Simultaneous use of multiplex ligation-dependent probe amplification assay and flow cytometric DNA ploidy analysis in patients with acute leukemia.

PubMed

Reyes-Núñez, Virginia; Galo-Hooker, Evelyn; Pérez-Romano, Beatriz; Duque, Ricardo E; Ruiz-Arguelles, Alejandro; Garcés-Eisele, Javier

2018-01-01

The aim of this work was to simultaneously use multiplex ligation-dependent probe amplification (MLPA) assay and flow cytometric DNA ploidy analysis (FPA) to detect aneuploidy in patients with newly diagnosed acute leukemia. MLPA assay and propidium iodide FPA were used to test samples from 53 consecutive patients with newly diagnosed acute leukemia referred to our laboratory for immunophenotyping. Results were compared by nonparametric statistics. The combined use of both methods significantly increased the rate of detection of aneuploidy as compared to that obtained by each method alone. The limitations of one method are somehow countervailed by the other and vice versa. MPLA and FPA yield different yet complementary information concerning aneuploidy in acute leukemia. The simultaneous use of both methods might be recommended in the clinical setting. © 2017 International Clinical Cytometry Society. © 2017 International Clinical Cytometry Society.

Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification

PubMed Central

Schouten, Jan P.; McElgunn, Cathal J.; Waaijer, Raymond; Zwijnenburg, Danny; Diepvens, Filip; Pals, Gerard

2002-01-01

We describe a new method for relative quantification of 40 different DNA sequences in an easy to perform reaction requiring only 20 ng of human DNA. Applications shown of this multiplex ligation-dependent probe amplification (MLPA) technique include the detection of exon deletions and duplications in the human BRCA1, MSH2 and MLH1 genes, detection of trisomies such as Down’s syndrome, characterisation of chromosomal aberrations in cell lines and tumour samples and SNP/mutation detection. Relative quantification of mRNAs by MLPA will be described elsewhere. In MLPA, not sample nucleic acids but probes added to the samples are amplified and quantified. Amplification of probes by PCR depends on the presence of probe target sequences in the sample. Each probe consists of two oligonucleotides, one synthetic and one M13 derived, that hybridise to adjacent sites of the target sequence. Such hybridised probe oligonucleotides are ligated, permitting subsequent amplification. All ligated probes have identical end sequences, permitting simultaneous PCR amplification using only one primer pair. Each probe gives rise to an amplification product of unique size between 130 and 480 bp. Probe target sequences are small (50–70 nt). The prerequisite of a ligation reaction provides the opportunity to discriminate single nucleotide differences. PMID:12060695

Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification.

PubMed

Schouten, Jan P; McElgunn, Cathal J; Waaijer, Raymond; Zwijnenburg, Danny; Diepvens, Filip; Pals, Gerard

2002-06-15

We describe a new method for relative quantification of 40 different DNA sequences in an easy to perform reaction requiring only 20 ng of human DNA. Applications shown of this multiplex ligation-dependent probe amplification (MLPA) technique include the detection of exon deletions and duplications in the human BRCA1, MSH2 and MLH1 genes, detection of trisomies such as Down's syndrome, characterisation of chromosomal aberrations in cell lines and tumour samples and SNP/mutation detection. Relative quantification of mRNAs by MLPA will be described elsewhere. In MLPA, not sample nucleic acids but probes added to the samples are amplified and quantified. Amplification of probes by PCR depends on the presence of probe target sequences in the sample. Each probe consists of two oligonucleotides, one synthetic and one M13 derived, that hybridise to adjacent sites of the target sequence. Such hybridised probe oligonucleotides are ligated, permitting subsequent amplification. All ligated probes have identical end sequences, permitting simultaneous PCR amplification using only one primer pair. Each probe gives rise to an amplification product of unique size between 130 and 480 bp. Probe target sequences are small (50-70 nt). The prerequisite of a ligation reaction provides the opportunity to discriminate single nucleotide differences.

Usefulness of MLPA in the detection of SHOX deletions.

PubMed

Funari, Mariana F A; Jorge, Alexander A L; Souza, Silvia C A L; Billerbeck, Ana E C; Arnhold, Ivo J P; Mendonca, Berenice B; Nishi, Mirian Y

2010-01-01

SHOX haploinsufficiency causes a wide spectrum of short stature phenotypes, such as Leri-Weill dyschondrosteosis (LWD) and disproportionate short stature (DSS). SHOX deletions are responsible for approximately two thirds of isolated haploinsufficiency; therefore, it is important to determine the most appropriate methodology for detection of gene deletion. In this study, three methodologies for the detection of SHOX deletions were compared: the fluorescence in situ hybridization (FISH), microsatellite analysis and multiplex ligation-dependent probe amplification (MLPA). Forty-four patients (8 LWD and 36 DSS) were analyzed. The cosmid LLNOYCO3'M'34F5 was used as a probe for the FISH analysis and microsatellite analysis were performed using three intragenic microsatellite markers. MLPA was performed using commercial kits. Twelve patients (8 LWD and 4 DSS) had deletions in SHOX area detected by MLPA and 2 patients generated discordant results with the other methodologies. In the first case, the deletion was not detected by FISH. In the second case, both FISH and microsatellite analyses were unable to identify the intragenic deletion. In conclusion, MLPA was more sensitive, less expensive and less laborious; therefore, it should be used as the initial molecular method for the detection of SHOX gene deletion. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Dosage analysis of cancer predisposition genes by multiplex ligation-dependent probe amplification

PubMed Central

Bunyan, D J; Eccles, D M; Sillibourne, J; Wilkins, E; Thomas, N Simon; Shea-Simonds, J; Duncan, P J; Curtis, C E; Robinson, D O; Harvey, J F; Cross, N C P

2004-01-01

Multiplex ligation-dependent probe amplification (MLPA) is a recently described method for detecting gross deletions or duplications of DNA sequences, aberrations which are commonly overlooked by standard diagnostic analysis. To determine the incidence of copy number variants in cancer predisposition genes from families in the Wessex region, we have analysed the hMLH1 and hMSH2 genes in patients with hereditary nonpolyposis colorectal cancer (HNPCC), BRCA1 and BRCA2 in families with hereditary breast/ovarian cancer (BRCA) and APC in patients with familial adenomatous polyposis coli (FAP). Hereditary nonpolyposis colorectal cancer (n=162) and FAP (n=74) probands were fully screened for small mutations, and cases for which no causative abnormality were found (HNPCC, n=122; FAP, n=24) were screened by MLPA. Complete or partial gene deletions were identified in seven cases for hMSH2 (5.7% of mutation-negative HNPCC; 4.3% of all HNPCC), no cases for hMLH1 and six cases for APC (25% of mutation negative FAP; 8% of all FAP). For BRCA1 and BRCA2, a partial mutation screen was performed and 136 mutation-negative cases were selected for MLPA. Five deletions and one duplication were found for BRCA1 (4.4% of mutation-negative BRCA cases) and one deletion for BRCA2 (0.7% of mutation-negative BRCA cases). Cost analysis indicates it is marginally more cost effective to perform MLPA prior to point mutation screening, but the main advantage gained by prescreening is a greatly reduced reporting time for the patients who are positive. These data demonstrate that dosage analysis is an essential component of genetic screening for cancer predisposition genes. PMID:15475941

Fluorescent Quantification of DNA Based on Core-Shell Fe3O4@SiO2@Au Nanocomposites and Multiplex Ligation-Dependent Probe Amplification.

PubMed

Fan, Jing; Yang, Haowen; Liu, Ming; Wu, Dan; Jiang, Hongrong; Zeng, Xin; Elingarami, Sauli; Ll, Zhiyang; Li, Song; Liu, Hongna; He, Nongyue

2015-02-01

In this research, a novel method for relative fluorescent quantification of DNA based on Fe3O4@SiO2@Au gold-coated magnetic nanocomposites (GMNPs) and multiplex ligation- dependent probe amplification (MLPA) has been developed. With the help of self-assembly, seed-mediated growth and chemical reduction method, core-shell Fe3O4@SiO2@Au GMNPs were synthesized. Through modified streptavidin on the GMNPs surface, we obtained a bead chip which can capture the biotinylated probes. Then we designed MLPA probes which were tagged with biotin or Cy3 and target DNA on the basis of human APP gene sequence. The products from the thermostable DNA ligase induced ligation reactions and PCR amplifications were incubated with SA-GMNPs. After washing, magnetic separation, spotting, the fluorescent scanning results showed our method can be used for the relative quantitative analysis of the target DNA in the concentration range of 03004~0.5 µM.

Use of next-generation sequencing to detect LDLR gene copy number variation in familial hypercholesterolemia.

PubMed

Iacocca, Michael A; Wang, Jian; Dron, Jacqueline S; Robinson, John F; McIntyre, Adam D; Cao, Henian; Hegele, Robert A

2017-11-01

Familial hypercholesterolemia (FH) is a heritable condition of severely elevated LDL cholesterol, caused predominantly by autosomal codominant mutations in the LDL receptor gene ( LDLR ). In providing a molecular diagnosis for FH, the current procedure often includes targeted next-generation sequencing (NGS) panels for the detection of small-scale DNA variants, followed by multiplex ligation-dependent probe amplification (MLPA) in LDLR for the detection of whole-exon copy number variants (CNVs). The latter is essential because ∼10% of FH cases are attributed to CNVs in LDLR ; accounting for them decreases false negative findings. Here, we determined the potential of replacing MLPA with bioinformatic analysis applied to NGS data, which uses depth-of-coverage analysis as its principal method to identify whole-exon CNV events. In analysis of 388 FH patient samples, there was 100% concordance in LDLR CNV detection between these two methods: 38 reported CNVs identified by MLPA were also successfully detected by our NGS method, while 350 samples negative for CNVs by MLPA were also negative by NGS. This result suggests that MLPA can be removed from the routine diagnostic screening for FH, significantly reducing associated costs, resources, and analysis time, while promoting more widespread assessment of this important class of mutations across diagnostic laboratories. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

[Molecular diagnosis of spinal muscular atrophy by multiplex ligation-dependent probe amplification].

PubMed

Zeng, Jian; Ke, Long-feng; Deng, Xiao-jun; Cai, Mei-ying; Tu, Xiang-dong; Lan, Feng-hua

2008-12-16

To investigate the effect of multiplex ligation-dependent probe amplification (MLPA) in molecular diagnosis of spinal muscular atrophy (SMA). Peripheral blood samples were collected from 13 SMA patients, 31 parents of SMA patients, 50 healthy individuals without family history of SMA, and 10 specimens of amniotic fluid from these families were collected too. Genomic DNA was analyzed by MLPA, conventional PCR-RFLP, and allele-specific PCR. In complete agreement with the results of conventional PCR-RFLP and allele-specific PCR, MLPA analysis showed that all of the 13 patients had homozygous deletion of the survival of motor neuron 1 (SMN1) gene, and there was significant difference between the SMA severity (type I to type III) and SMN2 copy number (P < 0.05). Of the 31 parents 29 (93.5%) had 1 copy of SMN1, 2 (6.5%) had 2 copies of SMN1. Of the 50 healthy individuals, 1 (2.0%) had 1 copy of SMN1, 48 (96.0%) had 2 copies of SMN1, and 1 (2.0%) had 3 copies. The SMN1 copy number of the parents was significantly higher than that of the healthy individuals (P < 0.01). Two of the 10 fetuses had homozygous deletion of SMN1. The MLPA technique has proved to be an accurate and reliable tool for the molecular diagnosis of SMA, both in patients and in healthy carriers.

Can we rely on the multiplex ligation-dependent probe amplification method (MLPA) for prenatal diagnosis?

PubMed Central

Omrani, Mir Davood; Azizi, Faezeh; Rajabibazl, Masoumeh; Safavi Naini, Niloufar; Omrani, Sara; Abbasi, Arezo Mona; Saleh Gargari, Soraya

2014-01-01

Background: The major aneuploidies that are diagnosed prenatally involve the autosomal chromosomes 13, 18, and 21, as well as sex chromosomes, X and Y. Because multiplex ligation-dependent probe amplification (MLPA) is rapid and non-invasive, it has replaced traditional culture methods for the screening and diagnosis of common aneuploidies in some countries. Objective: To evaluate the sensitivity and specificity of MLPA in a cross-sectional descriptive study for the detection of chromosomal aneuploidies in comparison to other methods. Materials and Methods: Genomic DNA was extracted from the peripheral blood samples of 10 normal controls and the amniotic fluid of 55 patients. Aneuploidies screening of chromosomes 13, 18, 21, X and Y were carried out using specific MLPA probe mixes (P095-A2). For comparison purposes, samples were also tested by Quantitative Fluorescent-PCR (QF-PCR) and routine chromosomal culture method. Results: Using this specific MLPA technique and data-analyzing software (Genemarker v1.85), one case was diagnosed with 45, X (e.g. Monosomy X or Turner’s Syndrome), and the remaining 54 cases revealed normal karyotypes. These results were concordant with routine chromosomal culture and QF-PCR findings. Conclusion: The experiment demonstrates that MLPA can provide a rapid and accurate clinical method for prenatal identification of common chromosomal aneuploidies with 100% sensitivity and 100% specificity. PMID:24976821

Genetic diagnosis of Duchenne and Becker muscular dystrophy using multiplex ligation-dependent probe amplification in Rwandan patients.

PubMed

Uwineza, Annette; Hitayezu, Janvier; Murorunkwere, Seraphine; Ndinkabandi, Janvier; Kalala Malu, Celestin Kaputu; Caberg, Jean Hubert; Dideberg, Vinciane; Bours, Vincent; Mutesa, Leon

2014-04-01

Duchenne and Becker muscular dystrophies are the most common clinical forms of muscular dystrophies. They are genetically X-linked diseases caused by a mutation in the dystrophin (DMD) gene. A genetic diagnosis was carried out in six Rwandan patients presenting a phenotype of Duchenne and Becker muscular dystrophies and six asymptomatic female carrier relatives using multiplex ligation-dependent probe amplification (MLPA). Our results revealed deletion of the exons 48-51 in one patient, an inherited deletion of the exons 8-21 in two brothers and a de novo deletion of the exons 46-50 in the fourth patient. No copy number variation was found in two patients. Only one female carrier presented exon deletion in the DMD gene. This is the first cohort of genetic analysis in Rwandan patients affected by Duchenne and Becker muscular dystrophies. This report confirmed that MLPA assay can be easily implemented in low-income countries.

Evaluation of two molecular techniques for rapid detection of the main dermatophytic agents of tinea capitis.

PubMed

Deng, S; Zhou, Z; de Hoog, G S; Wang, X; Abliz, P; Sun, J; Najafzadeh, M J; Pan, W; Lei, W; Zhu, S; Hasimu, H; Zhang, P; Guo, Y; Deng, D; Liao, W

2015-12-01

Tinea capitis is very common in Western China, with the most widespread aetiological agent being Trichophyton violaceum, while Microsporum canis is prevalent in the remainder of China. Conventional diagnostics and internal transcribed spacer (ITS) sequencing analyses have proven relatively limited due to the close phylogenetic relationship of anthropophilic dermatophytes. Therefore, alternative molecular tools with sufficient specificity, reproducibility and sensitivity are necessary. To evaluate two molecular techniques [multiplex ligation-dependent probe amplification (MLPA) and rolling circle amplification (RCA)] for rapid detection of the aetiological agents of tinea capitis, T. violaceum and M. canis. Probes of RCA and MLPA were designed with target sequences in the rDNA ITS gene region. Strains tested consist of 31 T. violaceum, 22 M. canis and 24 reference strains of species that are taxonomically close to the target species. The specificity and reproducibility of RCA and MLPA in detection of T. violaceum and M. canis were both 100% in both species. Sensitivity testing showed that RCA was positive at concentrations down to 1·68 × 10(6) copies of DNA in the TvioRCA probe, and 2·7 × 10(8) copies of DNA in McRCA. MLPA yielded positive results at concentrations of DNA down to 1·68 × 10(1) copies in the TvioMLPA probe and 2·7 × 10(2) in McMLPA. The two techniques were sufficiently specific and sensitive for discriminating the target DNA of T. violaceum and M. canis from that of closely related dermatophytes. RCA and MLPA are advantageous in their reliability and ease of operation compared with standard polymerase chain reaction and conventional methods. © 2015 British Association of Dermatologists.

[MLPA technique--principles and use in practice].

PubMed

Rusu, Cristina; Sireteanu, Adriana; Puiu, Maria; Skrypnyk, Cristina; Tomescu, E; Csep, Katalin; Creţ, Victoria; Barbarii, Ligia

2007-01-01

MLPA (Multiplex Ligation-dependent Probe Amplification) is a recently introduced method, based on PCR principle, useful for the detection of different genetic abnormalities (aneuploidies, gene deletions/duplications, subtelomeric rearrangements, methylation status etc). The technique is simple, reliable and cheap. We present this method to discuss its importance for a modern genetic service and to underline its multiple advantages.

Genetic heterogeneity in uveal melanoma assessed by multiplex ligation-dependent probe amplification.

PubMed

Dopierala, Justyna; Damato, Bertil E; Lake, Sarah L; Taktak, Azzam F G; Coupland, Sarah E

2010-10-01

To determine intratumor genetic heterogeneity in uveal melanoma (UM) by multiplex ligation-dependent probe amplification (MLPA) in formalin-fixed, paraffin-embedded (FFPE) tumor tissues. DNA was extracted from whole tumor sections and from two to nine different areas microdissected from 32 FFPE UMs. Thirty-one loci on chromosomes 1, 3, 6, and 8 were tested with MLPA for copy number changes. The tumor was considered heterogeneous at a locus if (1) the difference in dosage quotients (DQs) of any two areas was 0.2 or more, and (2) the DQs of the areas belonged to different ranges. Comparison of MLPA data obtained from microdissected areas of the UMs showed heterogeneity in 1 to 26 examined loci in 24 (75%) tumors, with only 25% of the tumors being homogeneous. Intratumor heterogeneity of 3p12.2, 6p21.2, and 8q11.23 was most common, occurring in >30% of the UMs. Gains of chromosome 3 were observed in four UMs, with three of these tumors showing the highest degree of heterogeneity. Copy number variation was associated with differences in tumor cell type, but not with differences in tumor pigmentation or reactive inflammation. UMs with genetic heterogeneity across multiple sample sites showed equivocal MLPA results when the whole tumor section was examined. These results suggest that different clones dilute MLPA results. Heterogeneity of chromosomal abnormalities of chromosomes 1, 3, 6, and 8 is present in most UMs. This heterogeneity causes equivocal MLPA results. One random tumor sample may not be representative of the whole tumor and, therefore, may be insufficient for prognostic testing.

The First Report of a 290-bp Deletion in β-Globin Gene in the South of Iran

PubMed Central

Hamid, Mohammad; Nejad, Ladan Dawoody; Shariati, Gholamreza; Galehdari, Hamid; Saberi, Alihossein; Mohammadi-Anaei, Marziye

2017-01-01

Background: β-thalassemia is one of the most widespread diseases in the world, including Iran. In this study, we reported, for the first time, a 290-bp β-globin gene deletion in the south of Iran. Methods: Four individuals from three unrelated families with Arabic ethnic background were studied in Khuzestan Province. Red blood cell indices and hemoglobin analysis were carried out according to the standard methods. Genomic DNA was obtained from peripheral blood cells by salting out procedures. β-globin gene amplification, multiplex ligation-dependent probe amplification (MLPA), and DNA sequencing were performed. Results: The PCR followed by sequencing and MLPA test of the β-globin gene confirmed the presence of a 290-bp deletion in the heterozygous form, along with -88C>A mutation. All the individuals had elevated hemoglobin A2 and normal fetal hemoglobin levels. Conclusions: This mutation causes β0-thalassemia and can be highly useful for prenatal diagnosis in compound heterozygous condition with different β-globin gene mutations. PMID:26948378

A modified multiplex ligation-dependent probe amplification method for the detection of 22q11.2 copy number variations in patients with congenital heart disease.

PubMed

Zhang, Xiaoqing; Xu, Yuejuan; Liu, Deyuan; Geng, Juan; Chen, Sun; Jiang, Zhengwen; Fu, Qihua; Sun, Kun

2015-05-08

Copy number variations (CNVs) of chromosomal region 22q11.2 are associated with a subset of patients with congenital heart disease (CHD). Accurate and efficient detection of CNV is important for genetic analysis of CHD. The aim of the study was to introduce a novel approach named CNVplex®, a high-throughput analysis technique designed for efficient detection of chromosomal CNVs, and to explore the prevalence of sub-chromosomal imbalances in 22q11.2 loci in patients with CHD from a single institute. We developed a novel technique, CNVplex®, for high-throughput detection of sub-chromosomal copy number aberrations. Modified from the multiplex ligation-dependent probe amplification (MLPA) method, it introduced a lengthening ligation system and four universal primer sets, which simplified the synthesis of probes and significantly improved the flexibility of the experiment. We used 110 samples, which were extensively characterized with chromosomal microarray analysis and MLPA, to validate the performance of the newly developed method. Furthermore, CNVplex® was used to screen for sub-chromosomal imbalances in 22q11.2 loci in 818 CHD patients consecutively enrolled from Shanghai Children's Medical Center. In the methodology development phase, CNVplex® detected all copy number aberrations that were previously identified with both chromosomal microarray analysis and MLPA, demonstrating 100% sensitivity and specificity. In the validation phase, 22q11.2 deletion and 22q11.2 duplication were detected in 39 and 1 of 818 patients with CHD by CNVplex®, respectively. Our data demonstrated that the frequency of 22q11.2 deletion varied among sub-groups of CHD patients. Notably, 22q11.2 deletion was more commonly observed in cases with conotruncal defect (CTD) than in cases with non-CTD (P<0.001). With higher resolution and more probes against selected chromosomal loci, CNVplex® also identified several individuals with small CNVs and alterations in other chromosomes. CNVplex® is sensitive and specific in its detection of CNVs, and it is an alternative to MLPA for batch screening of pathogenetic CNVs in known genomic loci.

Genetic analysis of PAX3 for diagnosis of Waardenburg syndrome type I.

PubMed

Matsunaga, Tatsuo; Mutai, Hideki; Namba, Kazunori; Morita, Noriko; Masuda, Sawako

2013-04-01

PAX3 genetic analysis increased the diagnostic accuracy for Waardenburg syndrome type I (WS1). Analysis of the three-dimensional (3D) structure of PAX3 helped verify the pathogenicity of a missense mutation, and multiple ligation-dependent probe amplification (MLPA) analysis of PAX3 increased the sensitivity of genetic diagnosis in patients with WS1. Clinical diagnosis of WS1 is often difficult in individual patients with isolated, mild, or non-specific symptoms. The objective of the present study was to facilitate the accurate diagnosis of WS1 through genetic analysis of PAX3 and to expand the spectrum of known PAX3 mutations. In two Japanese families with WS1, we conducted a clinical evaluation of symptoms and genetic analysis, which involved direct sequencing, MLPA analysis, quantitative PCR of PAX3, and analysis of the predicted 3D structure of PAX3. The normal-hearing control group comprised 92 subjects who had normal hearing according to pure tone audiometry. In one family, direct sequencing of PAX3 identified a heterozygous mutation, p.I59F. Analysis of PAX3 3D structures indicated that this mutation distorted the DNA-binding site of PAX3. In the other family, MLPA analysis and subsequent quantitative PCR detected a large, heterozygous deletion spanning 1759-2554 kb that eliminated 12-18 genes including a whole PAX3 gene.

Using a combination of MLPA kits to detect chromosomal imbalances in patients with multiple congenital anomalies and mental retardation is a valuable choice for developing countries.

