined by X-ray crystallography, is described. Lastly, the structure shares a high degree of similarity with previously determined prokaryoticMLH1homologs; however, this structure affords a more accurate platform for the classification ofMLH1variants.

Ras mutation cooperates with β-catenin activation to drive bladder tumourigenesis.

PubMed

Ahmad, I; Patel, R; Liu, Y; Singh, L B; Taketo, M M; Wu, X-R; Leung, H Y; Sansom, O J

2011-03-03

Mutations in the Ras family of proteins (predominantly in H-Ras) occur in approximately 40% of urothelial cell carcinoma (UCC). However, relatively little is known about subsequent mutations/pathway alterations that allow tumour progression. Indeed, expressing mutant H-Ras within the mouse bladder does not lead to tumour formation, unless this is expressed at high levels. The Wnt signalling pathway is deregulated in approximately 25% of UCC, so we examined if this correlated with the activation of MAPK signalling in human UCC and found a significant correlation. To test the functional significance of this association we examined the impact of combining Ras mutation (H-Ras(Q61L) or K-Ras(G12D)) with an activating β-catenin mutation within the mouse bladder using Cre-LoxP technology. Although alone, neither Ras mutation nor β-catenin activation led to UCC (within 12 months), mice carrying both mutations rapidly developed UCC. Mechanistically this was associated with reduced levels of p21 with dependence on the MAPK signalling pathway. Moreover, tumours from these mice were sensitive to MEK inhibition. Importantly, in human UCC there was a negative correlation between levels of p-ERK and p21 suggesting that p21 accumulation may block tumour progression following Ras mutation. Taken together these data definitively show Ras pathway activation strongly cooperates with Wnt signalling to drive UCC in vivo.

Surgical perspective of T1799A BRAF mutation diagnostic value in papillary thyroid carcinoma.

PubMed

Brahma, Bayu; Yulian, Erwin Danil; Ramli, Muchlis; Setianingsih, Iswari; Gautama, Walta; Brahma, Putri; Sastroasmoro, Sudigdo; Harimurti, Kuntjoro

2013-01-01

Throughout Indonesia, thyroid cancer is one of the ten commonest malignancies, with papillary thyroid carcinoma (PTC) in our hospital accounting for about 60% of all thyroid nodules. Although fine needle aspiration biopsy (FNAB) is the most reliable diagnostic tool, some nodules are diagnosed as indeterminate and second surgery is common for PTC. The aim of this study was to establish the diagnostic value and feasibility of testing the BRAF T1799A mutation on FNA specimens for improving PTC diagnosis. This prospective study enrolled 95 patients with thyroid nodules and future surgery planned. Results of mutational status were compared with surgical pathology diagnosis. Of the 70 cases included in the final analysis, 62.8% were PTC and the prevalence of BRAF mutation was 38.6%. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for BRAF mutation analysis were 36%, 100%, 100% and 48%, respectively. With other data findings, nodules with "onset less than 5 year" and "hard consistency" were proven as diagnostic determinants for BRAF mutation with a probability of 62.5%. This mutation was also a significant risk factor for extra-capsular extension. Molecular analysis of the BRAF T1799A mutation in FNAB specimens has high specificity and positive predictive value for PTC. It could be used in the selective patients with clinical characteristics to facilitate PTC diagnosis and for guidance regarding extent of thyroidectomy.

Biochemical and genetic diagnosis of the primary hyperoxalurias: a review.

PubMed

Rumsby, G

2000-01-01

The primary hyperoxalurias are a group of inherited disorders of endogenous oxalate overproduction. Diagnosis of the two best-characterized disorders, primary hyperoxaluria (PH) Types 1 and 2, is achieved by sequential measurement of alanine:glyoxylate aminotransferase and glyoxylate reductase enzyme activity in a single needle liver biopsy. While genetic analysis of PH2 is still at a relatively early stage, the AGXT gene defective in the Type 1 disorder is well characterized, and a number of mutations have been identified. To determine whether mutation analysis could replace enzymatic analysis for the diagnosis of PH1, DNA samples from 127 consecutive unrelated patients in whom there was a high clinical suspicion of primary hyperoxaluria were analyzed for the presence of the G630A and T853C mutations, which together account for approximately 34% of the mutant alleles in our patient cohort. The sensitivity of mutation detection was 47% in those patients with enzymologically confirmed Type 1 disease, showing that mutation analysis cannot effectively replace enzymology at the present time. However, there is little doubt of the value of genetic methods (mutation and linkage analysis) for diagnosing PH1 (and eventually PH2) in other family members and for prenatal diagnosis and carrier testing.

BRCA Mutation Frequency and Patterns of Treatment Response in BRCA Mutation–Positive Women With Ovarian Cancer: A Report From the Australian Ovarian Cancer Study Group

PubMed Central

Alsop, Kathryn; Fereday, Sian; Meldrum, Cliff; deFazio, Anna; Emmanuel, Catherine; George, Joshy; Dobrovic, Alexander; Birrer, Michael J.; Webb, Penelope M.; Stewart, Colin; Friedlander, Michael; Fox, Stephen; Bowtell, David; Mitchell, Gillian

2012-01-01

Purpose The frequency of BRCA1 and BRCA2 germ-line mutations in women with ovarian cancer is unclear; reports vary from 3% to 27%. The impact of germ-line mutation on response requires further investigation to understand its impact on treatment planning and clinical trial design. Patients and Methods Women with nonmucinous ovarian carcinoma (n = 1,001) enrolled onto a population-based, case-control study were screened for point mutations and large deletions in both genes. Survival outcomes and responses to multiple lines of chemotherapy were assessed. Results Germ-line mutations were found in 14.1% of patients overall, including 16.6% of serous cancer patients (high-grade serous, 22.6%); 44% had no reported family history of breast or ovarian cancer. Patients carrying germ-line mutations had improved rates of progression-free and overall survival. In the relapse setting, patients carrying mutations more frequently responded to both platin- and nonplatin-based regimens than mutation-negative patients, even in patients with early relapse after primary treatment. Mutation-negative patients who responded to multiple cycles of platin-based treatment were more likely to carry somatic BRCA1/2 mutations. Conclusion BRCA mutation status has a major influence on survival in ovarian cancer patients and should be an additional stratification factor in clinical trials. Treatment outcomes in BRCA1/2 carriers challenge conventional definitions of platin resistance, and mutation status may be able to contribute to decision making and systemic therapy selection in the relapse setting. Our data, together with the advent of poly(ADP-ribose) polymerase inhibitor trials, supports the recommendation that germ-line BRCA1/2 testing should be offered to all women diagnosed with nonmucinous, ovarian carcinoma, regardless of family history. PMID:22711857

Use of whole genome sequencing in surveillance of drug resistant tuberculosis.

PubMed

McNerney, Ruth; Zignol, Matteo; Clark, Taane G

2018-05-01

The threat of resistance to anti-tuberculosis drugs is of global concern. Current efforts to monitor resistance rely on phenotypic testing where cultured bacteria are exposed to critical concentrations of the drugs. Capacity for such testing is low in TB endemic countries. Drug resistance is caused by mutations in the Mycobacterium tuberculosis genome and whole genome sequencing to detect these mutations offers an alternative means of assessing resistance. Areas covered: The challenges of assessing TB drug resistance are discussed. Progress in elucidating the M. tuberculosis resistome and evidence of the accuracy of next generation sequencing for detecting resistance is reviewed. Expert Commentary: There are considerable advantages to using next generation sequencing for TB drug resistance surveillance. Accuracy is high for detecting resistance to the major first-line drugs but is currently lower for the second-line drugs due to our incomplete knowledge regarding resistance causing mutations. With the advances in sequencing technology and the opportunity to replace phenotypic drug susceptibility testing with safer and more cost effective methods it would appear that the question is when to implement. Current bottlenecks are sample extraction to allow whole genome sequencing directly from sputum and the lack of bioinformatics expertise in some TB endemic countries.