PubMed

Jehee, Fernanda Sarquis; Takamori, Jean Tetsuo; Medeiros, Paula F Vasconcelos; Pordeus, Ana Carolina B; Latini, Flavia Roche M; Bertola, Débora Romeo; Kim, Chong Ae; Passos-Bueno, Maria Rita

2011-01-01

Conventional karyotyping detects anomalies in 3-15% of patients with multiple congenital anomalies and mental retardation (MCA/MR). Whole-genome array screening (WGAS) has been consistently suggested as the first choice diagnostic test for this group of patients, but it is very costly for large-scale use in developing countries. We evaluated the use of a combination of Multiplex Ligation-dependent Probe Amplification (MLPA) kits to increase the detection rate of chromosomal abnormalities in MCA/MR patients. We screened 261 MCA/MR patients with two subtelomeric and one microdeletion kits. This would theoretically detect up to 70% of all submicroscopic abnormalities. Additionally we scored the de Vries score for 209 patients in an effort to find a suitable cut-off for MLPA screening. Our results reveal that chromosomal abnormalities were present in 87 (33.3%) patients, but only 57 (21.8%) were considered causative. Karyotyping detected 15 abnormalities (6.9%), while MLPA identified 54 (20.7%). Our combined MLPA screening raised the total detection number of pathogenic imbalances more than three times when compared to conventional karyotyping. We also show that using the de Vries score as a cut-off for this screening would only be suitable under financial restrictions. A decision analytic model was constructed with three possible strategies: karyotype, karyotype + MLPA and karyotype + WGAS. Karyotype + MLPA strategy detected anomalies in 19.8% of cases which account for 76.45% of the expected yield for karyotype + WGAS. Incremental Cost Effectiveness Ratio (ICER) of MLPA is three times lower than that of WGAS, which means that, for the same costs, we have three additional diagnoses with MLPA but only one with WGAS. We list all causative alterations found, including rare findings, such as reciprocal duplications of regions deleted in Sotos and Williams-Beuren syndromes. We also describe imbalances that were considered polymorphisms or rare variants, such as the new SNP that confounded the analysis of the 22q13.3 deletion syndrome. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Genetic Analyses of the NF1 Gene in Turkish Neurofibromatosis Type I Patients and Definition of three Novel Variants

PubMed Central

Ulusal, SD; Gürkan, H; Atlı, E; Özal, SA; Çiftdemir, M; Tozkır, H; Karal, Y; Güçlü, H; Eker, D; Görker, I

2017-01-01

Abstract Neurofibromatosis Type I (NF1) is a multi systemic autosomal dominant neurocutaneous disorder predisposing patients to have benign and/or malignant lesions predominantly of the skin, nervous system and bone. Loss of function mutations or deletions of the NF1 gene is responsible for NF1 disease. Involvement of various pathogenic variants, the size of the gene and presence of pseudogenes makes it difficult to analyze. We aimed to report the results of 2 years of multiplex ligation-dependent probe amplification (MLPA) and next generation sequencing (NGS) for genetic diagnosis of NF1 applied at our genetic diagnosis center. The MLPA, semiconductor sequencing and Sanger sequencing were performed in genomic DNA samples from 24 unrelated patients and their affected family members referred to our center suspected of having NF1. In total, three novel and 12 known pathogenic variants and a whole gene deletion were determined. We suggest that next generation sequencing is a practical tool for genetic analysis of NF1. Deletion/duplication analysis with MLPA may also be helpful for patients clinically diagnosed to carry NF1 but do not have a detectable mutation in NGS. PMID:28924536

Clinical utility of multiplex ligation-dependent probe amplification technique in identification of aetiology of unexplained mental retardation: a study in 203 Indian patients.

PubMed

Boggula, Vijay R; Shukla, Anju; Danda, Sumita; Hariharan, Sankar V; Nampoothiri, Sheela; Kumar, Rashmi; Phadke, Shubha R

2014-01-01

Developmental delay (DD)/mental retardation also described as intellectual disability (ID), is seen in 1-3 per cent of general population. Diagnosis continues to be a challenge at clinical level. With the advancement of new molecular cytogenetic techniques such as cytogenetic microarray (CMA), multiplex ligation-dependent probe amplification (MLPA) techniques, many microdeletion/microduplication syndromes with DD/ID are now delineated. MLPA technique can probe 40-50 genomic regions in a single reaction and is being used for evaluation of cases with DD/ID. In this study we evaluated the clinical utility of MLPA techniques with different probe sets to identify the aetiology of unexplained mental retardation in patients with ID/DD. A total of 203 randomly selected DD/ID cases with/without malformations were studied. MLPA probe sets for subtelomeric regions (P070/P036) and common microdeletions/microduplications (P245-A2) and X-chromosome (P106) were used. Positive cases with MLPA technique were confirmed using either fluorescence in situ hybridization (FISH) or follow up confirmatory MLPA probe sets. The overall detection rate was found to be 9.3 per cent (19 out of 203). The detection rates were 6.9 and 7.4 per cent for common microdeletion/microduplication and subtelomeric probe sets, respectively. No abnormality was detected with probe set for X-linked ID. The subtelomeric abnormalities detected included deletions of 1p36.33, 4p, 5p, 9p, 9q, 13q telomeric regions and duplication of 9pter. The deletions/duplications detected in non telomeric regions include regions for Prader Willi/Angelman regions, Williams syndrome, Smith Magenis syndrome and Velocardiofacial syndrome. Our results show that the use of P245-A2 and P070/P036-E1 probes gives good diagnostic yield. Though MLPA cannot probe the whole genome like cytogenetic microarray, due to its ease and relative low cost it is an important technique for evaluation of cases with DD/ID.

Molecular Analysis-Based Genetic Characterization of a Cohort of Patients with Duchenne and Becker Muscular Dystrophy in Eastern China.

PubMed

Zhao, Hui-Hui; Sun, Xue-Ping; Shi, Ming-Chao; Yi, Yong-Xiang; Cheng, Hong; Wang, Xing-Xia; Xu, Qing-Cheng; Ma, Hong-Ming; Wu, Hao-Quan; Jin, Qing-Wen; Niu, Qi

2018-04-05

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are common X-linked recessive neuromuscular disorders caused by mutations in dystrophin gene. Multiplex polymerase chain reaction (multiplex PCR) and multiplex ligation-dependent probe amplification (MLPA) are the most common methods for detecting dystrophin gene mutations. This study aimed to contrast the two methods and discern the genetic characterization of patients with DMD/BMD in Eastern China. We collected 121 probands, 64 mothers of probands, and 15 fetuses in our study. The dystrophin gene was detected by multiplex PCR primarily in 28 probands, and MLPA was used in multiplex PCR-negative cases subsequently. The dystrophin gene of the remaining 93 probands and 62 female potential carriers was tested by MLPA directly. In fetuses, multiplex PCR and MLPA were performed on 4 fetuses and 10 fetuses, respectively. In addition, sequencing was also performed in 4 probands with negative MLPA. We found that 61.98% of the subjects had genetic mutations including deletions (50.41%) and duplications (11.57%). There were 43.75% of mothers as carriers of the mutation. In 15 fetuses, 2 out of 7 male fetuses were found to be unhealthy and 2 out of 8 female fetuses were found to be carriers. Exons 3-26 and 45-52 have the maximum frequency in mutation regions. In the frequency of exons individually, exon 47 and exon 50 were the most common in deleted regions and exons 5, 6, and 7 were found most frequently in duplicated regions. MLPA has better productivity and sensitivity than multiplex PCR. Prenatal diagnosis should be applied in DMD high-risk fetuses to reduce the disease incidence. Furthermore, it is the responsibility of physicians to inform female carriers the importance of prenatal diagnosis.

Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype®MTBDRplus.

PubMed

Santos, Paula Fernanda Gonçalves Dos; Costa, Elis Regina Dalla; Ramalho, Daniela M; Rossetti, Maria Lucia; Barcellos, Regina Bones; Nunes, Luciana de Souza; Esteves, Leonardo Souza; Rodenbusch, Rodrigo; Anthony, Richard M; Bergval, Indra; Sengstake, Sarah; Viveiros, Miguel; Kritski, Afrânio; Oliveira, Martha M

2017-06-01

To cope with the emergence of multidrug-resistant tuberculosis (MDR-TB), new molecular methods that can routinely be used to screen for a wide range of drug resistance related genetic markers in the Mycobacterium tuberculosis genome are urgently needed. To evaluate the performance of multiplex ligaton-dependent probe amplification (MLPA) against Genotype® MTBDRplus to detect resistance to isoniazid (INHr) and rifampicin (RIFr). 96 culture isolates characterised for identification, drug susceptibility testing (DST) and sequencing of rpoB, katG, and inhA genes were evaluated by the MLPA and Genotype®MTBDRplus assays. With sequencing as a reference standard, sensitivity (SE) to detect INHr was 92.8% and 85.7%, and specificity (SP) was 100% and 97.5%, for MLPA and Genotype®MTBDRplus, respectively. In relation to RIFr, SE was 87.5% and 100%, and SP was 100% and 98.8%, respectively. Kappa value was identical between Genotype®MTBDRplus and MLPA compared with the standard DST and sequencing for detection of INHr [0.83 (0.75-0.91)] and RIFr [0.93 (0.88-0.98)]. Compared to Genotype®MTBDRplus, MLPA showed similar sensitivity to detect INH and RIF resistance. The results obtained by the MLPA and Genotype®MTBDRplus assays indicate that both molecular tests can be used for the rapid detection of drug-resistant TB with high accuracy. MLPA has the added value of providing information on the circulating M. tuberculosis lineages.

Duchenne Muscular Dystrophy and Becker Muscular Dystrophy Confirmed by Multiplex Ligation-Dependent Probe Amplification: Genotype-Phenotype Correlation in a Large Cohort.

PubMed

Vengalil, Seena; Preethish-Kumar, Veeramani; Polavarapu, Kiran; Mahadevappa, Manjunath; Sekar, Deepha; Purushottam, Meera; Thomas, Priya Treesa; Nashi, Saraswathi; Nalini, Atchayaram

2017-01-01

Studies of cases of Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) confirmed by multiplex ligation-dependent probe amplification (MLPA) have determined the clinical characteristics, genotype, and relations between the reading frame and phenotype for different countries. This is the first such study from India. A retrospective genotype-phenotype analysis of 317 MLPA-confirmed patients with DMD or BMD who visited the neuromuscular clinic of a quaternary referral center in southern India. The 317 patients comprised 279 cases of DMD (88%), 32 of BMD (10.1%), and 6 of intermediate phenotype (1.9%). Deletions accounted for 91.8% of cases, with duplications causing the remaining 8.2%. There were 254 cases of DMD (91%) with deletions and 25 (9%) due to duplications, and 31 cases (96.8%) of BMD with deletions and 1 (3.2%) due to duplication. All six cases of intermediate type were due to deletions. The most-common mutation was a single-exon deletion. Deletions of six or fewer exons constituted 68.8% of cases. The deletion of exon 50 was the most common. The reading-frame rule held in 90% of DMD and 94% of BMD cases. A tendency toward a lower IQ and earlier wheelchair dependence was observed with distal exon deletions, though a significant correlation was not found. The reading-frame rule held in 90% to 94% of children, which is consistent with reports from other parts of the world. However, testing by MLPA is a limitation, and advanced sequencing methods including analysis of the structure of mutant dystrophin is needed for more-accurate assessments of the genotype-phenotype correlation.

Use of the HPV MLPA assay in cervical cytology for the prediction of high grade lesions.

PubMed

Litjens, Rogier J N T M; Theelen, Wendy; van de Pas, Yvonne; Ossel, Jessica; Reijans, Martin; Simons, Guus; Speel, Ernst-Jan M; Slangen, Brigitte F M; Ramaekers, Frans C S; Kruitwagen, Roy F P M; Hopman, Anton H N

2013-08-01

Current screening methods for uterine cervical cancer such as Papanicolaou smears and/or high risk human Papillomavirus (HR-HPV) detection have a high negative predictive value but a low positive predictive value for the presence of high grade cervical lesions. Therefore, new parameters are needed to reduce the rate of unnecessary referrals for colposcopy. The predictive value of the HPV multiplex ligation-dependent probe amplification (MLPA) assay, which can assess simultaneously HPV16/18 viral load and viral integration, was evaluated. The assay was applied to 170 cervical cytological samples, and the results were correlated with the matching histological follow-up. The GP5+/6+ assay and qPCR were used as a control for HR-HPV typing. The MLPA assay classified a higher percentage of cases as high-risk (high-viral load and/or viral integration) with higher grades of dysplasia. There was a high correlation between the HPV MLPA assay and qPCR for viral load and HPV genotyping, and between the MLPA assay and the GP5+/6+ assay for HPV genotyping. The sensitivity and specificity of the HPV MLPA assay for the detection of high-grade lesions were 44% and 93%, respectively. This study demonstrates that the HPV MLPA assay can reliably detect HPV 16/18, viral load, and viral integration in cytological samples. Also, high-risk classification correlated well with the presence of high-grade dysplasia. However, for the implementation of the MLPA assay into clinical practice, additional HR-HPV types need to be included to increase the sensitivity of the assay, and thereby increase its negative predictive value. Copyright © 2013 Wiley Periodicals, Inc.

[PAX3 gene mutation analysis for two Waardenburg syndrome type Ⅰ families and their prenatal diagnosis].

PubMed

Bai, Y; Liu, N; Kong, X D; Yan, J; Qin, Z B; Wang, B

2016-12-07

Objective: To analyze the mutations of PAX3 gene in two Waardenburg syndrome type Ⅰ (WS1) pedigrees and make prenatal diagnosis for the high-risk 18-week-old fetus. Methods: PAX3 gene was first analyzed by Sanger sequencing and multiplex ligation-dependent probe amplification(MLPA) for detecting pathogenic mutation of the probands of the two pedigrees. The mutations were confirmed by MLPA and Sanger in parents and unrelated healthy individuals.Prenatal genetic diagnosis for the high-risk fetus was performed by amniotic fluid cell after genotyping. Results: A heterozygous PAX3 gene gross deletion (E7 deletion) was identified in all patients from WS1-01 family, and not found in 20 healthy individuals.Prenatal diagnosis in WS1-01 family indicated that the fetus was normal. Molecular studies identified a novel deletion mutation c. 1385_1386delCT within the PAX3 gene in all affected WS1-02 family members, but in none of the unaffected relatives and 200 healthy individuals. Conclusions: PAX3 gene mutation is etiological for two WS1 families. Sanger sequencing plus MLPA is effective and accurate for making gene diagnosis and prenatal diagnosis.

Identification of a novel large deletion in a patient with severe factor V deficiency using an in-house F5 MLPA assay.

PubMed

Nuzzo, F; Paraboschi, E M; Straniero, L; Pavlova, A; Duga, S; Castoldi, E

2015-01-01

Factor V (FV) deficiency is a rare autosomal recessive bleeding disorder caused by mutations in the F5 gene. FV-deficient patients in whom no mutation or only one mutation is found may harbour large gene rearrangements, which are not detected by conventional mutation screening strategies. The aim of this study was to develop and validate a multiplex ligation-dependent probe amplification (MLPA) assay for the detection of large deletions and duplications in the F5 gene. Twenty-two MLPA probes targeting 19 of the 25 exons and the upstream and downstream regions of the F5 gene were designed and tested in 10 normal controls, a patient with a known heterozygous deletion of F5 exons 1-7 (positive control) and 14 genetically unexplained FV-deficient patients. MLPA results were confirmed by digital PCR on a QuantStudio(™) 3D Digital PCR System. The F5-specific probes yielded a reproducible peak profile in normal controls, correctly detected the known deletion in the positive control and suggested the presence of a novel deletion of exons 9-10 in a patient with undetectable FV levels and only one identified mutation. Follow-up by chip-based digital PCR, long-range PCR and direct sequencing confirmed that this patient carried a heterozygous F5 deletion of 1823 bp extending from intron 8 to intron 10. Bioinformatics sequence analysis pinpointed repetitive elements that might have originated the deletion. In conclusion, we have developed and validated an MLPA assay for the detection of gross F5 gene rearrangements. This assay may represent a valuable tool for the molecular diagnosis of FV deficiency. © 2014 John Wiley & Sons Ltd.

Analyses of Genotypes and Phenotypes of Ten Chinese Patients with Wolf-Hirschhorn Syndrome by Multiplex Ligation-dependent Probe Amplification and Array Comparative Genomic Hybridization

PubMed Central

Yang, Wen-Xu; Pan, Hong; Li, Lin; Wu, Hai-Rong; Wang, Song-Tao; Bao, Xin-Hua; Jiang, Yu-Wu; Qi, Yu

2016-01-01

Background: Wolf-Hirschhorn syndrome (WHS) is a contiguous gene syndrome that is typically caused by a deletion of the distal portion of the short arm of chromosome 4. However, there are few reports about the features of Chinese WHS patients. This study aimed to characterize the clinical and molecular cytogenetic features of Chinese WHS patients using the combination of multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (array CGH). Methods: Clinical information was collected from ten patients with WHS. Genomic DNA was extracted from the peripheral blood of the patients. The deletions were analyzed by MLPA and array CGH. Results: All patients exhibited the core clinical symptoms of WHS, including severe growth delay, a Greek warrior helmet facial appearance, differing degrees of intellectual disability, and epilepsy or electroencephalogram anomalies. The 4p deletions ranged from 2.62 Mb to 17.25 Mb in size and included LETM1, WHSC1, and FGFR3. Conclusions: The combined use of MLPA and array CGH is an effective and specific means to diagnose WHS and allows for the precise identification of the breakpoints and sizes of deletions. The deletion of genes in the WHS candidate region is closely correlated with the core WHS phenotype. PMID:26960370

Familial cases of Norrie disease detected by copy number analysis.

PubMed

Arai, Eisuke; Fujimaki, Takuro; Yanagawa, Ai; Fujiki, Keiko; Yokoyama, Toshiyuki; Okumura, Akihisa; Shimizu, Toshiaki; Murakami, Akira

2014-09-01

Norrie disease (ND, MIM#310600) is an X-linked disorder characterized by severe vitreoretinal dysplasia at birth. We report the results of causative NDP gene analysis in three male siblings with Norrie disease and describe the associated phenotypes. Three brothers with suspected Norrie disease and their mother presented for clinical examination. After obtaining informed consent, DNA was extracted from the peripheral blood of the proband, one of his brothers and his unaffected mother. Exons 1-3 of the NDP gene were amplified by polymerase chain reaction (PCR), and direct sequencing was performed. Multiplex ligation-dependent probe amplification (MLPA) was also performed to search for copy number variants in the NDP gene. The clinical findings of the three brothers included no light perception, corneal opacity, shallow anterior chamber, leukocoria, total retinal detachment and mental retardation. Exon 2 of the NDP gene was not amplified in the proband and one brother, even when the PCR primers for exon 2 were changed, whereas the other two exons showed no mutations by direct sequencing. MLPA analysis showed deletion of exon 2 of the NDP gene in the proband and one brother, while there was only one copy of exon 2 in the mother. Norrie disease was diagnosed in three patients from a Japanese family by clinical examination and was confirmed by genetic analysis. To localize the defect, confirmation of copy number variation by the MLPA method was useful in the present study.

Expanding the mutational spectrum in Johanson-Blizzard syndrome: identification of whole exon deletions and duplications in the UBR1 gene by multiplex ligation-dependent probe amplification analysis.

PubMed

Sukalo, Maja; Schäflein, Eva; Schanze, Ina; Everman, David B; Rezaei, Nima; Argente, Jesús; Lorda-Sanchez, Isabel; Deshpande, Charu; Takahashi, Tsutomu; Kleger, Alexander; Zenker, Martin

2017-11-01

Johanson-Blizzard syndrome (JBS, MIM #243800) is a very rare autosomal recessive disorder characterized by exocrine pancreatic insufficiency, nasal wing hypoplasia, hypodontia, and other abnormalities. JBS is caused by mutations of the UBR1 gene (MIM *605981), encoding a ubiquitin ligase of the N-end rule pathway. Molecular findings in a total of 65 unrelated patients with a clinical diagnosis of JBS who were previously screened for UBR1 mutations by Sanger sequencing were reviewed and cases lacking a disease-causing UBR1 mutation on either one or both alleles were included in this study. In order to discover mutations that are not detectable by Sanger sequencing, we designed a probe set for multiplex ligation-dependent probe amplification (MLPA) analysis of the UBR1 gene and analyzed the copy number status of all 47 UBR1 exons. Our previous studies using Sanger sequencing could detect mutations in 93.1% of 130 disease-associated UBR1 alleles. Six patients with a highly suggestive clinical diagnosis of JBS and unsolved genotype were included in this study. MLPA analysis detected six alleles harboring exon deletions/duplications, thereby raising the mutation detection rate in the entire cohort to 97.7% (127/130 alleles). We conclude that single or multi-exon deletions or duplications account for a substantial proportion of JBS-associated UBR1 mutations. © 2017 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

Novel deletion of the E3A ubiquitin protein ligase gene detected by multiplex ligation-dependent probe amplification in a patient with Angelman syndrome

PubMed Central

Calì, Francesco; Ragalmuto, Alda; Chiavetta, Valeria; Calabrese, Giuseppe; Fichera, Marco; Vinci, Mirella; Ruggeri, Giuseppa; Schinocca, Pietro; Sturnio, Maurizio; Romano, Salvatore; Elia, Maurizio

2010-01-01

Angelman syndrome (AS) is a severe neurobehavioural disorder caused by failure of expression of the maternal copy of the imprinted domain located on 15q11-q13. There are different mechanisms leading to AS: maternal microdeletion, uniparental disomy, defects in a putative imprinting centre, mutations of the E3 ubiquitin protein ligase (UBE3A) gene. However, some of suspected cases of AS are still scored negative to all the latter mutations. Recently, it has been shown that a proportion of negative cases bear large deletions overlapping one or more exons of the UBE3A gene. These deletions are difficult to detect by conventional gene-scanning methods due to the masking effect by the non-deleted allele. In this study, we have used for the first time multiplex ligation-dependent probe amplification (MLPA) and comparative multiplex dosage analysis (CMDA) to search for large deletions affecting the UBE3A gene. Using this approach, we identified a novel causative deletion involving exon 8 in an affected sibling. Based on our results, we propose the use of MLPA as a fast, accurate and inexpensive test to detect large deletions in the UBE3A gene in a small but significant percentage of AS patients. PMID:21072004

Characterisation of ATM mutations in Slavic Ataxia telangiectasia patients.

PubMed

Soukupova, Jana; Pohlreich, Petr; Seemanova, Eva

2011-09-01

Ataxia telangiectasia (AT) is a genomic instability syndrome characterised, among others, by progressive cerebellar degeneration, oculocutaneous telangiectases, immunodeficiency, elevated serum alpha-phetoprotein level, chromosomal breakage, hypersensitivity to ionising radiation and increased cancer risk. This autosomal recessive disorder is caused by mutations in the ataxia telangiectasia mutated (ATM) gene coding for serine/threonine protein kinase with a crucial role in response to DNA double-strand breaks. We characterised genotype and phenotype of 12 Slavic AT patients from 11 families. Mutation analysis included sequencing of the entire coding sequence, adjacent intron regions, 3'UTR and 5'UTR of the ATM gene and multiplex ligation-dependent probe amplification (MLPA) for the detection of large deletions/duplications at the ATM locus. The high incidence of new and individual mutations demonstrates a marked mutational heterogeneity of AT in the Czech Republic. Our data indicate that sequence analysis of the entire coding region of ATM is sufficient for a high detection rate of mutations in ATM and that MLPA analysis for the detection of deletions/duplications seems to be redundant in the Slavic population.

[Application of droplet digital PCR technology for genetic testing and prenatal diagnosis of spinal muscular atrophy].

PubMed

Zou, Yang; Xu, Peiwen; Li, Jie; Huang, Sexin; Gao, Ming; Kang, Ranran; Gao, Xuan; Gao, Yuan

2016-10-01

To explore the clinical application of droplet digital PCR (ddPCR) for genetic testing and prenatal diagnosis of spinal muscular atrophy (SMA) with deletion of SMN1 gene exon 7. A total of 138 clinical samples, including 121 peripheral blood, 13 amniotic fluid, 2 umbilical cord blood and 2 chorionic villi from 56 SMA families, were tested by both ddPCR and multiplex ligation-dependent probe amplification (MLPA). Results of the two approaches were analyzed with commercial software QuantaSoft (ddPCR) and Coffalyser (MLPA), respectively. Among the 138 cases, 25 had two copies, 84 had one copy, and 29 had null copy of exon 7 of the SMN1 gene. The results of ddPCR and MLPA were completely consistent. As a rapid, precise and economically efficient method, ddPCR will provide a new choice for genetic testing of SMA.

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.

Detection of BRCA1 gross rearrangements by droplet digital PCR.

PubMed

Preobrazhenskaya, Elena V; Bizin, Ilya V; Kuligina, Ekatherina Sh; Shleykina, Alla Yu; Suspitsin, Evgeny N; Zaytseva, Olga A; Anisimova, Elena I; Laptiev, Sergey A; Gorodnova, Tatiana V; Belyaev, Alexey M; Imyanitov, Evgeny N; Sokolenko, Anna P

2017-10-01

Large genomic rearrangements (LGRs) constitute a significant share of pathogenic BRCA1 mutations. Multiplex ligation-dependent probe amplification (MLPA) is a leading method for LGR detection; however, it is entirely based on the use of commercial kits, includes relatively time-consuming hybridization step, and is not convenient for large-scale screening of recurrent LGRs. We developed and validated the droplet digital PCR (ddPCR) assay, which covers the entire coding region of BRCA1 gene and is capable to precisely quantitate the copy number for each exon. 141 breast cancer (BC) patients, who demonstrated evident clinical features of hereditary BC but turned out to be negative for founder BRCA1/2 mutations, were subjected to the LGR analysis. Four patients with LGR were identified, with three cases of exon 8 deletion and one women carrying the deletion of exons 5-7. Excellent concordance with MLPA test was observed. Exon 8 copy number was tested in additional 720 BC and 184 ovarian cancer (OC) high-risk patients, and another four cases with the deletion were revealed; MLPA re-analysis demonstrated that exon 8 loss was a part of a larger genetic alteration in two cases, while the remaining two patients had isolated defect of exon 8. Long-range PCR and next generation sequencing of DNA samples carrying exon 8 deletion revealed two types of recurrent LGRs. Droplet digital PCR is a reliable tool for the detection of large genomic rearrangements.

Design and Validation of a New MLPA-Based Assay for the Detection of RS1 Gene Deletions and Application in a Large Family with X-Linked Juvenile Retinoschisis.

PubMed

Nicoletti, Annalisa; Ziccardi, Lucia; Maltese, Paolo Enrico; Benedetti, Sabrina; Palumbo, Orazio; Rendina, Michelina; D'Agruma, Leonardo; Falsini, Benedetto; Wang, Xinjing; Bertelli, Matteo

2017-02-01

X-linked juvenile retinoschisis (XLRS) is a severe ocular disorder that can evolve to blindness. More than 200 different disease-causing mutations have been reported in the RS1 gene and approximately 10% of these are deletions. Since transmission is X-linked, males are always affected and females are usually carriers. The identification of female carriers is always important and poses a technical challenge. Therefore, we sought to develop a multiplex ligation dependent probe amplification (MLPA)-based method to identify deletions or duplications in this gene. We then used our assay to study a large XLRS family. We designed six probes specific for each RS1 exon and then optimized and validated our method using control samples with known gene deletions. In the XLRS family, RS1 gene copy number variation was assessed by "home-made" MLPA analysis and by single nucleotide polymorphism (SNP) array analysis using the CytoScan HD Array. Direct sequencing was used for deletion breakpoint mapping. Our assay detected all deletions in control samples. All affected males of the family were positive for a deletion of exon 2 of the RS1 gene (RS1:NM_000330:c.53-?_78+?del). Carrier females were also identified. Our method is easily replicated, reliable, and inexpensive and allows female carriers to be detected. This is the first report of deep characterization of a whole exon deletion in the RS1 gene.

Cost-Effectiveness Analysis of Diagnosis of Duchenne/Becker Muscular Dystrophy in Colombia.

PubMed

Atehortúa, Sara C; Lugo, Luz H; Ceballos, Mateo; Orozco, Esteban; Castro, Paula A; Arango, Juan C; Mateus, Heidi E

2018-03-09

To determine the cost-effectiveness ratio of different courses of action for the diagnosis of Duchenne or Becker muscular dystrophy in Colombia. The cost-effectiveness analysis was performed from the Colombian health system perspective. Decision trees were constructed, and different courses of action were compared considering the following tests: immunohistochemistry (IHC), Western blot (WB), multiplex polymerase chain reaction, multiplex ligation-dependent probe amplification (MLPA), and the complete sequencing of the dystrophin gene. The time horizon matched the duration of sample extraction and analysis. Transition probabilities were obtained from a systematic review. Costs were constructed with a type-case methodology using the consensus of experts and the valuation of resources from consulting laboratories and the 2001 Social Security Institute cost manual. Deterministic sensitivity and scenario analyses were performed with one or more unavailable alternatives. Costs were converted from Colombian pesos to US dollars using the 2014 exchange rate. In the base case, WB was the dominant strategy, with a cost of US $419.07 and a sensitivity of 100%. This approach remains the dominant strategy down to a 98.2% sensitivity and while costs do not exceed US $837.38. If WB was not available, IHC had the best cost-effectiveness ratio, followed by MLPA and sequencing. WB is a cost-effective alternative for the diagnosis of patients suspected of having Duchenne or Becker muscular dystrophy in the Colombian health system. The IHC test is rated as the second-best detection method. If these tests are not available, MLPA followed by sequencing would be the most cost-effective alternative. Copyright © 2018. Published by Elsevier Inc.

Identification of de novo mutations of Duchénnè/Becker muscular dystrophies in southern Spain.

PubMed

Garcia, Susana; de Haro, Tomás; Zafra-Ceres, Mercedes; Poyatos, Antonio; Gomez-Capilla, Jose A; Gomez-Llorente, Carolina

2014-01-01

Duchénnè/Becker muscular dystrophies (DMD/BMD) are X-linked diseases, which are caused by a de novo gene mutation in one-third of affected males. The study objectives were to determine the incidence of DMD/BMD in Andalusia (Spain) and to establish the percentage of affected males in whom a de novo gene mutation was responsible. Multiplex ligation-dependent probe amplification (MLPA) technology was applied to determine the incidence of DMD/BMD in 84 males with suspicion of the disease and 106 female relatives. Dystrophin gene exon deletion (89.5%) or duplication (10.5%) was detected in 38 of the 84 males by MLPA technology; de novo mutations account for 4 (16.7%) of the 24 mother-son pairs studied. MLPA technology is adequate for the molecular diagnosis of DMD/BMD and establishes whether the mother carries the molecular alteration responsible for the disease, a highly relevant issue for genetic counseling.

Subtelomeric Rearrangements and Copy Number Variations in People with Intellectual Disabilities

ERIC Educational Resources Information Center

Christofolini, D. M.; De Paula Ramos, M. A.; Kulikowski, L. D.; Da Silva Bellucco, F. T.; Belangero, S. I. N.; Brunoni, D.; Melaragno, M. I.

2010-01-01

Background: The most prevalent type of structural variation in the human genome is represented by copy number variations that can affect transcription levels, sequence, structure and function of genes. Method: In the present study, we used the multiplex ligation-dependent probe amplification (MLPA) technique and quantitative PCR for the detection…

Multiplex ligation-dependent probe amplification for genetic screening in autism spectrum disorders: Efficient identification of known microduplications and identification of a novel microduplication in ASMT

PubMed Central

Cai, Guiqing; Edelmann, Lisa; Goldsmith, Juliet E; Cohen, Ninette; Nakamine, Alisa; Reichert, Jennifer G; Hoffman, Ellen J; Zurawiecki, Danielle M; Silverman, Jeremy M; Hollander, Eric; Soorya, Latha; Anagnostou, Evdokia; Betancur, Catalina; Buxbaum, Joseph D

2008-01-01

Background It has previously been shown that specific microdeletions and microduplications, many of which also associated with cognitive impairment (CI), can present with autism spectrum disorders (ASDs). Multiplex ligation-dependent probe amplification (MLPA) represents an efficient method to screen for such recurrent microdeletions and microduplications. Methods In the current study, a total of 279 unrelated subjects ascertained for ASDs were screened for genomic disorders associated with CI using MLPA. Fluorescence in situ hybridization (FISH), quantitative polymerase chain reaction (Q-PCR) and/or direct DNA sequencing were used to validate potential microdeletions and microduplications. Methylation-sensitive MLPA was used to characterize individuals with duplications in the Prader-Willi/Angelman (PWA) region. Results MLPA showed two subjects with typical ASD-associated interstitial duplications of the 15q11-q13 PWA region of maternal origin. Two additional subjects showed smaller, de novo duplications of the PWA region that had not been previously characterized. Genes in these two novel duplications include GABRB3 and ATP10A in one case, and MKRN3, MAGEL2 and NDN in the other. In addition, two subjects showed duplications of the 22q11/DiGeorge syndrome region. One individual was found to carry a 12 kb deletion in one copy of the ASPA gene on 17p13, which when mutated in both alleles leads to Canavan disease. Two subjects showed partial duplication of the TM4SF2 gene on Xp11.4, previously implicated in X-linked non-specific mental retardation, but in our subsequent analyses such variants were also found in controls. A partial duplication in the ASMT gene, located in the pseudoautosomal region 1 (PAR1) of the sex chromosomes and previously suggested to be involved in ASD susceptibility, was observed in 6–7% of the cases but in only 2% of controls (P = 0.003). Conclusion MLPA proves to be an efficient method to screen for chromosomal abnormalities. We identified duplications in 15q11-q13 and in 22q11, including new de novo small duplications, as likely contributing to ASD in the current sample by increasing liability and/or exacerbating symptoms. Our data indicate that duplications in TM4SF2 are not associated with the phenotype given their presence in controls. The results in PAR1/PAR2 are the first large-scale studies of gene dosage in these regions, and the findings at the ASMT locus indicate that further studies of the duplication of the ASMT gene are needed in order to gain insight into its potential involvement in ASD. Our studies also identify some limitations of MLPA, where single base changes in probe binding sequences alter results. In summary, our studies indicate that MLPA, with a focus on accepted medical genetic conditions, may be an inexpensive method for detection of microdeletions and microduplications in ASD patients for purposes of genetic counselling if MLPA-identified deletions are validated by additional methods. PMID:18925931

Detection of SHOX gene aberrations in routine diagnostic practice and evaluation of phenotype scoring form effectiveness.

PubMed

Hirschfeldova, Katerina; Florianova, Martina; Kebrdlova, Vera; Urbanova, Marketa; Stekrova, Jitka

2017-02-01

Heterozygous aberrations of SHOX gene have been reported to be responsible for Léri-Weill dyschondrosteosis (LWD) and small portion of idiopathic short stature. The study was established to assess effectiveness of using phenotype 'scoring form' in patients indicated for SHOX gene defect analysis. The submitted study is based on a retrospective group of 352 unrelated patients enrolled as a part of the routine diagnostic practice and analyzed for aberrations affecting the SHOX gene. All participants were scanned for deletion/duplication within the main pseudoautosomal region (PAR1) using the multiplex ligation-dependent probe amplification (MLPA) method. The phenotype 'scoring form' is used in our laboratory practice to preselect patients for subsequent mutation analysis of SHOX gene-coding sequences. The overall detection rate was 11.1% but there was a significant increase in frequency of SHOX gene defect positive with increasing achieved score (P<0.0001). The most frequent aberration was a causal deletion within PAR1. In three probands, MLPA analysis indicated a more complex rearrangement. Madelung deformity or co-occurrence of disproportionate short stature, short forearm and muscular hypertrophy had represented the most potent markers to determine the likelihood of SHOX gene defect detection. We conclude that appliance of phenotype 'scoring form' had saved excessive sample analysis and enabled effective routine diagnostic testing.

Design and Validation of a New MLPA-Based Assay for the Detection of RS1 Gene Deletions and Application in a Large Family with X-Linked Juvenile Retinoschisis

PubMed Central

Nicoletti, Annalisa; Ziccardi, Lucia; Benedetti, Sabrina; Palumbo, Orazio; Rendina, Michelina; D'Agruma, Leonardo; Falsini, Benedetto; Wang, Xinjing; Bertelli, Matteo

2017-01-01

Aims: X-linked juvenile retinoschisis (XLRS) is a severe ocular disorder that can evolve to blindness. More than 200 different disease-causing mutations have been reported in the RS1 gene and approximately 10% of these are deletions. Since transmission is X-linked, males are always affected and females are usually carriers. The identification of female carriers is always important and poses a technical challenge. Therefore, we sought to develop a multiplex ligation dependent probe amplification (MLPA)-based method to identify deletions or duplications in this gene. We then used our assay to study a large XLRS family. Methods: We designed six probes specific for each RS1 exon and then optimized and validated our method using control samples with known gene deletions. In the XLRS family, RS1 gene copy number variation was assessed by “home-made” MLPA analysis and by single nucleotide polymorphism (SNP) array analysis using the CytoScan HD Array. Direct sequencing was used for deletion breakpoint mapping. Results: Our assay detected all deletions in control samples. All affected males of the family were positive for a deletion of exon 2 of the RS1 gene (RS1:NM_000330:c.53-?_78+?del). Carrier females were also identified. Conclusion: Our method is easily replicated, reliable, and inexpensive and allows female carriers to be detected. This is the first report of deep characterization of a whole exon deletion in the RS1 gene. PMID:27997221

Adult recurrent pilocytic astrocytoma: Clinical, histopathological and molecular study.

PubMed

Trabelsi, S; Mama, N; Ladib, M; Popov, S; Burford, A; Mokni, M; Tlili, K; Krifa, H; Varella-Garcia, M; Jones, C; Tahar Yacoubi, M; Saad, A; H'mida Ben Brahim, D

2015-12-01

PA is a grade I glial tumor that mostly occurs in children. However, although apparently similar to paediatric PA, adult PA presents a different clinical follow-up that could arise from specific molecular alterations. A variety of genetic alterations have been identified as diagnostic or prognostic glioma molecular markers. We describe a right infratentorial tumor that occurred in a 58-year-old man. Neuroimaging and neuropathological examination suggested PA as an initial diagnosis. The tumor was completely resected. Unexpectedly, two years later, a rapidly growing tumor on the operative site was observed with a second location in the pineal region. Immunohistochemical reactions (IHC), Multiplex ligation probe amplification (MLPA) and fluorescence in situ hybridization (FISH) was performed in both primary and relapse tumor. Neuroimaging and neuropathological examinations suggested an unusual diagnosis for adult patients: a recurrent PA. Both MLPA and FISH analysis contribute to diagnostic confirmation by KIAA1549: BRAF fusion detection. Additional genetic results revealed interesting findings that justified the tumor aggressivity. Molecular analysis of adult PA cases should be routinely combined with histopathological and neuroimaging examination to further refine prognostic diagnoses. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Improved multiplex ligation-dependent probe amplification analysis identifies a deleterious PMS2 allele generated by recombination with crossover between PMS2 and PMS2CL.

PubMed

Wernstedt, Annekatrin; Valtorta, Emanuele; Armelao, Franco; Togni, Roberto; Girlando, Salvatore; Baudis, Michael; Heinimann, Karl; Messiaen, Ludwine; Staehli, Noemie; Zschocke, Johannes; Marra, Giancarlo; Wimmer, Katharina

2012-09-01

Heterozygous PMS2 germline mutations are associated with Lynch syndrome. Up to one third of these mutations are genomic deletions. Their detection is complicated by a pseudogene (PMS2CL), which--owing to extensive interparalog sequence exchange--closely resembles PMS2 downstream of exon 12. A recently redesigned multiplex ligation-dependent probe amplification (MLPA) assay identifies PMS2 copy number alterations with improved reliability when used with reference DNAs containing equal numbers of PMS2- and PMS2CL-specific sequences. We selected eight such reference samples--all publicly available--and used them with this assay to study 13 patients with PMS2-defective colorectal tumors. Three presented deleterious alterations: an Alu-mediated exon deletion; a 125-kb deletion encompassing PMS2 and four additional genes (two with tumor-suppressing functions); and a novel deleterious hybrid PMS2 allele produced by recombination with crossover between PMS2 and PMS2CL, with the breakpoint in intron 10 (the most 5' breakpoint of its kind reported thus far). We discuss mechanisms that might generate this allele in different chromosomal configurations (and their diagnostic implications) and describe an allele-specific PCR assay that facilitates its detection. Our data indicate that the redesigned PMS2 MLPA assay is a valid first-line option. In our series, it identified roughly a quarter of all PMS2 mutations. Copyright © 2012 Wiley Periodicals, Inc.

Improved Multiplex Ligation-Dependent Probe Amplification Analysis Identifies a Deleterious PMS2 Allele Generated by Recombination with Crossover Between PMS2 and PMS2CL

PubMed Central

Wernstedt, Annekatrin; Valtorta, Emanuele; Armelao, Franco; Togni, Roberto; Girlando, Salvatore; Baudis, Michael; Heinimann, Karl; Messiaen, Ludwine; Staehli, Noemie; Zschocke, Johannes; Marra, Giancarlo; Wimmer, Katharina

2012-01-01

Heterozygous PMS2 germline mutations are associated with Lynch syndrome. Up to one third of these mutations are genomic deletions. Their detection is complicated by a pseudogene (PMS2CL), which – owing to extensive interparalog sequence exchange – closely resembles PMS2 downstream of exon 12. A recently redesigned multiplex ligation-dependent probe amplification (MLPA) assay identifies PMS2 copy number alterations with improved reliability when used with reference DNAs containing equal numbers of PMS2- and PMS2CL-specific sequences. We selected eight such reference samples – all publicly available – and used them with this assay to study 13 patients with PMS2-defective colorectal tumors. Three presented deleterious alterations: an Alu-mediated exon deletion; a 125-kb deletion encompassing PMS2 and four additional genes (two with tumor-suppressing functions); and a novel deleterious hybrid PMS2 allele produced by recombination with crossover between PMS2 and PMS2CL, with the breakpoint in intron 10 (the most 5′ breakpoint of its kind reported thus far). We discuss mechanisms that might generate this allele in different chromosomal configurations (and their diagnostic implications) and describe an allele-specific PCR assay that facilitates its detection. Our data indicate that the redesigned PMS2 MLPA assay is a valid first-line option. In our series, it identified roughly a quarter of all PMS2 mutations. © 2012 Wiley Periodicals, Inc. PMID:22585707

Prenatal diagnosis and molecular cytogenetic characterization of mosaicism for a small supernumerary marker chromosome derived from chromosome 22 associated with cat eye syndrome.

PubMed

Chen, Chih-Ping; Ko, Tsang-Ming; Chen, Yi-Yung; Su, Jun-Wei; Wang, Wayseen

2013-09-15

We present prenatal diagnosis of mosaicism for a small supernumerary marker chromosome (sSMC) derived from chromosome 22 associated with cat eye syndrome (CES) using cultured amniocytes in a pregnancy with fetal microcephaly, intrauterine growth restriction, left renal hypoplasia, total anomalous pulmonary venous return with dominant right heart and right ear deformity. The sSMC was bisatellited and dicentric, and was characterized by multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (aCGH). The SALSA MLPA P250-B1 DiGeorge Probemix showed duplication of gene dosage in the CES region. aCGH showed a 1.26-Mb duplication at 22q11.1-q11.21 encompassing CECR1-CECR7. The sSMC was likely inv dup(22) (q11.21). Prenatal diagnosis of an sSMC(22) at amniocentesis should alert CES. MLPA, aCGH and fetal ultrasound are useful for rapid diagnosis of CES in case of prenatally detected sSMC(22). Copyright © 2013 Elsevier B.V. All rights reserved.

The -(α)(5.2) Deletion Detected in a Uruguayan Family: First Case Report in the Americas.

PubMed

Soler, Ana María; Schelotto, Magdalena; de Oliveira Mota, Natalia; Dorta Ferreira, Roberta; Sonati, Maria de Fatima; da Luz, Julio Abayubá

2016-08-01

In Uruguay, α-thalassemia (α-thal) mutations were introduced predominantly by Mediterranean European immigrant populations and by slave trade of African populations. A patient with anemia with hypochromia and microcytosis, refractory to iron treatment and with normal hemoglobin (Hb) electrophoresis was analyzed for α-thal mutations by multiplex gap-polymerase chain reaction (gap-PCR), automated sequencing and multiplex ligation-dependent probe amplification (MLPA) analyses. Agarose gel electrophoresis of the multiplex gap-PCR showed a band of unexpected size (approximately 700 bp) in the samples from the proband and mother. Automated sequencing of the amplified fragment showed the presence of the -(α)(5.2) deletion (NG_000006.1: g.32867_38062del5196) [an α-thal-1 deletion of 5196 nucleotides (nts)]. The MLPA analysis of the proband's sample also showed the presence of the -(α)(5.2) deletion in heterozygous state. We report here the presence of the -(α)(5.2) deletion, for the first time in the Americas, in a Uruguayan family with Italian ancestry, detected with a previously described multiplex gap-PCR.

Intronic splicing mutations in PTCH1 cause Gorlin syndrome.

PubMed

Bholah, Zaynab; Smith, Miriam J; Byers, Helen J; Miles, Emma K; Evans, D Gareth; Newman, William G

2014-09-01

Gorlin syndrome is an autosomal dominant disorder characterized by multiple early-onset basal cell carcinoma, odontogenic keratocysts and skeletal abnormalities. It is caused by heterozygous mutations in the tumour suppressor PTCH1. Routine clinical genetic testing, by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA) to confirm a clinical diagnosis of Gorlin syndrome, identifies a mutation in 60-90 % of cases. We undertook RNA analysis on lymphocytes from ten individuals diagnosed with Gorlin syndrome, but without known PTCH1 mutations by exonic sequencing or MLPA. Two altered PTCH1 transcripts were identified. Genomic DNA sequence analysis identified an intron 7 mutation c.1068-10T>A, which created a strong cryptic splice acceptor site, leading to an intronic insertion of eight bases; this is predicted to create a frameshift p.(His358Alafs*12). Secondly, a deep intronic mutation c.2561-2057A>G caused an inframe insertion of 78 intronic bases in the cDNA transcript, leading to a premature stop codon p.(Gly854fs*3). The mutations are predicted to cause loss of function of PTCH1, consistent with its tumour suppressor function. The findings indicate the importance of RNA analysis to detect intronic mutations in PTCH1 not identified by routine screening techniques.

PWS/AS MS-MLPA Confirms Maternal Origin of 15q11.2 Microduplication.