The suitability of matrix assisted laser desorption/ionization time of flight mass spectrometry in a laboratory developed test using cystic fibrosis carrier screening as a model.

PubMed

Farkas, Daniel H; Miltgen, Nicholas E; Stoerker, Jay; van den Boom, Dirk; Highsmith, W Edward; Cagasan, Lesley; McCullough, Ron; Mueller, Reinhold; Tang, Lin; Tynan, John; Tate, Courtney; Bombard, Allan

2010-09-01

We designed a laboratory developed test (LDT) by using an open platform for mutation/polymorphism detection. Using a 108-member (mutation plus variant) cystic fibrosis carrier screening panel as a model, we completed the last phase of LDT validation by using matrix-assisted laser desorption/ionization time of flight mass spectrometry. Panel customization was accomplished via specific amplification primer and extension probe design. Amplified genomic DNA was subjected to allele specific, single base extension endpoint analysis by mass spectrometry for inspection of the cystic fibrosis transmembrane regulator gene (NM_000492.3). The panel of mutations and variants was tested against 386 blinded samples supplied by "authority" laboratories highly experienced in cystic fibrosis transmembrane regulator genotyping; >98% concordance was observed. All discrepant and discordant results were resolved satisfactorily. Taken together, these results describe the concluding portion of the LDT validation process and the use of mass spectrometry to detect a large number of complex reactions within a single run as well as its suitability as a platform appropriate for interrogation of scores to hundreds of targets.

Implementation and utilization of genetic testing in personalized medicine

PubMed Central

Abul-Husn, Noura S; Owusu Obeng, Aniwaa; Sanderson, Saskia C; Gottesman, Omri; Scott, Stuart A

2014-01-01

Clinical genetic testing began over 30 years ago with the availability of mutation detection for sickle cell disease diagnosis. Since then, the field has dramatically transformed to include gene sequencing, high-throughput targeted genotyping, prenatal mutation detection, preimplantation genetic diagnosis, population-based carrier screening, and now genome-wide analyses using microarrays and next-generation sequencing. Despite these significant advances in molecular technologies and testing capabilities, clinical genetics laboratories historically have been centered on mutation detection for Mendelian disorders. However, the ongoing identification of deoxyribonucleic acid (DNA) sequence variants associated with common diseases prompted the availability of testing for personal disease risk estimation, and created commercial opportunities for direct-to-consumer genetic testing companies that assay these variants. This germline genetic risk, in conjunction with other clinical, family, and demographic variables, are the key components of the personalized medicine paradigm, which aims to apply personal genomic and other relevant data into a patient’s clinical assessment to more precisely guide medical management. However, genetic testing for disease risk estimation is an ongoing topic of debate, largely due to inconsistencies in the results, concerns over clinical validity and utility, and the variable mode of delivery when returning genetic results to patients in the absence of traditional counseling. A related class of genetic testing with analogous issues of clinical utility and acceptance is pharmacogenetic testing, which interrogates sequence variants implicated in interindividual drug response variability. Although clinical pharmacogenetic testing has not previously been widely adopted, advances in rapid turnaround time genetic testing technology and the recent implementation of preemptive genotyping programs at selected medical centers suggest that personalized medicine through pharmacogenetics is now a reality. This review aims to summarize the current state of implementing genetic testing for personalized medicine, with an emphasis on clinical pharmacogenetic testing. PMID:25206309

Sensitive and reliable detection of Kit point mutation Asp 816 to Val in pathological material

PubMed Central

Kähler, Christian; Didlaukat, Sabine; Feller, Alfred C; Merz, Hartmut

2007-01-01

Background Human mastocytosis is a heterogenous disorder which is linked to a gain-of-function mutation in the kinase domain of the receptor tyrosine kinase Kit. This D816V mutation leads to constitutive activation and phosphorylation of Kit with proliferative disorders of mast cells in the peripheral blood, skin, and spleen. Most PCR applications used so far are labour-intensive and are not adopted to daily routine in pathological laboratories. The method has to be robust and working on such different materials like archival formalin-fixed, paraffin-embedded tissue (FFPE) and blood samples. Such a method is introduced in this publication. Methods The Kit point mutation Asp 816 to Val is heterozygous which means a problem in detection by PCR because the wild-type allele is also amplified and the number of cells which bear the point mutation is in most of the cases low. Most PCR protocols use probes to block the wild-type allele during amplification with more or less satisfying result. This is why point-mutated forward primers were designed and tested for efficiency in amplification of the mutated allele. Results One primer combination (A) fits the most for the introduced PCR assay. It was able just to amplify the mutated allele with high specificity from different patient's materials (FFPE or blood) of varying quality and quantity. Moreover, the sensitivity for this assay was convincing because 10 ng of DNA which bears the point mutation could be detected in a total volume of 200 ng of DNA. Conclusion The PCR assay is able to deal with different materials (blood and FFPE) this means quality and quantity of DNA and can be used for high-througput screening because of its robustness. Moreover, the method is easy-to-use, not labour-intensive, and easy to realise in a standard laboratory. PMID:17900365

Survey of familial glaucoma shows a high incidence of cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1) mutations in non-consanguineous congenital forms in a Spanish population

PubMed Central

Millá, Elena; Mañé, Begoña; Duch, Susana; Hernan, Imma; Borràs, Emma; Planas, Ester; Dias, Miguel de Sousa; Carballo, Miguel

2013-01-01

Purpose To identify myocilin (MYOC) and cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1) mutations in a Spanish population with different clinical forms of familial glaucoma or ocular hypertension (OHT). Methods Index patients from 226 families participated in this study. Patients were diagnosed with familial glaucoma or OHT by complete ophthalmologic examination. Screening for MYOC mutations was performed in 207 index patients: 96 with adult-onset primary open-angle glaucoma (POAG), 21 with primary congenital glaucoma (PCG), 18 with juvenile-onset open-angle glaucoma (JOAG), five with Axenfeld-Rieger syndrome (ARS), and 67 with other types of glaucoma. One hundred two of the families (including all those in whom a MYOC mutation was detected) were also screened for CYP1B1 mutations: 45 POAG, 25 PCG, 21 JOAG, four ARS, and seven others. Results We examined 292 individuals (patients and relatives) with a positive family history of glaucoma or OHT. We identified two novel MYOC variants, p.Lys39Arg and p.Glu218Lys, in two families with POAG, and six previously reported MYOC mutations in seven families with POAG (four), JOAG (one), PCG (one), and normotensive glaucoma (one). CYP1B1 mutations were found in 16 index patients with PCG (nine), POAG (three), JOAG (two), and ARS (two). Conclusions The high percentage (9/25=36%) of mutations in CYP1B1 found in non-consanguineous patients with congenital glaucoma mandates genetic testing. However, the percentage of mutations (9/207=4.4%) in MYOC associated with glaucoma is relatively low in our population. The variable phenotype expression of glaucoma, even in families, cannot be explained with a digenic mechanism between MYOC and CYP1B1. PMID:23922489

Evaluation of genotoxic activity of maleic hydrazide, ethyl methane sulfonate, and N-nitroso diethylamine in Tradescantia.