PubMed

Dawson, Angelika J; Cox, Janice; Hovanes, Karine; Spriggs, Elizabeth

2015-01-01

The proximal region of the long arm of chromosome 15q11.2-q13 is associated with various neurodevelopmental disorders, including Prader-Willi (PWS) and Angelman (AS) syndromes, autism, and other developmental abnormalities resulting from deletions and duplications. In addition, this region encompasses imprinted genes that cause PWS or AS, depending on the parent-of-origin. This imprinting allows for diagnosis of PWS or AS based on methylation status using methylation sensitive (MS) multiplex ligation dependent probe amplification (MLPA). Maternally derived microduplications at 15q11.2-q13 have been associated with autism and other neuropsychiatric disorders. Multiple methods have been used to determine the parent-of-origin for 15q11.2-q13 microdeletions and microduplications. In the present study, a four-year-old nondysmorphic female patient with developmental delay was found to have a de novo ~5 Mb duplication within 15q11.2 by oligonucleotide genomic array. In order to determine the significance of this microduplication to the clinical phenotype, the parent-of-origin needed to be identified. The PWS/AS MS-MLPA assay is generally used to distinguish between deletion and uniparental disomy (UPD) of 15q11.2-q13, resulting in either PWS or AS. However, our study shows that PWS/AS MS-MLPA can also efficiently distinguish the parental origin of duplications of 15q11.2-q13.

Mutation analysis of the MECP2 gene in patients of Slavic origin with Rett syndrome: novel mutations and polymorphisms.

PubMed

Zahorakova, Daniela; Rosipal, Robert; Hadac, Jan; Zumrova, Alena; Bzduch, Vladimir; Misovicova, Nadezda; Baxova, Alice; Zeman, Jiri; Martasek, Pavel

2007-01-01

Rett syndrome (RTT), an X-linked dominant neurodevelopmental disorder in females, is caused mainly by de novo mutations in the methyl-CpG-binding protein 2 gene (MECP2). Here we report mutation analysis of the MECP2 gene in 87 patients with RTT from the Czech and Slovak Republics, and Ukraine. The patients, all girls, with classical RTT were investigated for mutations using bi-directional DNA sequencing and conformation sensitive gel electrophoresis analysis of the coding sequence and exon/intron boundaries of the MECP2 gene. Restriction fragment length polymorphism analysis was performed to confirm the mutations that cause the creation or abolition of the restriction site. Mutation-negative cases were subsequently examined by multiple ligation-dependent probe amplification (MLPA) to identify large deletions. Mutation screening revealed 31 different mutations in 68 patients and 12 non-pathogenic polymorphisms. Six mutations have not been previously published: two point mutations (323T>A, 904C>T), three deletions (189_190delGA, 816_832del17, 1069delAGC) and one deletion/inversion (1063_1236del174;1189_1231inv43). MLPA analysis revealed large deletions in two patients. The detection rate was 78.16%. Our results confirm the high frequency of MECP2 mutations in females with RTT and provide data concerning the mutation heterogeneity in the Slavic population.

MLPA based detection of mutations in the dystrophin gene of 180 Polish families with Duchenne/Becker muscular dystrophy.

PubMed

Zimowski, Janusz G; Massalska, Diana; Holding, Mariola; Jadczak, Sylwia; Fidziańska, Elżbieta; Lusakowska, Anna; Kostera-Pruszczyk, Anna; Kamińska, Anna; Zaremba, Jacek

2014-01-01

Duchenne/Becker muscular dystrophy (DMD/BMD) is a recessive, X-linked disorder caused by a mutation in the dystrophin gene. Deletions account for approximately 60-65% of mutations, duplications for 5-10%. The remaining cases are mainly point mutations. According to Monaco theory clinical form of the disease depends on maintaining or disrupting the reading frame. The purpose of the study was to determine frequency and location of deletions and duplications in the dystrophin gene, to determine the compliance between maintaining/disrupting the reading frame and clinical form of the disease and to check the effectiveness of MLPA (multiplex ligation-dependent probe amplification) in the detection of these mutations in hemizygous patients and heterozygous female carriers. The material is composed of combined results of molecular diagnosis carried out in years 2009-2012 in 180 unrelated patients referred with the diagnosis of DMD/BMD tested by use of MLPA. We identified 110 deletions, 22 duplication (in one patient two different duplications were detected) and 2 point mutations. Deletions involved mainly exons 45-54 and 3-21, whereas most duplications involved exons 3-18. The compliance with Monaco theory was 95% for deletions and 76% for duplications. Most of mutations in the dystrophin gene were localized in the hot spots - different for deletions and duplications. MLPA enabled their quick identification, exact localization and determination whether or not they maintained or disrupted the reading frame. MLPA was also effective in detection of deletions and duplications in female carriers. Copyright © 2014 Polish Neurological Society. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Quantification of the methylation status of the PWS/AS imprinted region: comparison of two approaches based on bisulfite sequencing and methylation-sensitive MLPA.

PubMed

Dikow, Nicola; Nygren, Anders Oh; Schouten, Jan P; Hartmann, Carolin; Krämer, Nikola; Janssen, Bart; Zschocke, Johannes

2007-06-01

Standard methods used for genomic methylation analysis allow the detection of complete absence of either methylated or non-methylated alleles but are usually unable to detect changes in the proportion of methylated and unmethylated alleles. We compare two methods for quantitative methylation analysis, using the chromosome 15q11-q13 imprinted region as model. Absence of the non-methylated paternal allele in this region leads to Prader-Willi syndrome (PWS) whilst absence of the methylated maternal allele results in Angelman syndrome (AS). A proportion of AS is caused by mosaic imprinting defects which may be missed with standard methods and require quantitative analysis for their detection. Sequence-based quantitative methylation analysis (SeQMA) involves quantitative comparison of peaks generated through sequencing reactions after bisulfite treatment. It is simple, cost-effective and can be easily established for a large number of genes. However, our results support previous suggestions that methods based on bisulfite treatment may be problematic for exact quantification of methylation status. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) avoids bisulfite treatment. It detects changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in one simple reaction. Once established in a laboratory setting, the method is more accurate, reliable and less time consuming.

Identification of novel mutations in the α-galactosidase A gene in patients with Fabry disease: pitfalls of mutation analyses in patients with low α-galactosidase A activity.

PubMed

Yoshimitsu, Makoto; Higuchi, Koji; Miyata, Masaaki; Devine, Sean; Mattman, Andre; Sirrs, Sandra; Medin, Jeffrey A; Tei, Chuwa; Takenaka, Toshihiro

2011-05-01

Fabry disease is an X-linked lysosomal storage disorder caused by mutations of the α-galactosidase A (GLA) gene, and the disease is a relatively prevalent cause of left ventricular hypertrophy followed by conduction abnormalities and arrhythmias. Mutation analysis of the GLA gene is a valuable tool for accurate diagnosis of affected families. In this study, we carried out molecular studies of 10 unrelated families diagnosed with Fabry disease. Genetic analysis of the GLA gene using conventional genomic sequencing was performed in 9 hemizygous males and 6 heterozygous females. In patients with no mutations in coding DNA sequence, multiplex ligation-dependent probe amplification (MLPA) and/or cDNA sequencing were performed. We identified a novel exon 2 deletion (IVS1_IVS2) in a heterozygous female by MLPA, which was undetectable by conventional sequencing methods. In addition, the g.9331G>A mutation that has previously been found only in patients with cardiac Fabry disease was found in 3 unrelated, newly-diagnosed, cardiac Fabry patients by sequencing GLA genomic DNA and cDNA. Two other novel mutations, g.8319A>G and 832delA were also found in addition to 4 previously reported mutations (R112C, C142Y, M296I, and G373D) in 6 other families. We could identify GLA gene mutations in all hemizygotes and heterozygotes from 10 families with Fabry disease. Mutations in 4 out of 10 families could not be identified by classical genomic analysis, which focuses on exons and the flanking region. Instead, these data suggest that MLPA analysis and cDNA sequence should be considered in genetic testing surveys of patients with Fabry disease. Copyright © 2011 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.

Probe-specific mixed-model approach to detect copy number differences using multiplex ligation-dependent probe amplification (MLPA)

PubMed Central

González, Juan R; Carrasco, Josep L; Armengol, Lluís; Villatoro, Sergi; Jover, Lluís; Yasui, Yutaka; Estivill, Xavier

2008-01-01

Background MLPA method is a potentially useful semi-quantitative method to detect copy number alterations in targeted regions. In this paper, we propose a method for the normalization procedure based on a non-linear mixed-model, as well as a new approach for determining the statistical significance of altered probes based on linear mixed-model. This method establishes a threshold by using different tolerance intervals that accommodates the specific random error variability observed in each test sample. Results Through simulation studies we have shown that our proposed method outperforms two existing methods that are based on simple threshold rules or iterative regression. We have illustrated the method using a controlled MLPA assay in which targeted regions are variable in copy number in individuals suffering from different disorders such as Prader-Willi, DiGeorge or Autism showing the best performace. Conclusion Using the proposed mixed-model, we are able to determine thresholds to decide whether a region is altered. These threholds are specific for each individual, incorporating experimental variability, resulting in improved sensitivity and specificity as the examples with real data have revealed. PMID:18522760

Acquired resistance to an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) in an uncommon G719S EGFR mutation.

PubMed

Osoegawa, Atsushi; Hashimoto, Takafumi; Takumi, Yohei; Abe, Miyuki; Yamada, Tomonori; Kobayashi, Ryoji; Miyawaki, Michiyo; Takeuchi, Hideya; Okamoto, Tatsuro; Sugio, Kenji

2018-03-28

Background Acquired resistance (AR) to an epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) is a common event, and several underlying mechanisms, including T790 M, MET amplification and PTEN downregulation, have been reported for the common EGFR mutations. EGFR G719X is an uncommon mutation that has been reported to show sensitivity to EGFR-TKIs. However, no established cell lines harboring the EGFR G719X have been reported in the literature. Materials and Methods G719S-GR cells were established from malignant pleural effusion of a patient whose tumor developed AR from gefitinib treatment. G719S-GR cells were then genotyped and tested for drug sensitivities. Multiplex ligation-dependent probe amplification (MLPA) was used to compare the clinical tumor samples with G719S-GR. Results G719S-GR cells were resistant to EGFR-TKIs with an LC50 of around 10 μM. A genomic analysis showed that G719S-GR cells harbor the EGFR G719S mutation as well as the amplification of EGFR locus. The homozygous deletion of CDKN2A and the loss of PTEN and TSC1 were also detected. On comparing the copy number of tumor suppressor genes using MLPA, G719S-GR cells were found to lack one copy of PTEN, which was not observed in a tumor obtained before gefitinib treatment. Loss of PTEN may result in AKT activation. The mTORC1/2 inhibitor Torin-1 was able to inhibit the downstream signaling when combined with osimertinib. Discussion The newly established G719S-GR cell line may be useful for investigating the mechanism underlying the development of AR in the G719X mutation; the loss of PTEN may be one such mechanism.

Duplication of SOX9 associated with 46,XX ovotesticular disorder of sex development.

PubMed

López-Hernández, Berenice; Méndez, Juan Pablo; Coral-Vázquez, Ramón Mauricio; Benítez-Granados, Jesús; Zenteno, Juan Carlos; Villegas-Ruiz, Vanessa; Calzada-León, Raúl; Soderlund, Daniela; Canto, Patricia

2018-04-04

The purpose of the present study was to investigate whether ten unrelated SRY-negative individuals with this sex differentiation disorder presented a double dose of SOX9 as the cause of their disease. Ten unrelated SRY-negative 46,XX ovotesticular disorder of sexual development (DSD) subjects were molecularly studied. Multiplex-ligation dependent probe amplification (MLPA) and quantitative real-time PCR analysis (qRT-PCR) for SOX9 were performed. The MLPA analysis demonstrated that one patient presented a heterozygous duplication of the entire SOX9 coding region (above 1.3 value of peak ratio), as well as at least a ~ 483 kb upstream duplication. Moreover, no duplication of other SOX9 probes was observed corresponding to the region between -1007 and -1500 kb upstream. A qRT-PCR analysis showed a duplication of at least -581 kb upstream and ~1.63 kb of the coding region that encompasses exon 3. The limits of the duplication were mapped approximately from ~71539762 to 72122741 of Chr17. No molecular abnormalities were found in the remaining nine patients. This study is thought to be the first report regarding a duplication of SOX9 that is associated with the presence of 46,XX ovotesticular DSD, encompassing at least -581 kb upstream, and the almost entire coding region of the gene. Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Familial cleidocranial dysplasia misdiagnosed as rickets over three generations.

PubMed

Franceschi, Roberto; Maines, Evelina; Fedrizzi, Michela; Piemontese, Maria Rosaria; De Bonis, Patrizia; Agarwal, Nivedita; Bellizzi, Maria; Di Palma, Annunziata

2015-10-01

Cleidocranial dysplasia (CCD) is a rare autosomal dominant skeletal dysplasia characterized by hypoplastic clavicles, late closure of the fontanels, dental problems and other skeletal features. CCD is caused by mutations, deletions or duplications in runt-related transcription factor 2 (RUNX2), which encodes for a protein essential for osteoblast differentiation and chondrocyte maturation. We describe three familial cases of CCD, misdiagnosed as rickets over three generations. No mutations were detected on standard DNA sequencing of RUNX2, but a novel deletion was identified on quantitative polymerase chain reaction (qPCR) and multiple ligation-dependent probe amplification (MLPA). The present cases indicate that CCD could be misdiagnosed as rickets, leading to inappropriate treatment, and confirm that mutations in RUNX2 are not able to be identified on standard DNA sequencing in all CCD patients, but can be identified on qPCR and MLPA. © 2015 Japan Pediatric Society.

Evaluation of point mutations in dystrophin gene in Iranian Duchenne and Becker muscular dystrophy patients: introducing three novel variants.

PubMed

Haghshenas, Maryam; Akbari, Mohammad Taghi; Karizi, Shohreh Zare; Deilamani, Faravareh Khordadpoor; Nafissi, Shahriar; Salehi, Zivar

2016-06-01

Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked neuromuscular diseases characterized by progressive muscular weakness and degeneration of skeletal muscles. Approximately two-thirds of the patients have large deletions or duplications in the dystrophin gene and the remaining one-third have point mutations. This study was performed to evaluate point mutations in Iranian DMD/BMD male patients. A total of 29 DNA samples from patients who did not show any large deletion/duplication mutations following multiplex polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) screening were sequenced for detection of point mutations in exons 50-79. Also exon 44 was sequenced in one sample in which a false positive deletion was detected by MLPA method. Cycle sequencing revealed four nonsense, one frameshift and two splice site mutations as well as two missense variants.

Diagnostics of SHOX gene rearrangement in 46,XX women with idiopathic short stature.

PubMed

Mitka, Magdalena; Bednarek, Michał; Kałużewski, Bogdan

2016-01-01

The SHOX gene has been mapped at the pseudoautosomal region 1 (PAR1) of chromosomes X (Xp22.33) and Y (Yp11.32). The loss of SHOX gene functionality is assumed to be responsible for the Leri-Weill syndrome formation and the disproportionate short stature (DSS). The SHOX gene rearrangements constitute the majority of cases of gene functionality loss. Therefore, a practical application of the method, which allows for the diagnostics of the gene rearrangements, becomes a primary issue. With such an assumption, the MLPA technique (multiplex ligation - dependent probe amplification) becomes the method of choice. DNA samples were evaluated in the study by means of the MLPA method. The DNA was isolated from peripheral blood of sixty-three (63) 46,XX patients with short stature. Out of the examined patients, deletions within the SHOX gene were found in five (5) patients, and duplication at the PAR1 regulatory region of the SHOX gene in one (1) case. The obtained results confirm the opinion that the MLPA method, while enabling the diagnostics of the etiopathogenetic factor of short stature, identified in approximately 9.5% of cases, is a useful tool in the diagnostics of SHOX gene deletion and duplication. (Endokrynol Pol 2016; 67 (4): 397-402).

Optimizing the molecular diagnosis of CDKL5 gene-related epileptic encephalopathy in boys.

PubMed

Mei, Davide; Darra, Francesca; Barba, Carmen; Marini, Carla; Fontana, Elena; Chiti, Laura; Parrini, Elena; Dalla Bernardina, Bernardo; Guerrini, Renzo

2014-11-01

Mutations involving the cyclin-dependent kinase-like 5 (CDKL5) gene cause an early onset epileptic encephalopathy (EE) with severe neurologic impairment and a skewed 12:1 female-to-male ratio. To date, 18 mutations have been described in boys. We analyzed our cohort of boys with early onset EE to assess the diagnostic yield of our molecular approach. We studied 74 boys who presented early onset severe seizures, including infantile spasms and developmental delay, in the setting of EE, using Sanger sequencing, next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA). We identified alterations involving CDKL5 in four boys (5.4%) using NGS in one and MLPA in three. Three of four mutations were indicative of somatic mosaicism. CDKL5 gene mutations accounted for 5.4% of boys with early onset EE. Somatic mosaic mutations might be even more represented than germline mutations, probably because their less deleterious effect enhances viability of the male embryo. The molecular approach used for CDKL5 screening remarkably influences the diagnostic yield in boys. Diagnosis is optimized by Sanger sequencing combined with array-based methods or MLPA; alternatively, NGS targeted resequencing designed to also detect copy number alterations, may be performed. Wiley Periodicals, Inc. © 2014 International League Against Epilepsy.

Identification of a Novel De Novo Heterozygous Deletion in the SOX10 Gene in Waardenburg Syndrome Type II Using Next-Generation Sequencing.

PubMed

Li, Haonan; Jin, Peng; Hao, Qian; Zhu, Wei; Chen, Xia; Wang, Ping

2017-11-01

Waardenburg syndrome (WS) is a rare autosomal dominant disorder associated with pigmentation abnormalities and sensorineural hearing loss. In this study, we investigated the genetic cause of WSII in a patient and evaluated the reliability of the targeted next-generation exome sequencing method for the genetic diagnosis of WS. Clinical evaluations were conducted on the patient and targeted next-generation sequencing (NGS) was used to identify the candidate genes responsible for WSII. Multiplex ligation-dependent probe amplification (MLPA) and real-time quantitative polymerase chain reaction (qPCR) were performed to confirm the targeted NGS results. Targeted NGS detected the entire deletion of the coding sequence (CDS) of the SOX10 gene in the WSII patient. MLPA results indicated that all exons of the SOX10 heterozygous deletion were detected; no aberrant copy number in the PAX3 and microphthalmia-associated transcription factor (MITF) genes was found. Real-time qPCR results identified the mutation as a de novo heterozygous deletion. This is the first report of using a targeted NGS method for WS candidate gene sequencing; its accuracy was verified by using the MLPA and qPCR methods. Our research provides a valuable method for the genetic diagnosis of WS.

SOX2 anophthalmia syndrome: 12 new cases demonstrating broader phenotype and high frequency of large gene deletions.

PubMed

Bakrania, P; Robinson, D O; Bunyan, D J; Salt, A; Martin, A; Crolla, J A; Wyatt, A; Fielder, A; Ainsworth, J; Moore, A; Read, S; Uddin, J; Laws, D; Pascuel-Salcedo, D; Ayuso, C; Allen, L; Collin, J R O; Ragge, N K

2007-11-01

Developmental eye anomalies, which include anophthalmia (absent eye) or microphthalmia (small eye) are an important cause of severe visual impairment in infants and young children. Heterozygous mutations in SOX2, a SOX1B-HMG box transcription factor, have been found in up to 10% of individuals with severe microphthalmia or anophthalmia and such mutations could also be associated with a range of non-ocular abnormalities. We performed mutation analysis on a new cohort of 120 patients with congenital eye abnormalities, mainly anophthalmia, microphthalmia and coloboma. Multiplex ligation-dependent probe amplification (MLPA) and fluorescence in situ hybridisation (FISH) were used to detect whole gene deletion. We identified four novel intragenic SOX2 mutations (one single base deletion, one single base duplication and two point mutations generating premature translational termination codons) and two further cases with the previously reported c.70del20 mutation. Of 52 patients with severe microphthalmia or anophthalmia analysed by MLPA, 5 were found to be deleted for the whole SOX2 gene and 1 had a partial deletion. In two of these, FISH studies identified sub-microscopic deletions involving a minimum of 328 Kb and 550 Kb. The SOX2 phenotypes include a patient with anophthalmia, oesophageal abnormalities and horseshoe kidney, and a patient with a retinal dystrophy implicating SOX2 in retinal development. Our results provide further evidence that SOX2 haploinsufficiency is a common cause of severe developmental ocular malformations and that background genetic variation determines the varying phenotypes. Given the high incidence of whole gene deletion we recommend that all patients with severe microphthalmia or anophthalmia, including unilateral cases be screened by MLPA and FISH for SOX2 deletions.

Copy number variations in "classical" obesity candidate genes are not frequently associated with severe early-onset obesity in children.

PubMed

Windholz, Jan; Kovacs, Peter; Schlicke, Marina; Franke, Christin; Mahajan, Anubha; Morris, Andrew P; Lemke, Johannes R; Klammt, Jürgen; Kiess, Wieland; Schöneberg, Torsten; Pfäffle, Roland; Körner, Antje

2017-05-01

Obesity is genetically heterogeneous and highly heritable, although polymorphisms explain the phenotype in only a small proportion of obese children. We investigated the presence of copy number variations (CNVs) in "classical" genes known to be associated with (monogenic) early-onset obesity in children. In 194 obese Caucasian children selected for early-onset and severe obesity from our obesity cohort we screened for deletions and/or duplications by multiplex ligation-dependent probe amplification reaction (MLPA). As we found one MLPA probe to interfere with a polymorphism in SIM1 we investigated its association with obesity and other phenotypic traits in our extended cohort of 2305 children. In the selected subset of most severely obese children, we did not find CNV with MLPA in POMC, LEP, LEPR, MC4R, MC3R or MC2R genes. However, one SIM1 probe located at exon 9 gave signals suggestive for SIM1 insufficiency in 52 patients. Polymerase chain reaction (PCR) analysis identified this as a false positive result due to interference with single nucleotide polymorphism (SNP) rs3734354/rs3734355. We, therefore, investigated for associations of this polymorphism with obesity and metabolic traits in our extended cohort. We found rs3734354/rs3734355 to be associated with body mass index-standard deviation score (BMI-SDS) (p = 0.003), but not with parameters of insulin metabolism, blood pressure or food intake. In our modest sample of severely obese children, we were unable to find CNVs in well-established monogenic obesity genes. Nevertheless, we found an association of rs3734354 in SIM1 with obesity of early-onset type in children, although not with obesity-related traits.

Xp22.3 genomic deletions involving the CDKL5 gene in girls with early onset epileptic encephalopathy.

PubMed

Mei, Davide; Marini, Carla; Novara, Francesca; Bernardina, Bernardo D; Granata, Tiziana; Fontana, Elena; Parrini, Elena; Ferrari, Anna R; Murgia, Alessandra; Zuffardi, Orsetta; Guerrini, Renzo

2010-04-01

Mutations of the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) cause an X-linked encephalopathy with early onset intractable epilepsy, including infantile spasms and other seizure types, and a Rett syndrome (RTT)-like phenotype. Very limited information is available on the frequency and phenotypic spectrum associated with CDKL5 deletions/duplications. We investigated the role of CDKL5 deletions/duplications in causing early onset intractable epilepsy of unknown etiology in girls. We studied 49 girls with early onset intractable epilepsy, with or without infantile spasms, and developmental impairment, for whom no etiologic factors were obvious after clinical examination, brain magnetic resonance imaging (MRI) and expanded screening for inborn errors of metabolism. We performed CDKL5 gene mutation analysis in all and multiplex ligation dependent probe amplification assay (MLPA) in those who were mutation negative. Custom Array-comparative genomic hybridization (CGH), breakpoint polymerase chain reaction (PCR) analysis, and X-inactivation studies were performed in patients in whom MLPA uncovered a genomic alteration. We found CDKL5 mutations in 8.2% (4 of 49) of patients and genomic deletions in 8.2% (4 of 49). Overall, abnormalities of the CDKL5 gene accounted for 16.3% (8 of 49) of patients. CDKL5 gene deletions are an under-ascertained cause of early onset intractable epilepsy in girls. Genetic testing of CDKL5, including both mutation and deletion/duplication analysis, should be considered in this clinical subgroup.