PubMed

Alvarez-Moya, C; Santerre-Lucas, A; Zúñiga-González, G; Torres-Bugarín, O; Padilla-Camberos, E; Feria-Velasco, A

2001-01-01

To assess the genotoxic activity of N-nitroso diethylamine (NDEA), maleic hydrazide (MH), and ethyl methane sulfonate (EMS) using two systems: the comet assay on nuclei from Tradescantia, and the pink mutation test on Tradescantia staminal hairs (clone 4430). Tradescantia cups was obtained from Laboratorio de Citogenética y Mutagénesis del Centro de Ciencias de la Atmósfera de la Universidad Nacional Autónoma de México and treated with: N-nitroso diethylamine (NDEA) at 1, 5, 10 mM, maleic hydrazide (MH) at 1, 5, 10 mM and ethyl methane sulfonate (EMS) at 15, 30 and 45 mM; and used in both pink mutation assay and comet assay using cellular nuclei from Tradescantia staminal hairs. The observation of staminal hair was realized along eight days (6-14) after treatment), flowers produced day 14 after treatment were utilized done according to Underbrink. In previous reports on plants, were comet assay was used, breaking cellular wall and separating by centrifugation gradient are necessary. Here, nuclei from staminal hairs were obtained by squashing the cells (is not necessary to utilize to break special procedure cellular wall), collected using a nylon mesh of 80 Mm and next the comet assay was applied. Student's T test was the statistical test used for analyzing the comet assay data. Both assays showed a great sensitivity to the studied mutagens. A relationship between the dose-pink event and the dose-tail length was evident. Even though the Tradescantia mutation assay is a sensitive test with MH and EMS, low doses of NDEA were not able to induce a significant increase in the pink event frequencies; however, the comet assay was able to detect the mutagenic effect of NDEA at the same dose. Thus, it is clear that the comet assay is highly sensitive to the lowest dose of chemical mutagens. The comet assay on nuclei from Tradescantia staminal hairs is a useful tool to monitor genotoxic agents; it is simple, highly sensitive, and faster than the pink mutation test.

Stargardt macular dystrophy: common ABCA4 mutations in South Africa—establishment of a rapid genetic test and relating risk to patients

PubMed Central

Nossek, Christel A.; Greenberg, L. Jacquie; Ramesar, Rajkumar S.

2012-01-01

Purpose Based on the previous indications of founder ATP-binding cassette sub-family A member 4 gene (ABCA4) mutations in a South African subpopulation, the purpose was to devise a mechanism for identifying common disease-causing mutations in subjects with ABCA4-associated retinopathies (AARs). Facilitating patient access to this data and determining the frequencies of the mutations in the South African population would enhance the current molecular diagnostic service offered. Methods The majority of subjects in this study were of Caucasian ancestry and affected with Stargardt macular dystrophy. The initial cohort consisted of DNA samples from 181 patients, and was screened using the ABCR400 chip. An assay was then designed to screen a secondary cohort of 72 patients for seven of the most commonly occurring ABCA4 mutations in this population. A total of 269 control individuals were also screened for the seven ABCA4 mutations. Results Microarray screening results from a cohort of 181 patients affected with AARs revealed that seven ABCA4 mutations (p.Arg152*, c.768G>T, p.Arg602Trp, p.Gly863Ala, p.Cys1490Tyr, c.5461–10T>C, and p.Leu2027Phe) occurred at a relatively high frequency. The newly designed genetic assay identified two of the seven disease-associated mutations in 28/72 patients in a secondary patient cohort. In the control cohort, 12/269 individuals were found to be heterozygotes, resulting in an estimated background frequency of these mutations in this particular population of 4.46 per 100 individuals. Conclusions The relatively high detection rate of seven ABCA4 mutations in the primary patient cohort led to the design and subsequent utility of a multiplex assay. This assay can be used as a viable screening tool and to reduce costs and laboratory time. The estimated background frequency of the seven ABCA4 mutations, together with the improved diagnostic service, could be used by counselors to facilitate clinical and genetic management of South African families with AARs. PMID:22328824

Overcoming Cost Implications of Mutational Analysis in Patients with Gastrointestinal Stromal Tumors: A Pragmatic Approach.

PubMed

Schöffski, Patrick; Wozniak, Agnieszka; Schöffski, Oliver; van Eycken, Liesbet; Debiec-Rychter, Maria

2016-01-01

Genetic analysis of tissue derived from patients with advanced gastrointestinal stromal tumors (GISTs) is not uniformly applied on a national and international level, even though mutational data can provide clinically relevant prognostic and predictive information, especially in patients qualifying for treatment with expensive targeted agents. The current article describes the rationale for genetic testing of GIST tissue, looks at financial implications associated with such analysis and speculates on potential cost savings introduced by routine mutational testing and tailored use of tyrosine kinase inhibitors based on genotyping. This work is based on a hypothetical analysis of epidemiological data, drug costs, reimbursement criteria and market research figures. The cost burden for routine genotyping of important genes in GISTs, especially in patients at high risk for relapse after primary surgery and in advanced, inoperable metastatic disease, is relatively low. The early identification of GISTs with primary resistance mutations should be the basis for personalized GIST treatment and reimbursement of drugs. As illustrated by Belgian figures, the exclusive use of a drug such as imatinib in patients who are likely to benefit from the agent based on genetic information can lead to significant cost savings, which outweigh the costs for testing. Mutational analysis of GIST should be considered early in all patients at risk for relapse after curative surgery and in the case of advanced, inoperable, metastatic disease. The costs for the actual genotyping should not be used as an argument against profiling of the tumor. The adjuvant and palliative systemic treatment of GISTs should be personalized based on the genotype and other known prognostic and predictive factors. Reimbursement criteria for essential agents such as imatinib should be adapted accordingly. © 2016 S. Karger GmbH, Freiburg.

Characterizing biased cancer-related cognitive processing: relationships with BRCA1/2 genetic mutation status, personal cancer history, age, and prophylactic surgery.

PubMed

Carpenter, Kristen M; Eisenberg, Stacy; Weltfreid, Sharone; Low, Carissa A; Beran, Tammy; Stanton, Annette L

2014-09-01

This study evaluated associations of cancer-related cognitive processing with BRCA1/2 mutation carrier status, personal cancer history, age, and election of prophylactic surgery in women at high risk for breast cancer. In a 2 (BRCA1/2 mutation carrier status) × 2 (personal cancer history) matched-control design, with age as an additional predictor, participants (N = 115) completed a computerized cancer Stroop task. Dependent variables were response latency to cancer-related stimuli (reaction time [RT]) and cancer-related cognitive interference (cancer RT minus neutral RT). RT and interference were tested as predictors of prophylactic surgery in the subsequent four years. RT for cancer-related words was significantly slower than other word groups, indicating biased processing specific to cancer-related stimuli. Participants with a cancer history evidenced longer RT to cancer-related words than those without a history; moreover, a significant Cancer History × Age interaction indicated that, among participants with a cancer history, the typical advantage associated with younger age on Stroop tasks was absent. BRCA mutation carriers demonstrated more cancer-related cognitive interference than noncarriers. Again, the typical Stroop age advantage was absent among carriers. Exploratory analyses indicated that BRCA+ status and greater cognitive interference predicted greater likelihood of undergoing prophylactic surgery. Post hoc tests suggest that cancer-related distress does not account for these relationships. In the genetic testing context, younger women with a personal cancer history or who are BRCA1/2 mutation carriers might be particularly vulnerable to biases in cancer-related cognitive processing. Biased processing was associated marginally with greater likelihood of prophylactic surgery. (PsycINFO Database Record (c) 2014 APA, all rights reserved).