Rare genomic rearrangement in a boy with Williams-Beuren syndrome associated to XYY syndrome and intriguing behavior.

PubMed

Dutra, Roberta L; Piazzon, Flavia B; Zanardo, Évelin A; Costa, Thais Virginia Moura Machado; Montenegro, Marília M; Novo-Filho, Gil M; Dias, Alexandre T; Nascimento, Amom M; Kim, Chong Ae; Kulikowski, Leslie D

2015-12-01

Williams-Beuren syndrome (WBS) is caused by a hemizygous contiguous gene microdeletion of 1.55-1.84 Mb at 7q11.23 region. Approximately, 28 genes have been shown to contribute to classical phenotype of SWB with presence of dysmorphic facial features, supravalvular aortic stenosis (SVAS), intellectual disability, and overfriendliness. With the use of Microarray-based comparative genomic hybridization and other molecular cytogenetic techniques, is possible define with more accuracy partial or atypical deletion and refine the genotype-phenotype correlation. Here, we report on a rare genomic structural rearrangement in a boy with atypical deletion in 7q11.23 and XYY syndrome with characteristic clinical signs, but not sufficient for the diagnosis of WBS. Cytogenetic analysis of G-banding showed a karyotype 47,XYY. Analysis of DNA with the technique of MLPA (Multiplex Ligation-dependent Probe Amplification) using kits a combination of kits (P064, P036, P070, and P029) identified an atypical deletion on 7q11.23. In addition, high resolution SNP Oligonucleotide Microarray Analysis (SNP-array) confirmed the alterations found by MLPA and revealed others pathogenic CNVs, in the chromosomes 7 and X. The present report demonstrates an association not yet described in literature, between Williams-Beuren syndrome and 47,XYY. The identification of atypical deletion in 7q11.23 concomitant to additional pathogenic CNVs in others genomic regions allows a better comprehension of clinical consequences of atypical genomic rearrangements. © 2015 Wiley Periodicals, Inc.

Decisions on Further Research for Predictive Biomarkers of High-Dose Alkylating Chemotherapy in Triple-Negative Breast Cancer: A Value of Information Analysis.

PubMed

Miquel-Cases, Anna; Retèl, Valesca P; van Harten, Wim H; Steuten, Lotte M G

2016-06-01

To inform decisions about the design and priority of further studies of emerging predictive biomarkers of high-dose alkylating chemotherapy (HDAC) in triple-negative breast cancer (TNBC) using value-of-information analysis. A state transition model compared treating women with TNBC with current clinical practice and four biomarker strategies to personalize HDAC: 1) BRCA1-like profile by array comparative genomic hybridization (aCGH) testing; 2) BRCA1-like profile by multiplex ligation-dependent probe amplification (MLPA) testing; 3) strategy 1 followed by X-inactive specific transcript gene (XIST) and tumor suppressor p53 binding protein (53BP1) testing; and 4) strategy 2 followed by XIST and 53BP1 testing, from a Dutch societal perspective and a 20-year time horizon. Input data came from literature and expert opinions. We assessed the expected value of partial perfect information, the expected value of sample information, and the expected net benefit of sampling for potential ancillary studies of an ongoing randomized controlled trial (RCT; NCT01057069). The expected value of partial perfect information indicated that further research should be prioritized to the parameter group including "biomarkers' prevalence, positive predictive value (PPV), and treatment response rates (TRRs) in biomarker-negative patients and patients with TNBC" (€639 million), followed by utilities (€48 million), costs (€40 million), and transition probabilities (TPs) (€30 million). By setting up four ancillary studies to the ongoing RCT, data on 1) TP and MLPA prevalence, PPV, and TRR; 2) aCGH and aCGH/MLPA plus XIST and 53BP1 prevalence, PPV, and TRR; 3) utilities; and 4) costs could be simultaneously collected (optimal size = 3000). Further research on predictive biomarkers for HDAC should focus on gathering data on TPs, prevalence, PPV, TRRs, utilities, and costs from the four ancillary studies to the ongoing RCT. Copyright © 2016 International Society for Pharmacoeconomics and Outcomes Research (ISPOR). Published by Elsevier Inc. All rights reserved.

Branchio-otic syndrome caused by a genomic rearrangement: clinical findings and molecular cytogenetic studies in a patient with a pericentric inversion of chromosome 8.

PubMed

Schmidt, T; Bierhals, T; Kortüm, F; Bartels, I; Liehr, T; Burfeind, P; Shoukier, M; Frank, V; Bergmann, C; Kutsche, K

2014-01-01

Branchio-oto-renal (BOR) syndrome is an autosomal dominantly inherited developmental disorder, which is characterized by anomalies of the ears, the branchial arches and the kidneys. It is caused by mutations in the genes EYA1,SIX1 and SIX5. Genomic rearrangements of chromosome 8 affecting the EYA1 gene have also been described. Owing to this fact, methods for the identification of abnormal copy numbers such as multiplex ligation-dependent probe amplification (MLPA) have been introduced as routine laboratory techniques for molecular diagnostics of BOR syndrome. The advantages of these techniques are clear compared to standard cytogenetic and array approaches as well as Southern blot. MLPA detects deletions or duplications of a part or the entire gene of interest, but not balanced structural aberrations such as inversions and translocations. Consequently, disruption of a gene by a genomic rearrangement may escape detection by a molecular genetic analysis, although this gene interruption results in haploinsufficiency and, therefore, causes the disease. In a patient with clinical features of BOR syndrome, such as hearing loss, preauricular fistulas and facial dysmorphisms, but no renal anomalies, neither sequencing of the 3 genes linked to BOR syndrome nor array comparative genomic hybridization and MLPA were able to uncover a causative mutation. By routine cytogenetic analysis, we finally identified a pericentric inversion of chromosome 8 in the affected female. High-resolution multicolor banding confirmed the chromosome 8 inversion and narrowed down the karyotype to 46,XX,inv(8)(p22q13). By applying fluorescence in situ hybridization, we narrowed down both breakpoints on chromosome 8 and found the EYA1 gene in q13.3 to be directly disrupted. We conclude that standard karyotyping should not be neglected in the genetic diagnostics of BOR syndrome or other Mendelian disorders, particularly when molecular testing failed to detect any causative alteration in patients with a convincing phenotype. © 2013 S. Karger AG, Basel.

Epileptic encephalopathy in a girl with an interstitial deletion of Xp22 comprising promoter and exon 1 of the CDKL5 gene.

PubMed

Bahi-Buisson, Nadia; Girard, Benoit; Gautier, Agnes; Nectoux, Juliette; Fichou, Yann; Saillour, Yoann; Poirier, Karine; Chelly, Jamel; Bienvenu, Thierry

2010-01-05

We report a 2-year-old girl with early onset seizures variant of Rett syndrome with a deletion at Xp22 detected by multiplex ligation-dependent probe amplification (MLPA) technique. This patient presented with tonic seizures at 7 days of life. Subsequently, she developed infantile spasms at three months and finally refractory myoclonic epilepsy. She demonstrated severe encephalopathy with hypotonia, deceleration of head growth, with eye gaze but limited eye pursuit, no language, limited hand use, and intermittent hand stereotypies. This combination of clinical features, suggestive of early onset variant of Rett syndrome led us to screen the CDKL5 gene. In a first step, screening of the whole coding sequence of the CDKL5 gene revealed no point mutations. In a second step, we searched gross rearrangements by MLPA and identified a microdeletion affecting both the promoter and exon 1 in CDKL5. Subsequent analysis on a Nimblegen HD2 microarray confirmed a deletion of approximately 300 kb at Xp22, including the BEND2, SCML2, and CDKL5 genes. In conclusion, our report suggests that searching for large rearrangements in CDKL5 should be considered in girls with early onset seizures and Rett-like features. (c) 2009 Wiley-Liss, Inc.

Genomic analysis using high density SNP based oligonucleotide arrays and MLPA provides a comprehensive analysis of INI1/SMARCB1 in malignant rhabdoid tumors

PubMed Central

Jackson, Eric M.; Sievert, Angela J.; Gai, Xiaowu; Hakonarson, Hakon; Judkins, Alexander R; Tooke, Laura; Perin, Juan Carlos; Xie, Hongbo; Shaikh, Tamim H.; Biegel, Jaclyn A.

2009-01-01

Translational Relevance Previous reports suggested that abnormalities of INI1 could be detected in 70–75% of malignant rhabdoid tumors. The mechanism of inactivation in the other 25% remained unclear. The goal of this study was to perform a high-resolution genomic analysis of a large series of rhabdoid tumors with the expectation of identifying additional loci related to the initiation or progression of these malignancies. We also developed a comprehensive set of assays, including a new MLPA assay, to interrogate the INI1 locus in 22q11.2. Intragenic deletions could be detected using the Illumina 550K Beadchip, whereas single exon deletions could be detected using MLPA. The current study demonstrates that with a multi-platform approach, alterations at the INI1 locus can be detected in almost all cases. Thus, appropriate molecular genetic testing can be used as an aid in the diagnosis and for treatment planning for most patients. Purpose A high-resolution genomic profiling and comprehensive targeted analysis of INI1/SMARCB1 of a large series of pediatric rhabdoid tumors was performed. The aim was to identify regions of copy number change and loss of heterozygosity that might pinpoint additional loci involved in the development or progression of rhabdoid tumors, and define the spectrum of genomic alterations of INI1 in this malignancy. Experimental Design A multi-platform approach, utilizing Illumina single nucleotide polymorphism (SNP) based oligonucleotide arrays, multiplex ligation dependent probe amplification (MLPA), fluorescence in situ hybridization (FISH), and coding sequence analysis was used to characterize genome wide copy number changes, loss of heterozygosity, and genomic alterations of INI1/SMARCB1 in a series of pediatric rhabdoid tumors. Results The bi-allelic alterations of INI1 that led to inactivation were elucidated in 50 of 51 tumors. INI1 inactivation was demonstrated by a variety of mechanisms, including deletions, mutations, and loss of heterozygosity. The results from the array studies highlighted the complexity of rearrangements of chromosome 22, compared to the low frequency of alterations involving the other chromosomes. Conclusions The results from the genome wide SNP-array analysis suggest that INI1 is the primary tumor suppressor gene involved in the development of rhabdoid tumors with no second locus identified. In addition, we did not identify hot spots for the breakpoints in sporadic tumors with deletions of chromosome 22q11.2. By employing a multimodality approach, the wide spectrum of alterations of INI1 can be identified in the majority of patients, which increases the clinical utility of molecular diagnostic testing. PMID:19276269

Germline PMS2 mutation screened by mismatch repair protein immunohistochemistry of colorectal cancer in Japan.

PubMed

Sugano, Kokichi; Nakajima, Takeshi; Sekine, Shigeki; Taniguchi, Hirokazu; Saito, Shinya; Takahashi, Masahiro; Ushiama, Mineko; Sakamoto, Hiromi; Yoshida, Teruhiko

2016-11-01

Germline PMS2 gene mutations were detected by RT-PCR/direct sequencing of total RNA extracted from puromycin-treated peripheral blood lymphocytes (PBL) and multiplex ligation-dependent probe amplification (MLPA) analyses of Japanese patients with colorectal cancer (CRC) fulfilling either the revised Bethesda Guidelines or being an age at disease onset of younger than 70 years, and screened by mismatch repair protein immunohistochemistry of formalin-fixed paraffin embedded sections. Of the 501 subjects examined, 7 (1.40%) showed the downregulated expression of the PMS2 protein alone and were referred to the genetic counseling clinic. Germline PMS2 mutations were detected in 6 (85.7%), including 3 nonsense and 1 frameshift mutations by RT-PCR/direct sequencing and 2 genomic deletions by MLPA. No mutations were identified in the other MMR genes (i.e. MSH2, MLH1 and MSH6). The prevalence of the downregulated expression of the PMS2 protein alone was 1.40% among the subjects examined and IHC results predicted the presence of PMS2 germline mutations. RT-PCR from puromycin-treated PBL and MLPA may be employed as the first screening step to detect PMS2 mutations without pseudogene interference, followed by the long-range PCR/nested PCR validation using genomic DNA. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Current molecular genetics strategies for the diagnosis of lysosomal storage disorders.

PubMed

Giugliani, Roberto; Brusius-Facchin, Ana-Carolina; Pasqualim, Gabriela; Leistner-Segal, Sandra; Riegel, Mariluce; Matte, Ursula

2016-01-01

Lysosomal storage disorders (LSDs) are a group of almost 50 monogenic diseases characterized by mutations causing deficiency of lysosomal enzymes or non-enzyme proteins involved in transport across the lysosomal membrane, protein maturation or lysosomal biogenesis. Usually, affected patients are normal at birth and have a progressive and severe disease with high morbidity and reduced life expectancy. The overall incidence of LSDs is usually estimated as 1:5000, but newborn screening studies are indicating that it could be much higher. Specific therapies were already developed for selected LSDs, making the timely and correct diagnosis very important for successful treatment and also for genetic counseling. In most LSD cases the biochemical techniques provide a reliable diagnosis. However, the identification of pathogenic mutations by genetic analysis is being increasingly recommended to provide additional information. In this paper we discuss the conventional methods for genetic analysis used in the LSDs [restriction fragment length polymorphism (RFLP), amplification-refractory mutation system (ARMS), single strand conformation polymorphism (SSCP), denaturing high performance liquid chromatography (dHPLC), real-time polymerase chain reaction, high resolution melting (HRM), multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing] and also the newer approaches [massive parallel sequencing, array comparative genomic hybridization (CGH)].

Application of a molecular diagnostic algorithm for haemophilia A and B using next-generation sequencing of entire F8, F9 and VWF genes.

PubMed

Bastida, Jose Maria; González-Porras, Jose Ramon; Jiménez, Cristina; Benito, Rocio; Ordoñez, Gonzalo R; Álvarez-Román, Maria Teresa; Fontecha, M Elena; Janusz, Kamila; Castillo, David; Fisac, Rosa María; García-Frade, Luis Javier; Aguilar, Carlos; Martínez, María Paz; Bermejo, Nuria; Herrero, Sonia; Balanzategui, Ana; Martin-Antorán, Jose Manuel; Ramos, Rafael; Cebeiro, Maria Jose; Pardal, Emilia; Aguilera, Carmen; Pérez-Gutierrez, Belen; Prieto, Manuel; Riesco, Susana; Mendoza, Maria Carmen; Benito, Ana; Hortal Benito-Sendin, Ana; Jiménez-Yuste, Víctor; Hernández-Rivas, Jesus Maria; García-Sanz, Ramon; González-Díaz, Marcos; Sarasquete, Maria Eugenia

2017-01-05

Currently, molecular diagnosis of haemophilia A and B (HA and HB) highlights the excess risk-inhibitor development associated with specific mutations, and enables carrier testing of female relatives and prenatal or preimplantation genetic diagnosis. Molecular testing for HA also helps distinguish it from von Willebrand disease (VWD). Next-generation sequencing (NGS) allows simultaneous investigation of several complete genes, even though they may span very extensive regions. This study aimed to evaluate the usefulness of a molecular algorithm employing an NGS approach for sequencing the complete F8, F9 and VWF genes. The proposed algorithm includes the detection of inversions of introns 1 and 22, an NGS custom panel (the entire F8, F9 and VWF genes), and multiplex ligation-dependent probe amplification (MLPA) analysis. A total of 102 samples (97 FVIII- and FIX-deficient patients, and five female carriers) were studied. IVS-22 screening identified 11 out of 20 severe HA patients and one female carrier. IVS-1 analysis did not reveal any alterations. The NGS approach gave positive results in 88 cases, allowing the differential diagnosis of mild/moderate HA and VWD in eight cases. MLPA confirmed one large exon deletion. Only one case did have no pathogenic variants. The proposed algorithm had an overall success rate of 99 %. In conclusion, our evaluation demonstrates that this algorithm can reliably identify pathogenic variants and diagnose patients with HA, HB or VWD.

Identification and characterization of cryptic SHOX intragenic deletions in three Japanese patients with Léri-Weill dyschondrosteosis.

PubMed

Fukami, Maki; Dateki, Sumito; Kato, Fumiko; Hasegawa, Yukihiro; Mochizuki, Hiroshi; Horikawa, Reiko; Ogata, Tsutomu

2008-01-01

Although short-stature homeobox-containing gene (SHOX ) haploinsufficiency is responsible for Léri-Weill dyschondrosteosis (LWD), the molecular defect has not been identified in approximately 20% of Japanese LWD patients. Furthermore, although high prevalence of microdeletions affecting SHOX is primarily ascribed to the presence of repeat sequences such as Alu elements around SHOX, it remains to be determined whether microdeletions are actually mediated by repeat sequences. We performed multiple ligation probe amplification (MLPA) assay in six Japanese LWD patients with apparently normal SHOX, followed by fluorescent in situ hybridization (FISH) analysis and sequencing for polymerase chain reaction (PCR) products encompassing the deletion junctions in patients with abnormal MLPA patterns. Consequently, heterozygous intragenic deletions were identified in three cases, i.e., a 5,906-bp deletion involving exons 4-5 in case 1, a 5,594-bp deletion involving exons 4-6a in case 2, and a 50,199-bp deletion involving exons 4-6b in case 3. The deletion breakpoints of cases 1 and 2 were present in nonrepeat sequences, whereas those of case 3 resided within Alu elements. The results suggest that cryptic SHOX intragenic deletions account for a small fraction of LWD and that microdeletions affecting SHOX can be generated by repeat-sequence-mediated aberrant recombinations and by nonhomologous end joining.

BeeDoctor, a Versatile MLPA-Based Diagnostic Tool for Screening Bee Viruses

PubMed Central

De Smet, Lina; Ravoet, Jorgen; de Miranda, Joachim R.; Wenseleers, Tom; Mueller, Matthias Y.; Moritz, Robin F. A.; de Graaf, Dirk C.

2012-01-01

The long-term decline of managed honeybee hives in the world has drawn significant attention to the scientific community and bee-keeping industry. A high pathogen load is believed to play a crucial role in this phenomenon, with the bee viruses being key players. Most of the currently characterized honeybee viruses (around twenty) are positive stranded RNA viruses. Techniques based on RNA signatures are widely used to determine the viral load in honeybee colonies. High throughput screening for viral loads necessitates the development of a multiplex polymerase chain reaction approach in which different viruses can be targeted simultaneously. A new multiparameter assay, called “BeeDoctor”, was developed based on multiplex-ligation probe dependent amplification (MLPA) technology. This assay detects 10 honeybee viruses in one reaction. “BeeDoctor” is also able to screen selectively for either the positive strand of the targeted RNA bee viruses or the negative strand, which is indicative for active viral replication. Due to its sensitivity and specificity, the MLPA assay is a useful tool for rapid diagnosis, pathogen characterization, and epidemiology of viruses in honeybee populations. “BeeDoctor” was used for screening 363 samples from apiaries located throughout Flanders; the northern half of Belgium. Using the “BeeDoctor”, virus infections were detected in almost eighty percent of the colonies, with deformed wing virus by far the most frequently detected virus and multiple virus infections were found in 26 percent of the colonies. PMID:23144717

BeeDoctor, a versatile MLPA-based diagnostic tool for screening bee viruses.

PubMed

De Smet, Lina; Ravoet, Jorgen; de Miranda, Joachim R; Wenseleers, Tom; Mueller, Matthias Y; Moritz, Robin F A; de Graaf, Dirk C

2012-01-01

The long-term decline of managed honeybee hives in the world has drawn significant attention to the scientific community and bee-keeping industry. A high pathogen load is believed to play a crucial role in this phenomenon, with the bee viruses being key players. Most of the currently characterized honeybee viruses (around twenty) are positive stranded RNA viruses. Techniques based on RNA signatures are widely used to determine the viral load in honeybee colonies. High throughput screening for viral loads necessitates the development of a multiplex polymerase chain reaction approach in which different viruses can be targeted simultaneously. A new multiparameter assay, called "BeeDoctor", was developed based on multiplex-ligation probe dependent amplification (MLPA) technology. This assay detects 10 honeybee viruses in one reaction. "BeeDoctor" is also able to screen selectively for either the positive strand of the targeted RNA bee viruses or the negative strand, which is indicative for active viral replication. Due to its sensitivity and specificity, the MLPA assay is a useful tool for rapid diagnosis, pathogen characterization, and epidemiology of viruses in honeybee populations. "BeeDoctor" was used for screening 363 samples from apiaries located throughout Flanders; the northern half of Belgium. Using the "BeeDoctor", virus infections were detected in almost eighty percent of the colonies, with deformed wing virus by far the most frequently detected virus and multiple virus infections were found in 26 percent of the colonies.

BRCA mutation carrier detection. A model-based cost-effectiveness analysis comparing the traditional family history approach and the testing of all patients with breast cancer.

PubMed

Norum, Jan; Grindedal, Eli Marie; Heramb, Cecilie; Karsrud, Inga; Ariansen, Sarah Louise; Undlien, Dag Erik; Schlichting, Ellen; Mæhle, Lovise

2018-01-01

Identification of BRCA mutation carriers among patients with breast cancer (BC) involves costs and gains. Testing has been performed according to international guidelines, focusing on family history (FH) of breast and/or ovarian cancer. An alternative is testing all patients with BC employing sequencing of the BRCA genes and Multiplex Ligation Probe Amplification (MLPA). A model-based cost-effectiveness analysis, employing data from Oslo University Hospital, Ullevål (OUH-U) and a decision tree, was done. The societal and the healthcare perspectives were focused and a lifetime perspective employed. The comparators were the traditional FH approach used as standard of care at OUH-U in 2013 and the intervention (testing all patients with BC) performed in 2014 and 2015 at the same hospital. During the latter period, 535 patients with BC were offered BRCA testing with sequencing and MLPA. National 2014 data on mortality rates and costs were implemented, a 3% discount rate used and the costing year was 2015. The incremental cost-effectiveness ratio was calculated in euros (€) per life-year gained (LYG). The net healthcare cost (healthcare perspective) was €40 503/LYG. Including all resource use (societal perspective), the cost was €5669/LYG. The univariate sensitivity analysis documented the unit cost of the BRCA test and the number of LYGs the prominent parameters affecting the result.Diagnostic BRCA testing of all patients with BC was superior to the FH approach and cost-effective within the frequently used thresholds (healthcare perspective) in Norway (€60 000-€80 000/LYG).