Technical Evaluation: Identification of Pathogenic Mutations in PKD1 and PKD2 in Patients with Autosomal Dominant Polycystic Kidney Disease by Next-Generation Sequencing and Use of a Comprehensive New Classification System.

PubMed

Kinoshita, Moritoshi; Higashihara, Eiji; Kawano, Haruna; Higashiyama, Ryo; Koga, Daisuke; Fukui, Takafumi; Gondo, Nobuhisa; Oka, Takehiko; Kawahara, Kozo; Rigo, Krisztina; Hague, Tim; Katsuragi, Kiyonori; Sudo, Kimiyoshi; Takeshi, Masahiko; Horie, Shigeo; Nutahara, Kikuo

2016-01-01

Genetic testing of PKD1 and PKD2 is expected to play an increasingly important role in determining allelic influences in autosomal dominant polycystic kidney disease (ADPKD) in the near future. However, to date, genetic testing is not commonly employed because it is expensive, complicated because of genetic heterogeneity, and does not easily identify pathogenic variants. In this study, we developed a genetic testing system based on next-generation sequencing (NGS), long-range polymerase chain reaction, and a new software package. The new software package integrated seven databases and provided access to five cloud-based computing systems. The database integrated 241 polymorphic nonpathogenic variants detected in 140 healthy Japanese volunteers aged >35 years, who were confirmed by ultrasonography as having no cysts in either kidney. Using this system, we identified 60 novel and 30 known pathogenic mutations in 101 Japanese patients with ADPKD, with an overall detection rate of 89.1% (90/101) [95% confidence interval (CI), 83.0%-95.2%]. The sensitivity of the system increased to 93.1% (94/101) (95% CI, 88.1%-98.0%) when combined with multiplex ligation-dependent probe amplification analysis, making it sufficient for use in a clinical setting. In 82 (87.2%) of the patients, pathogenic mutations were detected in PKD1 (95% CI, 79.0%-92.5%), whereas in 12 (12.8%) patients pathogenic mutations were detected in PKD2 (95% CI, 7.5%-21.0%); this is consistent with previously reported findings. In addition, we were able to reconfirm our pathogenic mutation identification results using Sanger sequencing. In conclusion, we developed a high-sensitivity NGS-based system and successfully employed it to identify pathogenic mutations in PKD1 and PKD2 in Japanese patients with ADPKD.

[A preliminary study on p53 gene in lung cancer tissues of workers exposed to silica and welding fumes].

PubMed

Liu, B; Zhou, P; Miao, Q

1997-05-01

Mutations of suppressor gene p53 was studied in 36 cases of silica related lung cancer and 6 cases of welding fume related lung cancer with immunohistochemical and PCR-SSCP methods. Cancer tissues were embedded in paraffin and stored for 13.4 years in average. Results revealed that there was abnormal mobility shift of electrophoresis in 18 cases with 20 point mutations of 42 specimens tested, accounted for 42.9%, and 50% (10/20) of the mutations were clustered in exon 8. This finding differed from mutational spectrum of gene in non-occupational lung cancer, in which mutation frequency of exon 8 ranged from 17.5% to 23.5%. Gene mutation frequency in varied pathological categories of pneumoconiosis related lung cancer also differed from that in common lung cancer. In the latter, the highest one was in small cell lung cancer (70%) and the lowest in adenocarcinoma (33%), but in the former, the highest in adenocarcinoma (53.9%) and the lowest in small cell lung cancer (30.8%). Immunohistochemical observations also showed a very high prevalence of p53 gene mutation expression (46.9%). Sequencing, which was determined in two cases of this study, revealed that two point mutations all occurred in non-hotspot codon 144 of p53 gene. Difference in gene mutation spectrum suggests that there exist specific carcinogens and carcinogenesis in silica and welding fume related lung cancer.

Clinical utility of routine MPL exon 10 analysis in the diagnosis of essential thrombocythaemia and primary myelofibrosis.

PubMed

Boyd, Elaine M; Bench, Anthony J; Goday-Fernández, Andrea; Anand, Shubha; Vaghela, Krishna J; Beer, Phillip; Scott, Mike A; Bareford, David; Green, Anthony R; Huntly, Brian; Erber, Wendy N

2010-04-01

Approximately 50% of essential thrombocythaemia and primary myelo-fibrosis patients do not have a JAK2 V617F mutation. Up to 5% of these are reported to have a MPL exon 10 mutation but testing for MPL is not routine as there are multiple mutation types. The ability to routinely assess both JAK2 and MPL mutations would be beneficial in the differential diagnosis of unexplained thrombocytosis or myelofibrosis. We developed and applied a high resolution melt (HRM) assay, capable of detecting all known MPL mutations in a single analysis, for the detection of MPL exon 10 mutations. We assessed 175 ET and PMF patients, including 67 that were JAK2 V617F-negative by real time polymerase chain reaction (PCR). Overall, 19/175 (11%) patients had a MPL exon 10 mutation, of whom 16 were JAK2 V617F-negative (16/67; 24%). MPL mutation types were W515L (11), W515K (4), W515R (2) and W515A (1). One patient had both W515L and S505N MPL mutations and these were present in the same haemopoietic colonies. Real time PCR for JAK2 V617F analysis and HRM for MPL exon 10 status identified one or more clonal marker in 71% of patients. This combined genetic approach increases the sensitivity of meeting the World Health Organization diagnostic criteria for these myeloproliferative neoplasms.

Molecular Testing for miRNA, mRNA, and DNA on Fine-Needle Aspiration Improves the Preoperative Diagnosis of Thyroid Nodules With Indeterminate Cytology.

PubMed

Labourier, Emmanuel; Shifrin, Alexander; Busseniers, Anne E; Lupo, Mark A; Manganelli, Monique L; Andruss, Bernard; Wylie, Dennis; Beaudenon-Huibregtse, Sylvie

2015-07-01

Molecular testing for oncogenic mutations or gene expression in fine-needle aspirations (FNAs) from thyroid nodules with indeterminate cytology identifies a subset of benign or malignant lesions with high predictive value. This study aimed to evaluate a novel diagnostic algorithm combining mutation detection and miRNA expression to improve the diagnostic yield of molecular cytology. Surgical specimens and preoperative FNAs (n = 638) were tested for 17 validated gene alterations using the miRInform Thyroid test and with a 10-miRNA gene expression classifier generating positive (malignant) or negative (benign) results. Cross-sectional sampling of thyroid nodules with atypia of undetermined significance/follicular lesion of undetermined significance (AUS/FLUS) or follicular neoplasm/suspicious for a follicular neoplasm (FN/SFN) cytology (n = 109) was conducted at 12 endocrinology centers across the United States. Qualitative molecular results were compared with surgical histopathology to determine diagnostic performance and model clinical effect. Mutations were detected in 69% of nodules with malignant outcome. Among mutation-negative specimens, miRNA testing correctly identified 64% of malignant cases and 98% of benign cases. The diagnostic sensitivity and specificity of the combined algorithm was 89% (95% confidence interval [CI], 73-97%) and 85% (95% CI, 75-92%), respectively. At 32% cancer prevalence, 61% of the molecular results were benign with a negative predictive value of 94% (95% CI, 85-98%). Independently of variations in cancer prevalence, the test increased the yield of true benign results by 65% relative to mRNA-based gene expression classification and decreased the rate of avoidable diagnostic surgeries by 69%. Multiplatform testing for DNA, mRNA, and miRNA can accurately classify benign and malignant thyroid nodules, increase the diagnostic yield of molecular cytology, and further improve the preoperative risk-based management of benign nodules with AUS/FLUS or FN/SFN cytology.