BRCA mutation carrier detection. A model-based cost-effectiveness analysis comparing the traditional family history approach and the testing of all patients with breast cancer

PubMed Central

Norum, Jan; Grindedal, Eli Marie; Heramb, Cecilie; Karsrud, Inga; Ariansen, Sarah Louise; Undlien, Dag Erik; Schlichting, Ellen; Mæhle, Lovise

2018-01-01

Background Identification of BRCA mutation carriers among patients with breast cancer (BC) involves costs and gains. Testing has been performed according to international guidelines, focusing on family history (FH) of breast and/or ovarian cancer. An alternative is testing all patients with BC employing sequencing of the BRCA genes and Multiplex Ligation Probe Amplification (MLPA). Patients and methods A model-based cost-effectiveness analysis, employing data from Oslo University Hospital, Ullevål (OUH-U) and a decision tree, was done. The societal and the healthcare perspectives were focused and a lifetime perspective employed. The comparators were the traditional FH approach used as standard of care at OUH-U in 2013 and the intervention (testing all patients with BC) performed in 2014 and 2015 at the same hospital. During the latter period, 535 patients with BC were offered BRCA testing with sequencing and MLPA. National 2014 data on mortality rates and costs were implemented, a 3% discount rate used and the costing year was 2015. The incremental cost-effectiveness ratio was calculated in euros (€) per life-year gained (LYG). Results The net healthcare cost (healthcare perspective) was €40 503/LYG. Including all resource use (societal perspective), the cost was €5669/LYG. The univariate sensitivity analysis documented the unit cost of the BRCA test and the number of LYGs the prominent parameters affecting the result. Diagnostic BRCA testing of all patients with BC was superior to the FH approach and cost-effective within the frequently used thresholds (healthcare perspective) in Norway (€60 000–€80 000/LYG). PMID:29682331

CDKL5 gene status in female patients with epilepsy and Rett-like features: two new mutations in the catalytic domain

PubMed Central

2012-01-01

Background Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) located in the Xp22 region have been shown to cause a subset of atypical Rett syndrome with infantile spasms or early seizures starting in the first postnatal months. Methods We performed mutation screening of CDKL5 in 60 female patients who had been identified as negative for the methyl CpG-binding protein 2 gene (MECP2) mutations, but who had current or past epilepsy, regardless of the age of onset, type, and severity. All the exons in the CDKL5 gene and their neighbouring sequences were examined, and CDKL5 rearrangements were studied by multiplex ligation-dependent probe amplification (MLPA). Results Six previously unidentified DNA changes were detected, two of which were disease-causing mutations in the catalytic domain: a frameshift mutation (c.509_510insGT; p.Glu170GlyfsX36) and a complete deletion of exon 10. Both were found in patients with seizures that started in the first month of life. Conclusions This study demonstrated the importance of CDKL5 mutations as etiological factors in neurodevelopmental disorders, and indicated that a thorough analysis of the CDKL5 gene sequence and its rearrangements should be considered in females with Rett syndrome-like phenotypes, severe encephalopathy and epilepsy with onset before 5 months of age. This study also confirmed the usefulness of MLPA as a diagnostic screening method for use in clinical practice. PMID:22867051

CDKL5 gene status in female patients with epilepsy and Rett-like features: two new mutations in the catalytic domain.

PubMed

Maortua, Hiart; Martínez-Bouzas, Cristina; Calvo, María-Teresa; Domingo, Maria-Rosario; Ramos, Feliciano; García-Ribes, Ainhoa; Martínez, María-Jesús; López-Aríztegui, María-Asunción; Puente, Nerea; Rubio, Izaskun; Tejada, María-Isabel

2012-08-06

Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) located in the Xp22 region have been shown to cause a subset of atypical Rett syndrome with infantile spasms or early seizures starting in the first postnatal months. We performed mutation screening of CDKL5 in 60 female patients who had been identified as negative for the methyl CpG-binding protein 2 gene (MECP2) mutations, but who had current or past epilepsy, regardless of the age of onset, type, and severity. All the exons in the CDKL5 gene and their neighbouring sequences were examined, and CDKL5 rearrangements were studied by multiplex ligation-dependent probe amplification (MLPA). Six previously unidentified DNA changes were detected, two of which were disease-causing mutations in the catalytic domain: a frameshift mutation (c.509_510insGT; p.Glu170GlyfsX36) and a complete deletion of exon 10. Both were found in patients with seizures that started in the first month of life. This study demonstrated the importance of CDKL5 mutations as etiological factors in neurodevelopmental disorders, and indicated that a thorough analysis of the CDKL5 gene sequence and its rearrangements should be considered in females with Rett syndrome-like phenotypes, severe encephalopathy and epilepsy with onset before 5 months of age. This study also confirmed the usefulness of MLPA as a diagnostic screening method for use in clinical practice.

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.

Germline mutations in SUFU cause Gorlin syndrome-associated childhood medulloblastoma and redefine the risk associated with PTCH1 mutations.

PubMed

Smith, Miriam J; Beetz, Christian; Williams, Simon G; Bhaskar, Sanjeev S; O'Sullivan, James; Anderson, Beverley; Daly, Sarah B; Urquhart, Jill E; Bholah, Zaynab; Oudit, Deemesh; Cheesman, Edmund; Kelsey, Anna; McCabe, Martin G; Newman, William G; Evans, D Gareth R

2014-12-20

Heterozygous germline PTCH1 mutations are causative of Gorlin syndrome (naevoid basal cell carcinoma), but detection rates > 70% have rarely been reported. We aimed to define the causative mutations in individuals with Gorlin syndrome without PTCH1 mutations. We undertook exome sequencing on lymphocyte DNA from four unrelated individuals from families with Gorlin syndrome with no PTCH1 mutations found by Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), or RNA analysis. A germline heterozygous nonsense mutation in SUFU was identified in one of four exomes. Sanger sequencing of SUFU in 23 additional PTCH1-negative Gorlin syndrome families identified a SUFU mutation in a second family. Copy-number analysis of SUFU by MLPA revealed a large heterozygous deletion in a third family. All three SUFU-positive families fulfilled diagnostic criteria for Gorlin syndrome, although none had odontogenic jaw keratocysts. Each SUFU-positive family included a single case of medulloblastoma, whereas only two (1.7%) of 115 individuals with Gorlin syndrome and a PTCH1 mutation developed medulloblastoma. We demonstrate convincing evidence that SUFU mutations can cause classical Gorlin syndrome. Our study redefines the risk of medulloblastoma in Gorlin syndrome, dependent on the underlying causative gene. Previous reports have found a 5% risk of medulloblastoma in Gorlin syndrome. We found a < 2% risk in PTCH1 mutation-positive individuals, with a risk up to 20× higher in SUFU mutation-positive individuals. Our data suggest childhood brain magnetic resonance imaging surveillance is justified in SUFU-related, but not PTCH1-related, Gorlin syndrome. © 2014 by American Society of Clinical Oncology.

Mosaicism for maternal uniparental disomy 15 in a boy with some clinical features of Prader-Willi syndrome.

PubMed

Zilina, Olga; Kahre, Tiina; Talvik, Inga; Oiglane-Shlik, Eve; Tillmann, Vallo; Ounap, Katrin

2014-01-01

Prader-Willi syndrome (PWS) is caused by the lack of paternal expression of imprinted genes in the human chromosomal region 15q11.2-q13.2, which can be due to an interstitial deletion at 15q11.2-q13 of paternal origin (65-75%), maternal uniparental disomy (matUPD) of chromosome 15 (20-30%), or an imprinting defect (1-3%). The majority of PWS-associated matUPD15 cases represent a complete heterodisomy of chromosome 15 or a mixture of hetero- and isodisomic regions across the chromosome 15. Pure maternal isodisomy is observed in only a few matUPD15 patients. Here we report a case of an 18-year-old boy with some clinical features of Prader-Willi syndrome, such as overweight, muscular hypotonia, facial dysmorphism and psychiatric problems, but there was no reason to suspect PWS in the patient based solely on the phenotype estimation. However, chromosomal microarray analysis (CMA) revealed mosaic loss of heterozygosity of the entire chromosome 15. Methylation-specific multiplex ligation-dependant probe amplification (MS-MLPA) analysis showed hypermethylation of the SNRPN and NDN genes in the PWS/AS critical region of chromosome 15 in this patient. Taking into consideration the MS-MLPA results and the presence of PWS features in the patient, we concluded that it was matUPD15, although the patient's parents were not enrolled in the study. According to CMA and karyotyping, no trisomic or monosomic cells were present. To the best of our knowledge, only two PWS cases with mosaic maternal isodisomy 15 and without trisomic/monosomic cell lines have been reported so far. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

A large deletion in the succinate dehydrogenase B gene (SDHB) in a Japanese patient with abdominal paraganglioma and concomitant metastasis.

PubMed

Kodama, Hitomi; Iihara, Masatoshi; Nissato, Sumiko; Isobe, Kazumasa; Kawakami, Yasushi; Okamoto, Takahiro; Takekoshi, Kazuhiro

2010-01-01

Recently, mutations in nuclear genes encoding two mitochondrial complex II subunit proteins, Succinate dehydrogenase D (SDHD) and SDHB, have been found to be associated with the development of familial pheochromocytomas and paragangliomas (hereditary pheochromocytoma/paraganglioma syndrome: HPPS). Growing evidence suggests that the mutation of SDHB is highly associated with abdominal paraganglioma and the following distant metastasis (malignant paraganglioma). In the present study, we used multiplex ligation dependent probe amplification (MLPA) analysis to identify a large heterozygous SDHB gene deletion encompassing sequences corresponding to the promoter region, in addition to exon 1 and exon 2 malignant paraganglioma patient in whom previously characterized SDHB mutations were undetectable. This is the first Japanese case report of malignant paraganglioma, with a large SDHB deletions. Our present findings strongly support the notion that large deletions in the SDHB gene should be considered in patients lacking characterized SDHB mutations.

PubMed Central

Bello, Luca

2016-01-01

Accurate definition of genetic mutations causing Duchenne muscular dystrophy (DMD) has always been relevant in order to provide genetic counseling to patients and families, and helps to establish the prognosis in the case where the distinction between Duchenne, Becker, or intermediate muscular dystrophy is not obvious. As molecular treatments aimed at dystrophin restoration in DMD are increasingly available as commercialized drugs or within clinical trials, genetic diagnosis has become an indispensable tool in order to determine eligibility for these treatments. DMD patients in which multiplex ligation-dependent probe amplification (MLPA) or similar techniques show a deletion suitable to exon skipping of exons 44, 45, 51, or 53, may be currently treated with AONs targeting these exons, in the context of clinical trials, or, as is the case for exon 51 skipping in the United States, with the first commercialized drug (eteplirsen). Patients who test negative at MLPA, but in whom DMD gene sequencing shows a nonsense mutation, may be amenable for treatment with stop codon readthrough compounds such as ataluren. Novel molecular approaches such as CRISPR-Cas9 targeting of specific DMD mutations are still in the preclinical stages, but appear promising. In conclusion, an accurate genetic diagnosis represents the entrance into a new scenario of personalized medicine in DMD. PMID:28484312

Duane retraction syndrome in a patient with Duchenne muscular dystrophy.

PubMed

Bosley, Thomas M; Salih, Mustafa A; Alkhalidi, Hisham; Oystreck, Darren T; El Khashab, Heba Y; Kondkar, Altaf A; Abu-Amero, Khaled K

2016-09-01

We describe the clinical features of a boy with bilateral Duane retraction syndrome (DRS), Duchenne muscular dystrophy (DMD), and other medical problems. The child was followed-up for five years; his chart was reviewed, including the results of a muscle biopsy and genetic testing. Multiplex ligation-dependent probe amplification (MLPA) was used to interrogate deletions/duplications in the dystrophin gene. The proband had bilateral DRS with otherwise normal ocular motility; he also had developmental delay, mild mental retardation, and seizures. Clinical diagnosis of DMD included progressive proximal weakness, highly elevated creatine kinase levels, and a muscle biopsy showing significant dystrophic changes including contracted, degenerative, and regenerative fibers, and negative dystrophin immunostaining. MLPA documented duplication of exons 3 and 4 of the dystrophin gene. This boy is the third patient to be reported with DRS and DMD, the second with bilateral DRS and the only one with other neurologic features. Mutated dystrophin is present in extraocular muscles and in the central nervous system (CNS) in DMD, leaving open the question of whether this co-occurrence is the result of the genetic muscle abnormality, CNS effects caused by dystrophin mutations, or chance.

Digital Detection of Multiple Minority Mutants and Expression Levels of Multiple Colorectal Cancer-Related Genes Using Digital-PCR Coupled with Bead-Array

PubMed Central

Huang, Huan; Li, Shuo; Sun, Lizhou; Zhou, Guohua

2015-01-01

To simultaneously analyze mutations and expression levels of multiple genes on one detection platform, we proposed a method termed “multiplex ligation-dependent probe amplification–digital amplification coupled with hydrogel bead-array” (MLPA–DABA) and applied it to diagnose colorectal cancer (CRC). CRC cells and tissues were sampled to extract nucleic acid, perform MLPA with sequence-tagged probes, perform digital emulsion polymerase chain reaction (PCR), and produce a hydrogel bead-array to immobilize beads and form a single bead layer on the array. After hybridization with fluorescent probes, the number of colored beads, which reflects the abundance of expressed genes and the mutation rate, was counted for diagnosis. Only red or green beads occurred on the chips in the mixed samples, indicating the success of single-molecule PCR. When a one-source sample was analyzed using mixed MLPA probes, beads of only one color occurred, suggesting the high specificity of the method in analyzing CRC mutation and gene expression. In gene expression analysis of a CRC tissue from one CRC patient, the mutant percentage was 3.1%, and the expression levels of CRC-related genes were much higher than those of normal tissue. The highly sensitive MLPA–DABA succeeds in the relative quantification of mutations and gene expressions of exfoliated cells in stool samples of CRC patients on the same chip platform. MLPA–DABA coupled with hydrogel bead-array is a promising method in the non-invasive diagnosis of CRC. PMID:25880764

Novel glucokinase mutations in patients with monogenic diabetes - clinical outline of GCK-MD and potential for founder effect in Slavic population.

PubMed

Borowiec, M; Antosik, K; Fendler, W; Deja, G; Jarosz-Chobot, P; Mysliwiec, M; Zmyslowska, A; Malecki, M; Szadkowska, A; Mlynarski, W

2012-03-01

Glucokinase (GCK) gene mutations are the causative factor of GCK-MD (monogenic diabetes) characterized by a mild clinical phenotype and potential for insulin withdrawal. This study presents the results of a nationwide genetic screening for GCK-MD performed in Poland. A group of 194 patients with clinical suspicion of GCK-MD and 17 patients with neonatal diabetes were subjected to GCK sequencing. Patients negative for GCK mutations were subjected to multiplex ligation-dependent probe amplification (MLPA) to detect deletions or insertions. A total of 44 GCK heterozygous mutations were found in 68 probands (35%). Among those, 20 mutations were novel ones: A282fs, D198V, E158X, G246V, G249R, I348N, L165V, L315Q, M115I, N254S, P284fs, Q338P, R377L, R43C, R46S, S212fs, S212P, T255N, V406A and Y214D. No abnormalities were detected in MLPA analysis. Homozygous D278E mutation was found in one patient with neonatal diabetes. The most frequently observed combinations of symptoms typical for GCK-MD were mild diabetes and/or fasting hyperglycaemia (98.3%), positive C-peptide at diagnosis (76%) and dominant mode of inheritance (59%). This study outlines numerous novel mutations of the GCK gene present in white Caucasians of Slavic origin. Thorough clinical assessment of known factors associated with GCK-MD may facilitate patient selection. © 2011 John Wiley & Sons A/S.

Microduplications at 22q11.21 are associated with non-syndromic classic bladder exstrophy.

PubMed

Draaken, Markus; Reutter, Heiko; Schramm, Charlotte; Bartels, Enrika; Boemers, Thomas M; Ebert, Anne-Karoline; Rösch, Wolfgang; Schröder, Annette; Stein, Raimund; Moebus, Susanne; Stienen, Dietlinde; Hoffmann, Per; Nöthen, Markus M; Ludwig, Michael

2010-01-01

The exstrophy-epispadias complex (EEC) comprises a spectrum of urogenital anomalies in which part or all of the distal urinary tract fails to close. The present study aimed to identify microaberrations characterized by loss or gain of genomic material that contribute to the EEC at a genome-wide level. Molecular karyotyping, utilizing 549,839 single nucleotide polymorphisms (SNPs) with an average spacing of 5.7 kilobases, was performed to screen an initial cohort of 16 patients with non-syndromic EEC. A de novo microduplication involving chromosomal region 22q11.21 was identified in one patient with classic exstrophy of the bladder (CBE). Subsequent multiplex ligation-dependent probe amplification (MLPA) analysis was performed with an MLPA 22q11 kit in a further 50 non-syndromic EEC cases. We identified one CBE patient with an overlapping 22q11.21 duplication in whom the duplication had been transmitted from the unaffected mother. Chromosomal region 22q11 is well known for its susceptibility to genomic rearrangements, and these are associated with various syndromes including the velo-cardio-facial/DiGeorge syndrome (VCFS/DGS), the der(22) syndrome, and the cat-eye syndrome. Duplications in this region result in a wide and variable spectrum of clinical presentations that include features of the VCFS/DGS, while some carriers present with a completely normal phenotype. Our findings extend the phenotypic spectrum of the 22q11.2 duplication syndrome, and indicate that this aberration predisposes to CBE with incomplete penetrance. Copyright 2009 Elsevier Masson SAS. All rights reserved.

Investigation of 15q11-q13, 16p11.2 and 22q13 CNVs in Autism Spectrum Disorder Brazilian Individuals with and without Epilepsy

PubMed Central

Moreira, Danielle P.; Griesi-Oliveira, Karina; Bossolani-Martins, Ana L.; Lourenço, Naila C. V.; Takahashi, Vanessa N. O.; da Rocha, Kátia M.; Moreira, Eloisa S.; Vadasz, Estevão; Meira, Joanna Goes Castro; Bertola, Debora; Halloran, Eoghan O’; Magalhães, Tiago R.; Fett-Conte, Agnes C.; Passos-Bueno, Maria Rita

2014-01-01

Copy number variations (CNVs) are an important cause of ASD and those located at 15q11-q13, 16p11.2 and 22q13 have been reported as the most frequent. These CNVs exhibit variable clinical expressivity and those at 15q11-q13 and 16p11.2 also show incomplete penetrance. In the present work, through multiplex ligation-dependent probe amplification (MLPA) analysis of 531 ethnically admixed ASD-affected Brazilian individuals, we found that the combined prevalence of the 15q11-q13, 16p11.2 and 22q13 CNVs is 2.1% (11/531). Parental origin could be determined in 8 of the affected individuals, and revealed that 4 of the CNVs represent de novo events. Based on CNV prediction analysis from genome-wide SNP arrays, the size of those CNVs ranged from 206 kb to 2.27 Mb and those at 15q11-q13 were limited to the 15q13.3 region. In addition, this analysis also revealed 6 additional CNVs in 5 out of 11 affected individuals. Finally, we observed that the combined prevalence of CNVs at 15q13.3 and 22q13 in ASD-affected individuals with epilepsy (6.4%) was higher than that in ASD-affected individuals without epilepsy (1.3%; p<0.014). Therefore, our data show that the prevalence of CNVs at 15q13.3, 16p11.2 and 22q13 in Brazilian ASD-affected individuals is comparable to that estimated for ASD-affected individuals of pure or predominant European ancestry. Also, it suggests that the likelihood of a greater number of positive MLPA results might be found for the 15q13.3 and 22q13 regions by prioritizing ASD-affected individuals with epilepsy. PMID:25255310

Investigation of 15q11-q13, 16p11.2 and 22q13 CNVs in autism spectrum disorder Brazilian individuals with and without epilepsy.

PubMed

Moreira, Danielle P; Griesi-Oliveira, Karina; Bossolani-Martins, Ana L; Lourenço, Naila C V; Takahashi, Vanessa N O; da Rocha, Kátia M; Moreira, Eloisa S; Vadasz, Estevão; Meira, Joanna Goes Castro; Bertola, Debora; O'Halloran, Eoghan; Magalhães, Tiago R; Fett-Conte, Agnes C; Passos-Bueno, Maria Rita

2014-01-01

Copy number variations (CNVs) are an important cause of ASD and those located at 15q11-q13, 16p11.2 and 22q13 have been reported as the most frequent. These CNVs exhibit variable clinical expressivity and those at 15q11-q13 and 16p11.2 also show incomplete penetrance. In the present work, through multiplex ligation-dependent probe amplification (MLPA) analysis of 531 ethnically admixed ASD-affected Brazilian individuals, we found that the combined prevalence of the 15q11-q13, 16p11.2 and 22q13 CNVs is 2.1% (11/531). Parental origin could be determined in 8 of the affected individuals, and revealed that 4 of the CNVs represent de novo events. Based on CNV prediction analysis from genome-wide SNP arrays, the size of those CNVs ranged from 206 kb to 2.27 Mb and those at 15q11-q13 were limited to the 15q13.3 region. In addition, this analysis also revealed 6 additional CNVs in 5 out of 11 affected individuals. Finally, we observed that the combined prevalence of CNVs at 15q13.3 and 22q13 in ASD-affected individuals with epilepsy (6.4%) was higher than that in ASD-affected individuals without epilepsy (1.3%; p<0.014). Therefore, our data show that the prevalence of CNVs at 15q13.3, 16p11.2 and 22q13 in Brazilian ASD-affected individuals is comparable to that estimated for ASD-affected individuals of pure or predominant European ancestry. Also, it suggests that the likelihood of a greater number of positive MLPA results might be found for the 15q13.3 and 22q13 regions by prioritizing ASD-affected individuals with epilepsy.

Genetic diagnosis as a tool for personalized treatment of Duchenne muscular dystrophy.

PubMed

Bello, Luca; Pegoraro, Elena

2016-12-01

Accurate definition of genetic mutations causing Duchenne muscular dystrophy (DMD) has always been relevant in order to provide genetic counseling to patients and families, and helps to establish the prognosis in the case where the distinction between Duchenne, Becker, or intermediate muscular dystrophy is not obvious. As molecular treatments aimed at dystrophin restoration in DMD are increasingly available as commercialized drugs or within clinical trials, genetic diagnosis has become an indispensable tool in order to determine eligibility for these treatments. DMD patients in which multiplex ligation-dependent probe amplification (MLPA) or similar techniques show a deletion suitable to exon skipping of exons 44, 45, 51, or 53, may be currently treated with AONs targeting these exons, in the context of clinical trials, or, as is the case for exon 51 skipping in the United States, with the first commercialized drug (eteplirsen). Patients who test negative at MLPA, but in whom DMD gene sequencing shows a nonsense mutation, may be amenable for treatment with stop codon readthrough compounds such as ataluren. Novel molecular approaches such as CRISPR-Cas9 targeting of specific DMD mutations are still in the preclinical stages, but appear promising. In conclusion, an accurate genetic diagnosis represents the entrance into a new scenario of personalized medicine in DMD.

Identification of a novel large intragenic deletion in a family with Fanconi anemia: first molecular report from India and review of literature.

PubMed

Shukla, Pallavi; Rao, Anita; Ghosh, Kanjaksha; Vundinti, Babu Rao

2013-04-15

We report here an Indian case with Fanconi anemia (FA) presented with fever, pallor, short stature, hyperpigmentation and upper limb anomaly. Chromosome breakage analysis together with FANCD2 Western blot monoubiquitination assay confirmed the diagnosis as FA. Multiplex ligation-dependent probe amplification (MLPA) revealed a novel homozygous large intragenic deletion (exons 8-27 del) in the FANCA gene in the proband. His sib and parents were also analyzed and found to be heterozygous for the same mutation. We also reviewed the literature of FANCA large intragenic deletions found in FA patients from different countries and the mechanism involved in the formation of these deletions. To the best of our knowledge, this is the first molecular report from India on FA. The finding expands the mutation spectrum of the FANCA gene. Identification of the mutation confirms the diagnosis of FA at DNA level and helps in providing proper genetic counseling to the family. Copyright © 2013 Elsevier B.V. All rights reserved.

Insertion of an SVA element, a nonautonomous retrotransposon, in PMS2 intron 7 as a novel cause of Lynch syndrome.