Performance impact of mutation operators of a subpopulation-based genetic algorithm for multi-robot task allocation problems.

PubMed

Liu, Chun; Kroll, Andreas

2016-01-01

Multi-robot task allocation determines the task sequence and distribution for a group of robots in multi-robot systems, which is one of constrained combinatorial optimization problems and more complex in case of cooperative tasks because they introduce additional spatial and temporal constraints. To solve multi-robot task allocation problems with cooperative tasks efficiently, a subpopulation-based genetic algorithm, a crossover-free genetic algorithm employing mutation operators and elitism selection in each subpopulation, is developed in this paper. Moreover, the impact of mutation operators (swap, insertion, inversion, displacement, and their various combinations) is analyzed when solving several industrial plant inspection problems. The experimental results show that: (1) the proposed genetic algorithm can obtain better solutions than the tested binary tournament genetic algorithm with partially mapped crossover; (2) inversion mutation performs better than other tested mutation operators when solving problems without cooperative tasks, and the swap-inversion combination performs better than other tested mutation operators/combinations when solving problems with cooperative tasks. As it is difficult to produce all desired effects with a single mutation operator, using multiple mutation operators (including both inversion and swap) is suggested when solving similar combinatorial optimization problems.

Prevalence of the BRCA1 founder mutation c.5266dupin Brazilian individuals at-risk for the hereditary breast and ovarian cancer syndrome

PubMed Central

2011-01-01

About 5-10% of breast and ovarian carcinomas are hereditary and most of these result from germline mutations in the BRCA1 and BRCA2 genes. In women of Ashkenazi Jewish ascendance, up to 30% of breast and ovarian carcinomas may be attributable to mutations in these genes, where 3 founder mutations, c.68_69del (185delAG) and c.5266dup (5382insC) in BRCA1 and c.5946del (6174delT) in BRCA2, are commonly encountered. It has been suggested by some authors that screening for founder mutations should be undertaken in all Brazilian women with breast cancer. Thus, the goal of this study was to determine the prevalence of three founder mutations, commonly identified in Ashkenazi individuals in a sample of non-Ashkenazi cancer-affected Brazilian women with clearly defined risk factors for hereditary breast and ovarian cancer (HBOC) syndrome. Among 137 unrelated Brazilian women from HBOC families, the BRCA1c.5266dup mutation was identified in seven individuals (5%). This prevalence is similar to that encountered in non-Ashkenazi HBOC families in other populations. However, among patients with bilateral breast cancer, the frequency of c.5266dup was significantly higher when compared to patients with unilateral breast tumors (12.1% vs 1.2%, p = 0.023). The BRCA1 c.68_69del and BRCA2 c.5946del mutations did not occur in this sample. We conclude that screening non-Ashkenazi breast cancer-affected women from the ethnically heterogeneous Brazilian populations for the BRCA1 c.68_69del and BRCA2 c.5946del is not justified, and that screening for BRCA1c.5266dup should be considered in high risk patients, given its prevalence as a single mutation. In high-risk patients, a negative screening result should always be followed by comprehensive BRCA gene testing. The finding of a significantly higher frequency of BRCA1 c.5266dup in women with bilateral breast cancer, as well as existence of other as yet unidentified founder mutations in this population, should be further assessed in a larger well characterized high-risk cohort. PMID:22185575

Ultra-deep sequencing reveals high prevalence and broad structural diversity of hepatitis B surface antigen mutations in a global population

PubMed Central

Gencay, Mikael; Hübner, Kirsten; Gohl, Peter; Seffner, Anja; Weizenegger, Michael; Neofytos, Dionysios; Batrla, Richard; Woeste, Andreas; Kim, Hyon-suk; Westergaard, Gaston; Reinsch, Christine; Brill, Eva; Thu Thuy, Pham Thi; Hoang, Bui Huu; Sonderup, Mark; Spearman, C. Wendy; Pabinger, Stephan; Gautier, Jérémie; Brancaccio, Giuseppina; Fasano, Massimo; Santantonio, Teresa; Gaeta, Giovanni B.; Nauck, Markus; Kaminski, Wolfgang E.

2017-01-01

The diversity of the hepatitis B surface antigen (HBsAg) has a significant impact on the performance of diagnostic screening tests and the clinical outcome of hepatitis B infection. Neutralizing or diagnostic antibodies against the HBsAg are directed towards its highly conserved major hydrophilic region (MHR), in particular towards its “a” determinant subdomain. Here, we explored, on a global scale, the genetic diversity of the HBsAg MHR in a large, multi-ethnic cohort of randomly selected subjects with HBV infection from four continents. A total of 1553 HBsAg positive blood samples of subjects originating from 20 different countries across Africa, America, Asia and central Europe were characterized for amino acid variation in the MHR. Using highly sensitive ultra-deep sequencing, we found 72.8% of the successfully sequenced subjects (n = 1391) demonstrated amino acid sequence variation in the HBsAg MHR. This indicates that the global variation frequency in the HBsAg MHR is threefold higher than previously reported. The majority of the amino acid mutations were found in the HBV genotypes B (28.9%) and C (25.4%). Collectively, we identified 345 distinct amino acid mutations in the MHR. Among these, we report 62 previously unknown mutations, which extends the worldwide pool of currently known HBsAg MHR mutations by 22%. Importantly, topological analysis identified the “a” determinant upstream flanking region as the structurally most diverse subdomain of the HBsAg MHR. The highest prevalence of “a” determinant region mutations was observed in subjects from Asia, followed by the African, American and European cohorts, respectively. Finally, we found that more than half (59.3%) of all HBV subjects investigated carried multiple MHR mutations. Together, this worldwide ultra-deep sequencing based genotyping study reveals that the global prevalence and structural complexity of variation in the hepatitis B surface antigen have, to date, been significantly underappreciated. PMID:28472040

Ultra-deep sequencing reveals high prevalence and broad structural diversity of hepatitis B surface antigen mutations in a global population.

PubMed

Gencay, Mikael; Hübner, Kirsten; Gohl, Peter; Seffner, Anja; Weizenegger, Michael; Neofytos, Dionysios; Batrla, Richard; Woeste, Andreas; Kim, Hyon-Suk; Westergaard, Gaston; Reinsch, Christine; Brill, Eva; Thu Thuy, Pham Thi; Hoang, Bui Huu; Sonderup, Mark; Spearman, C Wendy; Pabinger, Stephan; Gautier, Jérémie; Brancaccio, Giuseppina; Fasano, Massimo; Santantonio, Teresa; Gaeta, Giovanni B; Nauck, Markus; Kaminski, Wolfgang E

2017-01-01

The diversity of the hepatitis B surface antigen (HBsAg) has a significant impact on the performance of diagnostic screening tests and the clinical outcome of hepatitis B infection. Neutralizing or diagnostic antibodies against the HBsAg are directed towards its highly conserved major hydrophilic region (MHR), in particular towards its "a" determinant subdomain. Here, we explored, on a global scale, the genetic diversity of the HBsAg MHR in a large, multi-ethnic cohort of randomly selected subjects with HBV infection from four continents. A total of 1553 HBsAg positive blood samples of subjects originating from 20 different countries across Africa, America, Asia and central Europe were characterized for amino acid variation in the MHR. Using highly sensitive ultra-deep sequencing, we found 72.8% of the successfully sequenced subjects (n = 1391) demonstrated amino acid sequence variation in the HBsAg MHR. This indicates that the global variation frequency in the HBsAg MHR is threefold higher than previously reported. The majority of the amino acid mutations were found in the HBV genotypes B (28.9%) and C (25.4%). Collectively, we identified 345 distinct amino acid mutations in the MHR. Among these, we report 62 previously unknown mutations, which extends the worldwide pool of currently known HBsAg MHR mutations by 22%. Importantly, topological analysis identified the "a" determinant upstream flanking region as the structurally most diverse subdomain of the HBsAg MHR. The highest prevalence of "a" determinant region mutations was observed in subjects from Asia, followed by the African, American and European cohorts, respectively. Finally, we found that more than half (59.3%) of all HBV subjects investigated carried multiple MHR mutations. Together, this worldwide ultra-deep sequencing based genotyping study reveals that the global prevalence and structural complexity of variation in the hepatitis B surface antigen have, to date, been significantly underappreciated.