PubMed

van der Klift, Heleen M; Tops, Carli M; Hes, Frederik J; Devilee, Peter; Wijnen, Juul T

2012-07-01

Heterozygous germline mutations in the mismatch repair gene PMS2 predispose carriers for Lynch syndrome, an autosomal dominant predisposition to cancer. Here, we present a LINE-1-mediated retrotranspositional insertion in PMS2 as a novel mutation type for Lynch syndrome. This insertion, detected with Southern blot analysis in the genomic DNA of the patient, is characterized as a 2.2 kb long 5' truncated SVA_F element. The insertion is not detectable by current diagnostic testing limited to MLPA and direct Sanger sequencing on genomic DNA. The molecular nature of this insertion could only be resolved in RNA from cultured lymphocytes in which nonsense-mediated RNA decay was inhibited. Our report illustrates the technical problems encountered in the detection of this mutation type. Especially large heterozygous insertions will remain unnoticed because of preferential amplification of the smaller wild-type allele in genomic DNA, and are probably underreported in the mutation spectra of autosomal dominant disorders. © 2012 Wiley Periodicals, Inc.

Prevalence and Spectrum of Large Deletions or Duplications in the Major Long QT Syndrome-Susceptibility Genes and Implications for Long QT Syndrome Genetic Testing

PubMed Central

Tester, David J.; Benton, Amber J.; Train, Laura; Deal, Barbara; Baudhuin, Linnea M.; Ackerman, Michael J.

2010-01-01

Long QT Syndrome (LQTS) is a cardiac channelopathy associated with syncope, seizures, and sudden death. Approximately 75% of LQTS is due to mutations in genes encoding for three cardiac ion channel alpha-subunits (LQT1-3). However, traditional mutational analyses have limited detection capabilities for atypical mutations such as large gene rearrangements. Here, we set out to determine the prevalence and spectrum of large deletions/duplications in the major LQTS-susceptibility genes among unrelated patients who were mutation-negative following point mutation analysis of LQT1-12-susceptibility genes. Forty-two unrelated clinically strong LQTS patients were analyzed using multiplex ligation-dependent probe amplification (MLPA), a quantitative fluorescent technique for detecting multiple exon deletions and duplications. The SALSA-MLPA LQTS Kit from MRC-Holland was used to analyze the three major LQTS-associated genes: KCNQ1, KCNH2, and SCN5A and the two minor genes: KCNE1 and KCNE2. Overall, 2 gene rearrangements were found in 2/42 (4.8%, CI, 1.7–11%) unrelated patients. A deletion of KCNQ1 exon 3 was identified in a 10 year-old Caucasian boy with a QTc of 660 milliseconds (ms), a personal history of exercise-induced syncope, and a family history of syncope. A deletion of KCNQ1 exon 7 was identified in a 17 year-old Caucasian girl with a QTc of 480 ms, a personal history of exercise-induced syncope, and a family history of sudden cardiac death. In conclusion, since nearly 5% of patients with genetically elusive LQTS had large genomic rearrangements involving the canonical LQTS-susceptibility genes, reflex genetic testing to investigate genomic rearrangements may be of clinical value. PMID:20920651

Double-labelling immunohistochemistry for MGMT and a “cocktail” of non-tumourous elements is a reliable, quick and easy technique for inferring methylation status in glioblastomas and other primary brain tumours

PubMed Central

2013-01-01

Background Our aim was to develop a new protocol for MGMT immunohistochemistry with good agreement between observers and good correlation with molecular genetic tests of tumour methylation. We examined 40 primary brain tumours (30 glioblastomas and 10 oligodendroglial tumours) with our new technique, namely double-labelling immunohistochemistry for MGMT and a "cocktail" of non-tumour antigens (CD34, CD45 and CD68). We compared the results with single-labelling immunohistochemistry for MGMT and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA, a recognised molecular genetic technique which we applied as the gold-standard for the methylation status). Results Double-labelling immunohistochemistry for MGMT produced a visual separation of tumourous and non-tumourous elements on the same histological slide, making it quick and easy to determine whether tumour cell nuclei were MGMT-positive or MGMT-negative (and thereby infer the methylation status of the tumour). We found good agreement between observers (kappa 0.76) and within observer (kappa 0.84). Furthermore, double-labelling showed good specificity (80%), sensitivity (73.33%), positive predictive value (PPV, 83.33%) and negative predictive value (NPV, 68.75%) compared to MS-MLPA. Double-labelling was quicker and easier to assess than single-labelling and it outperformed quantitative computerised image analysis of MGMT single-labelling in terms of sensitivity, specificity, PPV and NPV. Conclusions Double-labelling immunohistochemistry for MGMT and a cocktail of non-tumourous elements provides a "one look" method for determining whether tumour cell nuclei are MGMT-positive or MGMT-negative. This can be used to infer the methylation status of the tumour. There is good observer agreement and good specificity, sensitivity, PPV and NPV compared to a molecular gold-standard. PMID:24252243

Double-labelling immunohistochemistry for MGMT and a "cocktail" of non-tumourous elements is a reliable, quick and easy technique for inferring methylation status in glioblastomas and other primary brain tumours.

PubMed

Burke, Elinor; Grobler, Mariana; Elderfield, Kay; Bond, Frances; Crocker, Matthew; Taylor, Rohan; Bridges, Leslie R

2013-06-10

Our aim was to develop a new protocol for MGMT immunohistochemistry with good agreement between observers and good correlation with molecular genetic tests of tumour methylation. We examined 40 primary brain tumours (30 glioblastomas and 10 oligodendroglial tumours) with our new technique, namely double-labelling immunohistochemistry for MGMT and a "cocktail" of non-tumour antigens (CD34, CD45 and CD68). We compared the results with single-labelling immunohistochemistry for MGMT and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA, a recognised molecular genetic technique which we applied as the gold-standard for the methylation status). Double-labelling immunohistochemistry for MGMT produced a visual separation of tumourous and non-tumourous elements on the same histological slide, making it quick and easy to determine whether tumour cell nuclei were MGMT-positive or MGMT-negative (and thereby infer the methylation status of the tumour). We found good agreement between observers (kappa 0.76) and within observer (kappa 0.84). Furthermore, double-labelling showed good specificity (80%), sensitivity (73.33%), positive predictive value (PPV, 83.33%) and negative predictive value (NPV, 68.75%) compared to MS-MLPA. Double-labelling was quicker and easier to assess than single-labelling and it outperformed quantitative computerised image analysis of MGMT single-labelling in terms of sensitivity, specificity, PPV and NPV. Double-labelling immunohistochemistry for MGMT and a cocktail of non-tumourous elements provides a "one look" method for determining whether tumour cell nuclei are MGMT-positive or MGMT-negative. This can be used to infer the methylation status of the tumour. There is good observer agreement and good specificity, sensitivity, PPV and NPV compared to a molecular gold-standard.

Characterization of the Chromosome 1q41q42.12 region, and the Candidate Gene DISP1, in Patients with CDH

PubMed Central

Kantarci, Sibel; Ackerman, Kate G; Russell, Meaghan N; Longoni, Mauro; Sougnez, Carrie; Noonan, Kristin M; Hatchwell, Eli; Zhang, Xiaoyun; Vanmarcke, Rafael Pieretti; Anyane-Yeboa, Kwame; Dickman, Paul; Wilson, Jay; Donahoe, Patricia K; Pober, Barbara R

2010-01-01

Cytogenetic and molecular cytogenetic studies demonstrate association between congenital diaphragmatic hernia (CDH) and chromosome 1q41q42 deletions. In this study, we screened a large CDH cohort (N=179) for microdeletions in this interval by the multiplex ligation-dependent probe amplification (MLPA) technique, and also sequenced two candidate genes located therein, dispatched 1 (DISP1) and homo sapiens H2.0-like homeobox (HLX). MLPA analysis verified deletions of this region in two cases, an unreported patient with a 46,XY,del(1)(q41q42.13) karyotype and a previously reported patient with a Fryns syndrome phenotype [Kantarci et al., 2006]. HLX sequencing showed a novel but maternally inherited single nucleotide variant (c.27C>G) in a patient with isolated CDH, while DISP1 sequencing revealed a mosaic de novo heterozygous substitution (c.4412C>G; p.Ala1471Gly) in a male with a left-sided Bochdalek hernia plus multiple other anomalies. Pyrosequencing demonstrated the mutant allele was present in 43%, 12%, and 4.5% of the patient’s lymphoblastoid, peripheral blood lymphocytes, and saliva cells, respectively. We examined Disp1 expression at day E11.5 of mouse diaphragm formation and confirmed its presence in the pleuroperitoneal fold, as well as the nearby lung which also expresses Sonic hedgehog (Shh). Our report describes the first de novo DISP1 point mutation in a patient with complex CDH. Combining this finding with Disp1 embryonic mouse diaphragm and lung tissue expression, as well as previously reported human chromosome 1q41q42 aberrations in patients with CDH, suggests that DISP1 may warrant further consideration as a CDH candidate gene. PMID:20799323

A systematic variant screening in familial cases of congenital heart defects demonstrates the usefulness of molecular genetics in this field

PubMed Central

El Malti, Rajae; Liu, Hui; Doray, Bérénice; Thauvin, Christel; Maltret, Alice; Dauphin, Claire; Gonçalves-Rocha, Miguel; Teboul, Michel; Blanchet, Patricia; Roume, Joëlle; Gronier, Céline; Ducreux, Corinne; Veyrier, Magali; Marçon, François; Acar, Philippe; Lusson, Jean-René; Levy, Marilyne; Beyler, Constance; Vigneron, Jacqueline; Cordier-Alex, Marie-Pierre; Heitz, François; Sanlaville, Damien; Bonnet, Damien; Bouvagnet, Patrice

2016-01-01

The etiology of congenital heart defect (CHD) combines environmental and genetic factors. So far, there were studies reporting on the screening of a single gene on unselected CHD or on familial cases selected for specific CHD types. Our goal was to systematically screen a proband of familial cases of CHD on a set of genetic tests to evaluate the prevalence of disease-causing variant identification. A systematic screening of GATA4, NKX2-5, ZIC3 and Multiplex ligation-dependent probe amplification (MLPA) P311 Kit was setup on the proband of 154 families with at least two cases of non-syndromic CHD. Additionally, ELN screening was performed on families with supravalvular arterial stenosis. Twenty-two variants were found, but segregation analysis confirmed unambiguously the causality of 16 variants: GATA4 (1 ×), NKX2-5 (6 ×), ZIC3 (3 ×), MLPA (2 ×) and ELN (4 ×). Therefore, this approach was able to identify the causal variant in 10.4% of familial CHD cases. This study demonstrated the existence of a de novo variant even in familial CHD cases and the impact of CHD variants on adult cardiac condition even in the absence of CHD. This study showed that the systematic screening of genetic factors is useful in familial CHD cases with up to 10.4% elucidated cases. When successful, it drastically improved genetic counseling by discovering unaffected variant carriers who are at risk of transmitting their variant and are also exposed to develop cardiac complications during adulthood thus prompting long-term cardiac follow-up. This study provides an important baseline at dawning of the next-generation sequencing era. PMID:26014430

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.

Genetic findings in treatment-naïve and proton-beam-radiated iris melanomas.

PubMed

Krishna, Yamini; Kalirai, Helen; Thornton, Sophie; Damato, Bertil E; Heimann, Heinrich; Coupland, Sarah E

2016-07-01

Iris melanomas (IM) are rare and have a lower mortality than posterior uveal melanomas (UM). Our aims were to determine the prevalence of genetic changes associated with prognosis of posterior UM, in both treated and non-treated IM. Retrospective database review and molecular analysis of all patients diagnosed with IM at the Liverpool Ocular Oncology Centre (LOOC) between 1993 and 2015. Archival pathology specimens of confirmed IM cases were analysed for chromosomal alterations, using multiplex ligation-dependent probe amplification (MLPA) or microsatellite analysis (MSA) depending on DNA yield, and BRAF mutation status. 5189 patients were diagnosed with intraocular melanoma at LOOC from 1993 to 2015. Of these, 303 (5.8%) patients were diagnosed with IM. Tissue samples were available for 26 IM cases. Twelve of these cases had biopsies taken post-proton beam radiotherapy (PBR). Histological subtyping showed 14 IM being spindle, 2 epithelioid and 10 were of mixed cell type. Twenty of the 26 IM cases (77%) analysed genetically were classified as either disomy 3 (n=16) or monosomy 3 (n=4). Chromosome 6p gain was detected in 4/18 (22%) IM, and polysomy 8q in 6%. BRAF mutations were not detected in any of the four IM cases examined. One patient with IM died from metastatic disease: this tumour was disomy 3 with 6p and 8q gains. All other patients were alive with no evidence of metastases at study closure. Chromosomal aberrations seen in posterior UM can also be demonstrated using MLPA or MSA in both treatment naïve and PBR-treated IM. Most IM display a low-metastatic risk chromosomal profile. 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/.

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.

Characteristic face: a key indicator for direct diagnosis of 22q11.2 deletions in Chinese velocardiofacial syndrome patients.

PubMed

Wu, Dandan; Chen, Yang; Xu, Chen; Wang, Ke; Wang, Huijun; Zheng, Fengyun; Ma, Duan; Wang, Guomin

2013-01-01

Velocardiofacial syndrome (VCFS) is a disease in human with an expansive phenotypic spectrum and diverse genetic mechanisms mainly associated with copy number variations (CNVs) on 22q11.2 or other chromosomes. However, the correlations between CNVs and phenotypes remain ambiguous. This study aims to analyze the types and sizes of CNVs in VCFS patients, to define whether correlations exist between CNVs and clinical manifestations in Chinese VCFS patients. In total, 55 clinically suspected Chinese VCFS patients and 100 normal controls were detected by multiplex ligation-dependent probe amplification (MLPA). The data from MLPA and all the detailed clinical features of the objects were documented and analyzed. A total of 44 patients (80.0%) were diagnosed with CNVs on 22q11.2. Among them, 43 (78.2%) presented with 22q11.2 heterozygous deletions, of whom 40 (93.0%) had typical 3-Mb deletion, and 3 (7.0%) exhibited proximal 1.5-Mb deletion; no patient was found with atypical deletion on 22q11.2. One patient (1.8%) presented with a 3-Mb duplication mapping to the typical 3-Mb region on 22q11.2, while none of the chromosomal abnormalities in the MLPA kit were found in the other 11 patients and 100 normal controls. All the 43 patients with 22q11.2 deletions displayed characteristic face and palatal anomalies; 37 of them (86.0%) had cognitive or behavioral disorders, and 23 (53.5%) suffered from immune deficiencies; 10 patients (23.3%) manifested congenital heart diseases. Interestingly, all patients with the characteristic face had 22q11.2 heterozygous deletions, but no difference in phenotypic spectrum was observed between 3-Mb and 1.5-Mb deletions. Our data suggest that the characteristic face can be used as a key indicator for direct diagnosis of 22q11.2 deletions in Chinese VCFS patients.

Rates of loss of heterozygosity and mitotic recombination in NF2 schwannomas, sporadic vestibular schwannomas and schwannomatosis schwannomas.

PubMed

Hadfield, K D; Smith, M J; Urquhart, J E; Wallace, A J; Bowers, N L; King, A T; Rutherford, S A; Trump, D; Newman, W G; Evans, D G

2010-11-25

Biallelic inactivation of the NF2 gene occurs in the majority of schwannomas. This usually involves a combination of a point mutation or multiexon deletion, in conjunction with either a second point mutation or loss of heterozygosity (LOH). We have performed DNA sequence and dosage analysis of the NF2 gene in a panel of 239 schwannoma tumours: 97 neurofibromatosis type 2 (NF2)-related schwannomas, 104 sporadic vestibular schwannomas (VS) and 38 schwannomatosis-related schwannomas. In total, we identified germline NF2 mutations in 86 out of 97 (89%) NF2 patients and a second mutational event in 77 out of 97 (79%). LOH was by far the most common form of second hit. A combination of microsatellite analysis with either conventional comparative genomic hybridization (CGH) or multiplex ligation-dependent probe amplification (MLPA) identified mitotic recombination (MR) as the cause of LOH in 14 out of 72 (19%) total evaluable tumours. Among sporadic VS, at least one NF2 mutation was identified by sequence analysis or MLPA in 65 out of 98 (66%) tumours. LOH occurred in 54 out of 96 (56%) evaluable tumours, but MR only accounted for 5 out of 77 (6%) tested. LOH was present in 28 out of 34 (82%) schwannomatosis-related schwannomas. In all eight patients who had previously tested positive for a germline SMARCB1 mutation, this involved loss of the whole, or part of the long arm, of chromosome 22. In contrast, 5 out of 22 (23%) tumours from patients with no germline SMARCB1 mutation exhibited MR. High-resolution Affymetrix SNP6 genotyping and copy number (CN) analysis (Affymetrix, Santa Clara, CA, USA) were used to determine the chromosomal breakpoint locations in tumours with MR. A range of unique recombination sites, spanning approximately 11.4 Mb, were identified. This study shows that MR is a mechanism of LOH in NF2 and SMARCB1-negative schwannomatosis-related schwannomas, occurring less frequently in sporadic VS. We found no evidence of MR in SMARCB1-positive schwannomatosis, suggesting that susceptibility to MR varies according to the disease context.

Meningeal Hemangiopericytomas and Meningomas: a Comparative Immunohistochemical and Genetic Study.

PubMed

Trabelsi, Saoussen; Mama, Nadia; Chourabi, Maroua; Mastouri, Maroua Haddaji; Ladib, Mohamed; Popov, Sergey; Burford, Anna; Mokni, Moncef; Tlili, Kalthoum; Krifa, Hedi; Jones, Chris; Yacoubi, Mohamed Tahar; Saad, Ali; Brahim, Dorra H'mida-Ben

2015-01-01

The meningeal hemangiopericytoma (MHPC) is a vascular tumor arising from pericytes. Most intracranial MHPCs resemble meningiomas (MNGs) in their clinical presentation and histological features and may therefore be misdiagnosed, despite important differences in prognosis. We report 8 cases of MHPC and 5 cases of MNG collected from 2007 to 2011 from the Neuro-Surgery and Histopathology departments. All 13 samples were re reviewed by two independent pathologists and investigated by immunohistochemistry (IHC) using mesenchymal, epithelial and neuro-glial markers. Additionally, we screened all tumors for a large panel of chromosomal alterations using multiplex ligation probe amplification (MLPA). Presence of the NAB2-STAT6 fusion gene was inferred by immunohistochemical staining for STAT6. Compared with MNG, MHPCs showed strong VIM (100% of cases), CD99 (62%), bcl-2 (87%), and p16 (75%) staining but only focal positivity with EMA (33%) and NSE (37%). The p21 antibody was positive in 62% of MHPC and less than 1% in all MNGs. MLPA data did not distinguish HPC from MNG, with PTEN loss and ERBB2 gain found in both. By contrast, STAT6 nuclear staining was observed in 3 MHPC cases and was absent from MNG. MNG and MHPC comprise a spectrum of tumors that cannot be easily differentiated based on histopathology. The presence of STAT6 nuclear positivity may however be a useful diagnostic marker.

Avoidance of pseudogene interference in the detection of 3' deletions in PMS2.

PubMed

Vaughn, Cecily P; Hart, Kimberly J; Samowitz, Wade S; Swensen, Jeffrey J

2011-09-01

Lynch syndrome is characterized by mutations in the mismatch repair genes MLH1, MSH2, MSH6, and PMS2. In PMS2, detection of mutations is confounded by numerous pseudogenes. Detection of 3' deletions is particularly complicated by the pseudogene PMS2CL, which has strong similarity to PMS2 exons 9 and 11-15, due to extensive gene conversion. A newly designed multiplex ligation-dependent probe amplification (MLPA) kit incorporates probes for variants found in both PMS2 and PMS2CL. This provides detection of deletions, but does not allow localization of deletions to the gene or pseudogene. To address this, we have developed a methodology incorporating reference samples with known copy numbers of variants, and paired MLPA results with sequencing of PMS2 and PMS2CL. We tested a subset of clinically indicated samples for which mutations were either unidentified or not fully characterized using existing methods. We identified eight unrelated patients with deletions encompassing exons 9-15, 11-15, 13-15, 14-15, and 15. By incorporating specific, characterized reference samples and sequencing the gene and pseudogene it is possible to identify deletions in this region of PMS2 and provide clinically relevant results. This methodology represents a significant advance in the diagnosis of patients with Lynch syndrome caused by PMS2 mutations. © 2011 Wiley-Liss, Inc.

Methylation profile analysis of DNA repair genes in hepatocellular carcinoma with MS-MLPA.

PubMed

Ozer, Ozge; Bilezikci, Banu; Aktas, Sema; Sahin, Feride I

2013-12-01

Hepatocellular carcinoma (HCC) is one of the rare tumors with well-defined risk factors. The multifactorial etiology of HCC can be explained by its complex molecular pathogenesis. In the current study, the methylation status of 7 genes involved in DNA repair mechanisms, namely MLH1, PMS2, MSH6, MSH2, MGMT, MSH3, and MLH3, was investigated in tumor samples from HCC patients, using the methylation-specific-multiplex ligated probe amplification method and the results were correlated with available clinical findings. The most common etiological factor in these cases was the presence of hepatitis B alone (47.2%). Among the 56 cases that were studied, promoter methylation was detected in at least one of the genes in 27 (48.2%) cases, only in 1 gene in 13 (23.2%) cases, and in >1 gene in 14 (25%) cases. Of the 7 genes investigated, methylation was most frequently observed in MSH3, in 14 (25%) cases. Methylation of at least 1 gene was significantly more frequent in patients with single tumors than multifocal tumors. There were significant differences regarding hepatitis B status, Child Class, tumor number, grade, and TNM stage in cases where PMS2 methylation was detected. Our results suggest that methylation of genes involved in mismatch repair may be responsible in the pathogenesis of HCC, and evaluating changes in multiple genes in these pathways simultaneously would be more informative. Despite being a robust and relatively inexpensive method, the methylation-specific-multiplex ligated probe amplification assay could be more extensively applied with improvements in the currently intricate data analysis component.

PROGNOSTIC SIGNIFICANCE OF CLINICAL, HISTOPATHOLOGICAL, AND MOLECULAR CHARACTERISTICS OF MEDULLOBLASTOMAS IN THE PROSPECTIVE HIT2000 MULTICENTER CLINICAL TRIAL COHORT

PubMed Central

Pietsch, Torsten; Schmidt, Rene; Remke, Marc; Korshunov, Andrey; Hovestadt, Volker; Jones, David TW; Felsberg, Jörg; Kaulich, Kerstin; Goschzik, Tobias; Kool, Marcel; Northcott, Paul A.; von Hoff, Katja; von Bueren, André O.; Friedrich, Carsten; Skladny, Heyko; Fleischhack, Gudrun; Taylor, Michael D.; Cremer, Friedrich; Lichter, Peter; Faldum, Andreas; Reifenberger, Guido; Rutkowski, Stefan; Pfister, Stefan M.

2014-01-01

BACKGROUND: This study aimed to prospectively evaluate clinical, histopathological and molecular variables for outcome prediction in medulloblastoma patients. METHODS: Patients from the HIT2000 cooperative clinical trial were prospectively enrolled based on the availability of sufficient tumor material and complete clinical information. This revealed a cohort of 184 patients (median age 7.6 years), which was randomly split at a 2:1 ratio into a training (n = 127), and a validation (n = 57) dataset. All samples were subjected to thorough histopathological investigation, CTNNB1 mutation analysis, quantitative PCR, MLPA and FISH analyses for cytogenetic variables, and methylome analysis. RESULTS: By univariable analysis, clinical factors (M-stage), histopathological variables (large cell component, endothelial proliferation, synaptophysin pattern), and molecular features (chromosome 6q status, MYC amplification, TOP2A copy-number, subgrouping) were found to be prognostic. Molecular consensus subgrouping (WNT, SHH, Group 3, Group 4) was validated as an independent feature to stratify patients into different risk groups. When comparing methods for the identification of WNT-driven medulloblastoma, this study identified CTNNB1 sequencing and methylation profiling to most reliably identify these patients. After removing patients with particularly favorable (CTNNB1 mutation, extensive nodularity) or unfavorable (MYC amplification) markers, a risk score for the remaining “intermediate molecular risk” population dependent on age, M-stage, pattern of synaptophysin expression, and MYCN copy-number status was identified and validated, with speckled synaptophysin expression indicating worse outcome. CONCLUSIONS: Methylation subgrouping and CTNNB1 mutation status represent robust tools for the risk-stratification of medulloblastoma. A simple clinico-pathological risk score for “intermediate molecular risk” patients was identified, which deserves further validation. SECONDARY CATEGORY: Pediatrics.