Next-generation sequencing of circulating tumor DNA to predict recurrence in triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy.

PubMed

Chen, Yu-Hsiang; Hancock, Bradley A; Solzak, Jeffrey P; Brinza, Dumitru; Scafe, Charles; Miller, Kathy D; Radovich, Milan

2017-01-01

Next-generation sequencing to detect circulating tumor DNA is a minimally invasive method for tumor genotyping and monitoring therapeutic response. The majority of studies have focused on detecting circulating tumor DNA from patients with metastatic disease. Herein, we tested whether circulating tumor DNA could be used as a biomarker to predict relapse in triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy. In this study, we analyzed samples from 38 early-stage triple-negative breast cancer patients with matched tumor, blood, and plasma. Extracted DNA underwent library preparation and amplification using the Oncomine Research Panel consisting of 134 cancer genes, followed by high-coverage sequencing and bioinformatics. We detected high-quality somatic mutations from primary tumors in 33 of 38 patients. TP53 mutations were the most prevalent (82%) followed by PIK3CA (16%). Of the 33 patients who had a mutation identified in their primary tumor, we were able to detect circulating tumor DNA mutations in the plasma of four patients (three TP53 mutations, one AKT1 mutation, one CDKN2A mutation). All four patients had recurrence of their disease (100% specificity), but sensitivity was limited to detecting only 4 of 13 patients who clinically relapsed (31% sensitivity). Notably, all four patients had a rapid recurrence (0.3, 4.0, 5.3, and 8.9 months). Patients with detectable circulating tumor DNA had an inferior disease free survival ( p  < 0.0001; median disease-free survival: 4.6 mos. vs. not reached; hazard ratio = 12.6, 95% confidence interval: 3.06-52.2). Our study shows that next-generation circulating tumor DNA sequencing of triple-negative breast cancer patients with residual disease after neoadjuvant chemotherapy can predict recurrence with high specificity, but moderate sensitivity. For those patients where circulating tumor DNA is detected, recurrence is rapid.

Germline whole exome sequencing and large-scale replication identifies FANCM as a likely high grade serous ovarian cancer susceptibility gene.

PubMed

Dicks, Ed; Song, Honglin; Ramus, Susan J; Oudenhove, Elke Van; Tyrer, Jonathan P; Intermaggio, Maria P; Kar, Siddhartha; Harrington, Patricia; Bowtell, David D; Group, Aocs Study; Cicek, Mine S; Cunningham, Julie M; Fridley, Brooke L; Alsop, Jennifer; Jimenez-Linan, Mercedes; Piskorz, Anna; Goranova, Teodora; Kent, Emma; Siddiqui, Nadeem; Paul, James; Crawford, Robin; Poblete, Samantha; Lele, Shashi; Sucheston-Campbell, Lara; Moysich, Kirsten B; Sieh, Weiva; McGuire, Valerie; Lester, Jenny; Odunsi, Kunle; Whittemore, Alice S; Bogdanova, Natalia; Dürst, Matthias; Hillemanns, Peter; Karlan, Beth Y; Gentry-Maharaj, Aleksandra; Menon, Usha; Tischkowitz, Marc; Levine, Douglas; Brenton, James D; Dörk, Thilo; Goode, Ellen L; Gayther, Simon A; Pharoah, D P Paul

2017-08-01

We analyzed whole exome sequencing data in germline DNA from 412 high grade serous ovarian cancer (HGSOC) cases from The Cancer Genome Atlas Project and identified 5,517 genes harboring a predicted deleterious germline coding mutation in at least one HGSOC case. Gene-set enrichment analysis showed enrichment for genes involved in DNA repair (p = 1.8×10 -3 ). Twelve DNA repair genes - APEX1, APLF, ATX, EME1, FANCL, FANCM, MAD2L2, PARP2, PARP3, POLN, RAD54L and SMUG1 - were prioritized for targeted sequencing in up to 3,107 HGSOC cases, 1,491 cases of other epithelial ovarian cancer (EOC) subtypes and 3,368 unaffected controls of European origin. We estimated mutation prevalence for each gene and tested for associations with disease risk. Mutations were identified in both cases and controls in all genes except MAD2L2 , where we found no evidence of mutations in controls. In FANCM we observed a higher mutation frequency in HGSOC cases compared to controls (29/3,107 cases, 0.96 percent; 13/3,368 controls, 0.38 percent; P=0.008) with little evidence for association with other subtypes (6/1,491, 0.40 percent; P=0.82). The relative risk of HGSOC associated with deleterious FANCM mutations was estimated to be 2.5 (95% CI 1.3 - 5.0; P=0.006). In summary, whole exome sequencing of EOC cases with large-scale replication in case-control studies has identified FANCM as a likely novel susceptibility gene for HGSOC, with mutations associated with a moderate increase in risk. These data may have clinical implications for risk prediction and prevention approaches for high-grade serous ovarian cancer in the future and a significant impact on reducing disease mortality.

A family with the Arg103Pro mutation in the NEUROD1 gene detected by next-generation sequencing - Clinical characteristics of mutation carriers.

PubMed

Szopa, Magdalena; Ludwig-Galezowska, Agnieszka H; Radkowski, Piotr; Skupien, Jan; Machlowska, Julita; Klupa, Tomasz; Wolkow, Pawel; Borowiec, Maciej; Mlynarski, Wojciech; Malecki, Maciej T

2016-02-01

Until now only a few families with early onset autosomal diabetes due to the NEUROD1 gene mutations have been identified. Moreover, only some of them meet strict MODY (maturity-onset diabetes of the young) criteria. Next-generation sequencing (NGS) provides an opportunity to detect more pathogenic mutations in this gene. Here, we evaluated the segregation of the Arg103Pro mutation in the NEUROD1 gene in a pedigree in which it was detected, and described the clinical characteristics of the mutation carriers. We included 156 diabetic probands of MODY families, among them 52 patients earlier tested for GCK-MODY and/or HNF1A-MODY by Sanger sequencing with negative results. Genetic testing was performed by targeted NGS sequencing using a panel of 28 monogenic diabetes genes. As detected by NGS, one patient had the missense Arg103Pro (CGC/CCC) mutation in the gene NEUROD1 changing the amino-acid structure of the DNA binding domain of this transcription factor. We confirmed this sequence difference by Sanger sequencing. This family had previously been tested with negative results for HNF1A gene mutations. 17 additional members of this family were invited for further testing. We confirmed the presence of the mutation in 11 subjects. Seven adult mutation carriers (all but one) from three generations had been already diagnosed with diabetes. There were 3 individuals with the Arg103Pro mutation diagnosed before the age of 30 years in the family. The range of age of the four unaffected mutation carriers (3 minors and 1 adult) was 3-48 years. Interestingly, one mutation carrier had a history of transient neonatal hypoglycemia, of which the clinical course resembled episodes typical for HNF4A-MODY. We report a family with autosomal dominant diabetes related to a new NEUROD1 mutation, one of very few meeting MODY criteria. The use of the NGS method will facilitate identification of more families with rare forms of MODY. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

High-resolution melting analysis for noninvasive prenatal diagnosis of IVS-II-I (G-A) fetal DNA in minor beta-thalassemia mothers.

PubMed

Zafari, Mandana; Gill, Pooria; Kowsaryan, Mehrnoush; Alipour, Abbass; Banihashemi, Ali

2016-10-01

The high-resolution melting (HRM) technique is fast, effective and successful method for mutation detection. The aim of this study was to determine the sensitivity and specificity of the HRM method for detection of a paternally inherited mutation in a fetus as a noninvasive prenatal diagnosis of β-thalassemia. Genomic DNAs were prepared from 50 β-thalassemia minor couples whose pregnancy was at risk for homozygous β-thalassemia. Ten milliliters of the maternal blood from each pregnant woman were collected and after separating plasma stored at -80 °C until analysis. The extracted DNAs were analyzed by HRM real-time PCR for detection of IVS-II-I (G-A) as a paternally inherited mutation. The gold standard was the result of a chorionic villus sampling by a standard reverse dot blotting test. The sensitivity and specificity of HRM real-time PCR were 92.6% and 82.6%, respectively. Also, the positive and negative predictive values were 86.2% and 90.47%, respectively. HRM real-time PCR was a sensitive and specific method for determining the paternally inherited mutation in the fetus at risk with thalassemia major.

Sexual selection on spontaneous mutations strengthens the between-sex genetic correlation for fitness.

PubMed

Allen, Scott L; McGuigan, Katrina; Connallon, Tim; Blows, Mark W; Chenoweth, Stephen F

2017-10-01

A proposed benefit to sexual selection is that it promotes purging of deleterious mutations from populations. For this benefit to be realized, sexual selection, which is usually stronger on males, must purge mutations deleterious to both sexes. Here, we experimentally test the hypothesis that sexual selection on males purges deleterious mutations that affect both male and female fitness. We measured male and female fitness in two panels of spontaneous mutation-accumulation lines of the fly, Drosophila serrata, each established from a common ancestor. One panel of mutation accumulation lines limited both natural and sexual selection (LS lines), whereas the other panel limited natural selection, but allowed sexual selection to operate (SS lines). Although mutation accumulation caused a significant reduction in male and female fitness in both the LS and SS lines, sexual selection had no detectable effect on the extent of the fitness reduction. Similarly, despite evidence of mutational variance for fitness in males and females of both treatments, sexual selection had no significant impact on the amount of mutational genetic variance for fitness. However, sexual selection did reshape the between-sex correlation for fitness: significantly strengthening it in the SS lines. After 25 generations, the between-sex correlation for fitness was positive but considerably less than one in the LS lines, suggesting that, although most mutations had sexually concordant fitness effects, sex-limited, and/or sex-biased mutations contributed substantially to the mutational variance. In the SS lines this correlation was strong and could not be distinguished from unity. Individual-based simulations that mimick the experimental setup reveal two conditions that may drive our results: (1) a modest-to-large fraction of mutations have sex-limited (or highly sex-biased) fitness effects, and (2) the average fitness effect of sex-limited mutations is larger than the average fitness effect of mutations that affect both sexes similarly. © 2017 The Author(s). Evolution © 2017 The Society for the Study of Evolution.

The population genetics of human disease: The case of recessive, lethal mutations

PubMed Central

Gao, Ziyue; Baker, Zachary; Diesel, José Francisco; Simons, Yuval B.; Haque, Imran S.; Pickrell, Joseph; Przeworski, Molly

2017-01-01

Do the frequencies of disease mutations in human populations reflect a simple balance between mutation and purifying selection? What other factors shape the prevalence of disease mutations? To begin to answer these questions, we focused on one of the simplest cases: recessive mutations that alone cause lethal diseases or complete sterility. To this end, we generated a hand-curated set of 417 Mendelian mutations in 32 genes reported to cause a recessive, lethal Mendelian disease. We then considered analytic models of mutation-selection balance in infinite and finite populations of constant sizes and simulations of purifying selection in a more realistic demographic setting, and tested how well these models fit allele frequencies estimated from 33,370 individuals of European ancestry. In doing so, we distinguished between CpG transitions, which occur at a substantially elevated rate, and three other mutation types. Intriguingly, the observed frequency for CpG transitions is slightly higher than expectation but close, whereas the frequencies observed for the three other mutation types are an order of magnitude higher than expected, with a bigger deviation from expectation seen for less mutable types. This discrepancy is even larger when subtle fitness effects in heterozygotes or lethal compound heterozygotes are taken into account. In principle, higher than expected frequencies of disease mutations could be due to widespread errors in reporting causal variants, compensation by other mutations, or balancing selection. It is unclear why these factors would have a greater impact on disease mutations that occur at lower rates, however. We argue instead that the unexpectedly high frequency of disease mutations and the relationship to the mutation rate likely reflect an ascertainment bias: of all the mutations that cause recessive lethal diseases, those that by chance have reached higher frequencies are more likely to have been identified and thus to have been included in this study. Beyond the specific application, this study highlights the parameters likely to be important in shaping the frequencies of Mendelian disease alleles. PMID:28957316

Mutational spectrum in breast cancer associated BRCA1 and BRCA2 genes in Colombia

PubMed Central

Gómez-Gutiérrez, Alberto; Díaz-Dussán, Natalia Andrea; Noguera-Santamaría, María Claudia; Díaz-Rincón, Diego; Casas-Gómez, María Consuelo

2017-01-01

Abstract Introduction: The risk of developing breast and ovarian cancer is higher in families that carry mutations in BRCA1 or BRCA2 genes, and timely mutation detection is critical. Objective: To identify the presence of mutations in the Colombian population and evaluate two testing strategies. Methods: From a total universe of 853 individual blood samples referred for BRCA1 and BRCA2 typing, 256 cases were analyzed by complete direct sequencing of both genes in Myriad Genetics, and the remaining 597 cases were studied by partial sequencing based on founder mutations in a PCR test designed by ourselves ("Profile Colombia"). Results: We found 107 patients carrying deleterious mutations in this group of patients, 69 (64.5%) located in BRCA1, and 38 (35.5%) in BRCA2. Overall, we detected 39 previously unreported mutations in Colombia (22 in BRCA1 and 17 in BRCA2) and only 4 out of the 6 previously reported founder mutations. Sixty four out of 597 patients (10.7%) studied by "Profile Colombia" showed mutations in BRCA1 or BRCA2, and 41/256 patients (16%) showed mutations by complete BRCA1-BRCA2 sequencing. Conclusions: The spectrum of 44 different mutations in Colombia as detected in our study is broader than the one previously reported for this country. "Profile Colombia" is a useful screening test to establish both founder and new mutations (detection rate of 10.7%) in cases with family history of breast cancer. Complete sequencing shows a detection rate of 16.0%, and should complement the study of the genetic basis of this disease. PMID:29021639

[Analysis of genetic polymorphisms and mutations of 20 frequently used STR loci among ethnic Hans from Henan].

PubMed

Wang, Hongdan; Kang, Bing; Gao, Yue; Huo, Xiaodong; Li, Tao; Guo, Qiannan; Zhu, Bofeng; Liao, Shixiu

2017-04-10

To study the genetic polymorphisms and mutations of 20 frequently used autosomal microsatellites among ethnic Hans from Henan. Peripheral blood samples of 2604 individuals were collected. DNA was amplified and genotyped using a PowerPlex(TM) 21 system. The frequencies, forensic parameters and mutation rates of the 20 short tandem repeat (STR) loci were analyzed. A total of 323 alleles were found in this population and the allelic frequencies have ranged from 0.0003 to 0.5144. Except for D3S1358, TH01 and TPOX, mutations have been found in all of the remaining 17 STR loci, which totaled 47, with mutation rates ranging from 0 to 3.46 × 10 -3 . The 20 STR loci selected by the PowerPlex(TM) 21 system are highly polymorphic among ethnic Hans from Henan, and may be of great value in forensic and human population studies. As no similar study has been carried out previously, above results may be of great value for individual discrimination and paternal testing.

A novel NDUFV1 gene mutation in complex I deficiency in consanguineous siblings with brainstem lesions and Leigh syndrome.

PubMed

Vilain, C; Rens, C; Aeby, A; Balériaux, D; Van Bogaert, P; Remiche, G; Smet, J; Van Coster, R; Abramowicz, M; Pirson, I

2012-09-01

Although deficiency of complex I of the mitochondrial respiratory chain is a frequent cause of encephalopathy in children, only a few mutations have been reported in each of its subunits. In the absence of families large enough for conclusive segregation analysis and of robust functional testing, it is difficult to unequivocally show the causality of the observed mutations and to delineate genotype-phenotype correlations, making additional observations necessary. We observed two consanguineous siblings with an early-onset encephalopathy, medulla, brainstem and mesencephalon lesions on brain magnetic resonance imaging and death before 8 months of age, caused by a complex I deficiency. We used a homozygosity mapping approach and identified a missense mutation in the NDUFV1 gene. The mutation, p.Arg386His, affects a highly conserved residue, contiguous to a cysteine residue known to coordinate an Fe ion. This observation adds to our understanding of complex I deficiency disease. It validates the important role of Arg386 and therefore supports the current molecular model of iron-sulfur clusters in NDUFV1. © 2011 John Wiley & Sons A/S.

Mutagenesis during plant responses to UVB radiation.

PubMed

Holá, M; Vágnerová, R; Angelis, K J

2015-08-01

We tested an idea that induced mutagenesis due to unrepaired DNA lesions, here the UV photoproducts, underlies the impact of UVB irradiation on plant phenotype. For this purpose we used protonemal culture of the moss Physcomitrella patens with 50% of apical cells, which mimics actively growing tissue, the most vulnerable stage for the induction of mutations. We measured the UVB mutation rate of various moss lines with defects in DNA repair (pplig4, ppku70, pprad50, ppmre11), and in selected clones resistant to 2-Fluoroadenine, which were mutated in the adenosine phosphotrasferase gene (APT), we analysed induced mutations by sequencing. In parallel we followed DNA break repair and removal of cyclobutane pyrimidine dimers with a half-life τ = 4 h 14 min determined by comet assay combined with UV dimer specific T4 endonuclease V. We show that UVB induces massive, sequence specific, error-prone bypass repair that is responsible for a high mutation rate owing to relatively slow, though error-free, removal of photoproducts by nucleotide excision repair (NER). Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Reverse cascade screening of newborns for hereditary haemochromatosis: a model for other late onset diseases?

PubMed

Cadet, E; Capron, D; Gallet, M; Omanga-Léké, M-L; Boutignon, H; Julier, C; Robson, K J H; Rochette, J

2005-05-01

Genetic testing can determine those at risk for hereditary haemochromatosis (HH) caused by HFE mutations before the onset of symptoms. However, there is no optimum screening strategy, mainly owing to the variable penetrance in those who are homozygous for the HFE Cys282Tyr (C282Y) mutation. The objective of this study was to identify the majority of individuals at serious risk of developing HFE haemochromatosis before they developed life threatening complications. We first estimated the therapeutic penetrance of the C282Y mutation in people living in la Somme, France, using genetic, demographic, biochemical, and follow up data. We examined the benefits of neonatal screening on the basis of increased risk to relatives of newborns carrying one or two copies of the C282Y mutation. Between 1999 and 2002, we screened 7038 newborns from two maternity hospitals in the north of France for the C282Y and His63Asp (H63D) mutations in the HFE gene, using bloodspots collected on Guthrie cards. Family studies and genetic counselling were undertaken, based on the results of the baby's genotype. In la Somme, we found that 24% of the adults homozygous for the C282Y mutation required at least 5 g iron to be removed to restore normal iron parameters (that is, the therapeutic penetrance). In the reverse cascade screening study, we identified 19 C282Y homozygotes (1/370), 491 heterozygotes (1/14) and 166 compound heterozygotes (1/42) in 7038 newborns tested. The reverse cascade screening strategy resulted in 80 adults being screened for both mutations. We identified 10 previously unknown C282Y homozygotes of whom six (four men and two women) required venesection. Acceptance of neonatal screening was high; parents understood the risks of having HH and the benefits of early detection, but a number of parents were reluctant to take the test themselves. Neonatal screening for HH is straightforward. Reverse cascade screening increased the efficiency of detecting affected adults with undiagnosed haemochromatosis. This strategy allows almost complete coverage for HH and could be a model for efficient screening for other late onset genetic diseases.

Non-ventricular, Clinical, and Functional Features of the RyR2(R420Q) Mutation Causing Catecholaminergic Polymorphic Ventricular Tachycardia.

PubMed

Domingo, Diana; Neco, Patricia; Fernández-Pons, Elena; Zissimopoulos, Spyros; Molina, Pilar; Olagüe, José; Suárez-Mier, M Paz; Lai, F Anthony; Gómez, Ana M; Zorio, Esther

2015-05-01

Catecholaminergic polymorphic ventricular tachycardia is a malignant disease, due to mutations in proteins controlling Ca(2+) homeostasis. While the phenotype is characterized by polymorphic ventricular arrhythmias under stress, supraventricular arrhythmias may occur and are not fully characterized. Twenty-five relatives from a Spanish family with several sudden deaths were evaluated with electrocardiogram, exercise testing, and optional epinephrine challenge. Selective RyR2 sequencing in an affected individual and cascade screening in the rest of the family was offered. The RyR2(R420Q) mutation was generated in HEK-293 cells using site-directed mutagenesis to conduct in vitro functional studies. The exercise testing unmasked catecholaminergic polymorphic ventricular tachycardia in 8 relatives (sensitivity = 89%; positive predictive value = 100%; negative predictive value = 93%), all of them carrying the heterozygous RyR2(R420Q) mutation, which was also present in the proband and a young girl without exercise testing, a 91% penetrance at the end of the follow-up. Remarkably, sinus bradycardia, atrial and junctional arrhythmias, and/or giant post-effort U-waves were identified in patients. Upon permeabilization and in intact cells, the RyR2(R420Q) expressing cells showed a smaller peak of Ca(2+) release than RyR2 wild-type cells. However, at physiologic intracellular Ca(2+) concentration, equivalent to the diastolic cytosolic concentration, the RyR2(R420Q) released more Ca(2+) and oscillated faster than RyR2 wild-type cells. The missense RyR2(R420Q) mutation was identified in the N-terminus of the RyR2 gene in this highly symptomatic family. Remarkably, this mutation is associated with sinus bradycardia, atrial and junctional arrhythmias, and giant U-waves. Collectively, functional heterologous expression studies suggest that the RyR2(R420Q) behaves as an aberrant channel, as a loss- or gain-of-function mutation depending on cytosolic intracellular Ca(2+) concentration. Copyright © 2014 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.