Identifying CNVs in 15q11q13 and 16p11.2 of Patients with Seizures Increases the Rates of Detecting Pathogenic Changes

PubMed Central

Vianna, Gabrielle S.; Freitas, Mariana L.; Oliveira, Valdirene T.de; Pietra, Rafaella X.; Gonçalves, Michele da S.; Rocha, Patrícia P.O.; Monteiro, Rejane A.C.; Ferreira, Luana C.A.; Xavier, Rosana R.; Carvalho, Andréia M.; Lima, Patrícia R. de M.; Monteiro, Maria Augusta N.P.; Mateo, Elvis C.; Giannetti, Juliana G.; César, Giovana da C.; Lima, Joziele de S.; Medeiros, Paula F.V.; Jehee, Fernanda S.

2016-01-01

Chromosomal changes are frequently observed in patients with syndromic seizures. Understanding the genetic etiology of this pathology is crucial for the guidance and genetic counseling of families as well as for the establishment of appropriate treatment. A combination of MLPA kits was used to identify pathogenic CNVs in a group of 70 syndromic patients with seizures. Initially, a screening was performed for subtelomeric changes (MLPA P036 and P070 kits) and for the regions most frequently related to microdeletion/microduplication syndromes (MLPA P064). Subsequently, the MLPA P343 was used to identify alterations in the 15q11q13, 16p11.2, and 22q13 regions. Screening with MLPA P343 allowed a 10-15.7% increase in the detection rate of CNVs reinforcing the importance of investigating changes in 15q11q13 and 16p11.2 in syndromic patients with seizures. We also demonstrated that the MLPA technique is an alternative with a great diagnostic potential, and we proposed its use as part of the initial assessment of syndromic patients with seizures. PMID:27920636

A Streamlined Protocol for Molecular Testing of the DMD Gene within a Diagnostic Laboratory: A Combination of Array Comparative Genomic Hybridization and Bidirectional Sequence Analysis

PubMed Central

Marquis-Nicholson, Renate; Lai, Daniel; Love, Jennifer M.; Love, Donald R.

2013-01-01

Purpose. The aim of this study was to develop a streamlined mutation screening protocol for the DMD gene in order to confirm a clinical diagnosis of Duchenne or Becker muscular dystrophy in affected males and to clarify the carrier status of female family members. Methods. Sequence analysis and array comparative genomic hybridization (aCGH) were used to identify mutations in the dystrophin DMD gene. We analysed genomic DNA from six individuals with a range of previously characterised mutations and from eight individuals who had not previously undergone any form of molecular analysis. Results. We successfully identified the known mutations in all six patients. A molecular diagnosis was also made in three of the four patients with a clinical diagnosis who had not undergone prior genetic screening, and testing for familial mutations was successfully completed for the remaining four patients. Conclusion. The mutation screening protocol described here meets best practice guidelines for molecular testing of the DMD gene in a diagnostic laboratory. The aCGH method is a superior alternative to more conventional assays such as multiplex ligation-dependent probe amplification (MLPA). The combination of aCGH and sequence analysis will detect mutations in 98% of patients with the Duchenne or Becker muscular dystrophy. PMID:23476807

Molecular analysis of holoprosencephaly in South America

PubMed Central

Savastano, Clarice Pagani; El-Jaick, Kênia Balbi; Costa-Lima, Marcelo Aguiar; Abath, Cristina Maria Batista; Bianca, Sebastiano; Cavalcanti, Denise Pontes; Félix, Têmis Maria; Scarano, Gioacchino; Llerena, Juan Clinton; Vargas, Fernando Regla; Moreira, Miguel Ângelo Martins; Seuánez, Hector N.; Castilla, Eduardo Enrique; Orioli, Iêda Maria

2014-01-01

Holoprosencephaly (HPE) is a spectrum of brain and facial malformations primarily reflecting genetic factors, such as chromosomal abnormalities and gene mutations. Here, we present a clinical and molecular analysis of 195 probands with HPE or microforms; approximately 72% of the patients were derived from the Latin American Collaborative Study of Congenital Malformations (ECLAMC), and 82% of the patients were newborns. Alobar HPE was the predominant brain defect in almost all facial defect categories, except for patients without oral cleft and median or lateral oral clefts. Ethmocephaly, cebocephaly, and premaxillary agenesis were primarily observed among female patients. Premaxillary agenesis occurred in six of the nine diabetic mothers. Recurrence of HPE or microform was approximately 19%. The frequency of microdeletions, detected using Multiplex Ligation-dependant Probe Amplification (MLPA) was 17% in patients with a normal karyotype. Cytogenetics or QF-PCR analyses revealed chromosomal anomalies in 27% of the probands. Mutational analyses in genes SHH, ZIC2, SIX3 and TGIF were performed in 119 patients, revealing eight mutations in SHH, two mutations in SIX3 and two mutations in ZIC2. Thus, a detailed clinical description of new HPE cases with identified genetic anomalies might establish genotypic and phenotypic correlations and contribute to the development of additional strategies for the analysis of new cases. PMID:24764759

A rapid NGS strategy for comprehensive molecular diagnosis of Birt-Hogg-Dubé syndrome in patients with primary spontaneous pneumothorax.

PubMed

Zhang, Xinxin; Ma, Dehua; Zou, Wei; Ding, Yibing; Zhu, Chengchu; Min, Haiyan; Zhang, Bin; Wang, Wei; Chen, Baofu; Ye, Minhua; Cai, Minghui; Pan, Yanqing; Cao, Lei; Wan, Yueming; Jin, Yu; Gao, Qian; Yi, Long

2016-05-27

Primary spontaneous pneumothorax (PSP) or pulmonary cysts is one of the manifestations of Birt-Hogg-Dube syndrome (BHDS) that is caused by heterozygous mutations in FLCN gene. Most of the mutations are SNVs and small indels, and there are also approximately 10 % large intragenic deletions and duplications of the mutations. These molecular findings are generally obtained by disparate methods including Sanger sequencing and Multiple Ligation-dependent Probe Amplification in the clinical laboratory. In addition, as a genetically heterogeneous disorder, PSP may be caused by mutations in multiple genes include FBN1, COL3A1, CBS, SERPINA1 and TSC1/TSC2 genes. For differential diagnosis, these genes should also be screened which makes the diagnostic procedure more time-consuming and labor-intensive. Forty PSP patients were divided into 2 groups. Nineteen patients with different pathogenic mutations of FLCN previously identified by conventional Sanger sequencing and MLPA were included in test group, 21 random PSP patients without any genetic screening were included in blinded sample group. 7 PSP genes including FLCN, FBN1, COL3A1, CBS, SERPINA1 and TSC1/TSC2 were designed and enriched by Haloplex system, sequenced on a Miseq platform and analyzed in the 40 patients to evaluate the performance of the targeted-NGS method. We demonstrated that the full spectrum of genes associated with pneumothorax including FLCN gene mutations can be identified simultaneously in multiplexed sequence data. Noteworthy, by our in-house copy number analysis of the sequence data, we could not only detect intragenic deletions, but also determine approximate deletion junctions simultaneously. NGS based Haloplex target enrichment technology is proved to be a rapid and cost-effective screening strategy for the comprehensive molecular diagnosis of BHDS in PSP patients, as it can replace Sanger sequencing and MLPA by simultaneously detecting exonic and intronic SNVs, small indels, large intragenic deletions and determining deletion junctions in PSP-related genes.

Molecular analysis of brazilian patients with combined pituitary hormone deficiency and orthotopic posterior pituitary lobe reveals eight different PROP1 alterations with three novel mutations.

PubMed

Madeira, Joao Lo; Nishi, Mirian Y; Nakaguma, Marilena; Benedetti, Anna F; Biscotto, Isabela Peixoto; Fernandes, Thamiris; Pequeno, Thiago; Figueiredo, Thalita; Franca, Marcela M; Correa, Fernanda A; Otto, Aline P; Abrão, Milena; Miras, Mirta B; Santos, Silvana; Jorge, Alexander Al; Costalonga, Everlayny F; Mendonca, Berenice B; Arnhold, Ivo Jp; Carvalho, Luciani R

2017-12-01

Mutations in PROP1, HESX1 and LHX3 are associated with combined pituitary hormone deficiency (CPHD) and orthotopic posterior pituitary lobe (OPP). To identify mutations in PROP1, HESX1 and LHX3 in a large cohort of patients with CPHD and OPP (35 Brazilian, two Argentinian). We studied 23 index patients with CPHD and OPP (six familial and 17 sporadic) as well as 14 relatives. PROP1 was sequenced by the Sanger method in all except one sporadic case studied using a candidate gene panel. Multiplex ligation-dependent probe amplification (MLPA) was applied to one familial case in whom PROP1 failed to amplify by PCR. In the 13 patients without PROP1 mutations, HESX1 and LHX3 were sequenced by the Sanger method. We identified PROP1 mutations in 10 index cases. Three mutations were novel: one affecting the initiation codon (c.1A>G) and two affecting splicing sites, c.109+1G>A and c.342+1G>C. The known mutations, c.150delA (p.Arg53Aspfs*112), c.218G>A (p.Arg73His), c.263T>C (p.Phe88Ser) and c.301_302delAG (p.Leu102Cysfs*8), were also detected. MLPA confirmed complete PROP1 deletion in one family. We did not identify HESX1 and LHX3 mutations by Sanger. PROP1 mutations are a prevalent cause of congenital CPHD with OPP, and therefore, PROP1 sequencing must be the first step of molecular investigation in patients with CPHD and OPP, especially in populations with a high frequency of PROP1 mutations. In the absence of mutations, massively parallel sequencing is a promising approach. The high prevalence and diversity of PROP1 mutations is associated with the ethnic background of this cohort. © 2017 John Wiley & Sons Ltd.

Screening and Familial Characterization of Copy-Number Variations in NR5A1 in 46,XY Disorders of Sex Development and Premature Ovarian Failure

PubMed Central

Harrison, Steven M.; Campbell, Ian M.; Keays, Melise; Granberg, Candace F.; Villanueva, Carlos; Tannin, Grace; Zinn, Andrew R.; Castrillon, Diego H.; Shaw, Chad A.; Stankiewicz, Paweł; Baker, Linda A.

2013-01-01

The NR5A1 gene encodes for steroidogenic factor 1, a nuclear receptor that regulates proper adrenal and gonadal development and function. Mutations identified by NR5A1 sequencing have been associated with disorders of sex development (DSD), ranging from sex reversal to severe hypospadias in 46,XY patients and premature ovarian failure (POF) in 46,XX patients. Previous reports have identified four families with a history of both 46,XY DSD and 46,XX POF carrying segregating NR5A1 sequence mutations. Recently, three 46,XY DSD sporadic cases with NR5A1 microdeletions have been reported. Here, we identify the first NR5A1 microdeletion transmitted in a pedigree with both 46,XY DSD and 46,XX POF. A 46,XY individual with DSD due to gonadal dysgenesis was born to a young mother who developed POF. Array CGH analysis revealed a maternally inherited 0.23 Mb microdeletion of chromosome 9q33.3, including the NR5A1 gene. Based on this finding, we screened patients with unexplained 46,XY DSD (n=11), proximal hypospadias (n=21) and 46,XX POF (n=36) for possible NR5A1 copy-number variations (CNVs) via multiplex ligation-dependent probe amplification (MLPA), but did not identify any additional CNVs involving NR5A1. These data suggest that NR5A1 CNVs are an infrequent cause of these disorders but that array CGH and MLPA are useful genomic screening tools to uncover the genetic basis of such unexplained cases. This case is the first report of a familial NR5A1 CNV transmitting in a pedigree, causing both the male and female phenotypes associated with NR5A1 mutations, and the first report of a NR5A1 CNV associated with POF. PMID:23918653

Association of an α-globin gene cluster duplication and heterozygous β-thalassemia in a patient with a severe thalassemia syndrome.

PubMed

Jiang, Hua; Liu, Sha; Zhang, Yong-Ling; Wan, Jun-Hui; Li, Ru; Li, Dong-Zhi

2015-01-01

We describe a new case of a β-thalassemia (β-thal) heterozygote with the mutation IVS-II-654 (C>T) presenting with a transfusion-dependent phenotype. Multiplex ligation-dependent probe amplification (MLPA) and array comparative genomic hybridization (CGH) analyses of the α-globin gene cluster revealed a full duplication of the α-globin genes including the upstream regulatory element. The duplicated allele and the normal allele in trans resulted in a total of six active α-globin genes. The severe clinical phenotype seemed to be related to the considerable excess of the α- and β-globin deficit caused by the presence of the β-thal. α-Globin cluster duplication should be considered in patients heterozygous for β-thal who show a more severe phenotype than β-thal trait.

CYP1B1 copy number variation is not a major contributor to primary congenital glaucoma.

PubMed

Souzeau, Emmanuelle; Hayes, Melanie; Ruddle, Jonathan B; Elder, James E; Staffieri, Sandra E; Kearns, Lisa S; Mackey, David A; Zhou, Tiger; Ridge, Bronwyn; Burdon, Kathryn P; Dubowsky, Andrew; Craig, Jamie E

2015-01-01

To evaluate the prevalence and the diagnostic utility of testing for CYP1B1 copy number variation (CNV) in primary congenital glaucoma (PCG) cases unexplained by CYP1B1 point mutations in The Australian and New Zealand Registry of Advanced Glaucoma. In total, 50 PCG cases either heterozygous for disease-causing variants or with no CYP1B1 sequence variants were included in the study. CYP1B1 CNV was analyzed by Multiplex Ligation-dependent Probe Amplification (MLPA). No deletions or duplications were found in any of the cases. This is the first study to report on CYP1B1 CNV in PCG cases. Our findings show that this mechanism is not a major contributor to the phenotype and is of limited diagnostic utility.

Contribution of Rare Copy Number Variants to Isolated Human Malformations

PubMed Central

Serra-Juhé, Clara; Rodríguez-Santiago, Benjamín; Cuscó, Ivon; Vendrell, Teresa; Camats, Núria; Torán, Núria; Pérez-Jurado, Luis A.

2012-01-01

Background Congenital malformations are present in approximately 2–3% of liveborn babies and 20% of stillborn fetuses. The mechanisms underlying the majority of sporadic and isolated congenital malformations are poorly understood, although it is hypothesized that the accumulation of rare genetic, genomic and epigenetic variants converge to deregulate developmental networks. Methodology/Principal Findings We selected samples from 95 fetuses with congenital malformations not ascribed to a specific syndrome (68 with isolated malformations, 27 with multiple malformations). Karyotyping and Multiplex Ligation-dependent Probe Amplification (MLPA) discarded recurrent genomic and cytogenetic rearrangements. DNA extracted from the affected tissue (46%) or from lung or liver (54%) was analyzed by molecular karyotyping. Validations and inheritance were obtained by MLPA. We identified 22 rare copy number variants (CNV) [>100 kb, either absent (n = 7) or very uncommon (n = 15, <1/2,000) in the control population] in 20/95 fetuses with congenital malformations (21%), including 11 deletions and 11 duplications. One of the 9 tested rearrangements was de novo while the remaining were inherited from a healthy parent. The highest frequency was observed in fetuses with heart hypoplasia (8/17, 62.5%), with two events previously related with the phenotype. Double events hitting candidate genes were detected in two samples with brain malformations. Globally, the burden of deletions was significantly higher in fetuses with malformations compared to controls. Conclusions/Significance Our data reveal a significant contribution of rare deletion-type CNV, mostly inherited but also de novo, to human congenital malformations, especially heart hypoplasia, and reinforce the hypothesis of a multifactorial etiology in most cases. PMID:23056206

TIMP3 Promoter Methylation Represents an Epigenetic Marker of BRCA1ness Breast Cancer Tumours.

PubMed

Maleva Kostovska, Ivana; Jakimovska, Milena; Popovska-Jankovic, Katerina; Kubelka-Sabit, Katerina; Karagjozov, Mitko; Plaseska-Karanfilska, Dijana

2018-03-09

Tumours presenting BRCAness profile behave more aggressively and are more invasive as a consequence of their complex genetic and epigenetic alterations, caused by impaired fidelity of the DNA repair processes. Methylation of promoter CpG islands represents an alternative mechanism to inactivate DNA repair and tumour suppressor genes. In our study, we analyzed the frequency of methylation changes of 24 tumour suppressor genes and explored their association with BRCAness profile. BRCA1ness profile and aberrant methylation were studied in 233 fresh frozen breast tumour tissues by Multiplex Ligation-dependent Probe Amplification (MLPA) and Methylation Specific (MS)-MLPA methods, respectively. Our analyses revealed that 12.4% of the breast cancer (BC) patients had tumours with a BRCA1ness profile. TIMP3 showed significantly higher (p = 5.8х10 -5 ) methylation frequency in tumours with BRCA1ness, while methylation of APC, GSTP1 and RASSF1 promoters was negatively associated with BRCA1ness (р = 0.0017, р = 0.007 and р = 0.046, respectively). TIMP3 methylation was also associated with triple negative (TN) BC. Furthermore, TN tumours showing BRCA1ness showed stronger association with TIMP3 methylation (p = 0.0008) in comparison to TN tumours without BRCA1ness (p = 0.009). In conclusion, we confirmed that TIMP3 methylation is a marker for TN tumours and furthermore we showed for the first time that TIMP3 promoter methylation is an epigenetic marker of BRCA1ness tumours.

Recognizing the tenascin-X deficient type of Ehlers-Danlos syndrome: a cross-sectional study in 17 patients.

PubMed

Demirdas, S; Dulfer, E; Robert, L; Kempers, M; van Beek, D; Micha, D; van Engelen, B G; Hamel, B; Schalkwijk, J; Loeys, B; Maugeri, A; Voermans, N C

2017-03-01

The tenascin-X (TNX) deficient type Ehlers-Danlos syndrome (EDS) is similar to the classical type of EDS. Because of the limited awareness among geneticists and the challenge of the molecular analysis of the TNXB gene, the TNX-deficient type EDS is probably to be under diagnosed. We therefore performed an observational, cross-sectional study. History and physical examination were performed. Results of serum TNX measurements were collected and mutation analysis was performed by a combination of next-generation sequencing (NGS), Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Included were 17 patients of 11 families with autosomal recessive inheritance and childhood onset. All patients had hyperextensible skin without atrophic scarring. Hypermobility of the joints was observed in 16 of 17 patients. Deformities of the hands and feet were observed frequently. TNX serum level was tested and absent in 11 patients (seven families). Genetic testing was performed in all families; 12 different mutations were detected, most of which are suspected to lead to non-sense mRNA mediated decay. In short, patients with the TNX-deficient type EDS typically have generalized joint hypermobility, skin hyperextensibility and easy bruising. In contrast to the classical type, the inheritance pattern is autosomal recessive and atrophic scarring is absent. Molecular analysis of TNXB in a diagnostic setting is challenging. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Novel deletions involving the USH2A gene in patients with Usher syndrome and retinitis pigmentosa.

PubMed

García-García, Gema; Aller, Elena; Jaijo, Teresa; Aparisi, Maria J; Larrieu, Lise; Faugère, Valérie; Blanco-Kelly, Fiona; Ayuso, Carmen; Roux, Anne-Francoise; Millán, José M

2014-01-01

The aim of the present work was to identify and characterize large rearrangements involving the USH2A gene in patients with Usher syndrome and nonsyndromic retinitis pigmentosa. The multiplex ligation-dependent probe amplification (MLPA) technique combined with a customized array-based comparative genomic hybridization (aCGH) analysis was applied to 40 unrelated patients previously screened for point mutations in the USH2A gene in which none or only one pathologic mutation was identified. We detected six large deletions involving USH2A in six out of the 40 cases studied. Three of the patients were homozygous for the deletion, and the remaining three were compound heterozygous with a previously identified USH2A point mutation. In five of these cases, the patients displayed Usher type 2, and the remaining case displayed nonsyndromic retinitis pigmentosa. The exact breakpoint junctions of the deletions found in USH2A in four of these cases were characterized. Our study highlights the need to develop improved efficient strategies of mutation screening based upon next generation sequencing (NGS) that reduce cost, time, and complexity and allow simultaneous identification of all types of disease-causing mutations in diagnostic procedures.

Genomic imbalances in syndromic congenital heart disease.

PubMed

Molck, Miriam Coelho; Simioni, Milena; Paiva Vieira, Társis; Sgardioli, Ilária Cristina; Paoli Monteiro, Fabíola; Souza, Josiane; Fett-Conte, Agnes Cristina; Félix, Têmis Maria; Lopes Monlléo, Isabella; Gil-da-Silva-Lopes, Vera Lúcia

To identify pathogenic genomic imbalances in patients presenting congenital heart disease (CHD) with extra cardiac anomalies and exclusion of 22q11.2 deletion syndrome (22q11.2 DS). 78 patients negative for the 22q11.2 deletion, previously screened by fluorescence in situ hybridization (FISH) and/or multiplex ligation probe amplification (MLPA) were tested by chromosomal microarray analysis (CMA). Clinically significant copy number variations (CNVs ≥300kb) were identified in 10% (8/78) of cases. In addition, potentially relevant CNVs were detected in two cases (993kb duplication in 15q21.1 and 706kb duplication in 2p22.3). Genes inside the CNV regions found in this study, such as IRX4, BMPR1A, SORBS2, ID2, ROCK2, E2F6, GATA4, SOX7, SEMAD6D, FBN1, and LTPB1 are known to participate in cardiac development and could be candidate genes for CHD. These data showed that patients presenting CHD with extra cardiac anomalies and exclusion of 22q11.2 DS should be investigated by CMA. The present study emphasizes the possible role of CNVs in CHD. Copyright © 2017 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

APC promoter 1B deletion in seven American families with familial adenomatous polyposis.

PubMed

Snow, A K; Tuohy, T M F; Sargent, N R; Smith, L J; Burt, R W; Neklason, D W

2015-10-01

Familial adenomatous polyposis (FAP) is a colorectal cancer predisposition syndrome caused by mutations in the adenomatous polyposis coli (APC) gene. Clinical genetic testing fails to identify disease causing mutations in up to 20% of clinically apparent FAP cases. Following the inclusion of multiplex ligation-dependent probe amplification (MLPA) probes specific for APC promoter 1B, seven probands were identified with a deletion of promoter 1B. Using haplotype analysis spanning the APC locus, the seven families appear to be identical by descent from a common founder. The clinical phenotype of 19 mutation carriers is classical FAP with colectomy at an average age of 24. The majority of cases had a large number of duodenal and gastric polyps. Measurements of allele-specific expression of APC mRNA using TaqMan assay confirmed that relative expression in the allele containing the promoter 1B deletion was reduced 42-98%, depending on tissue type. This study confirms the importance of APC promoter deletions as a cause of FAP and identifies a founder mutation in FAP patients from the United States. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

Investigation of FANCA gene in Fanconi anaemia patients in Iran

PubMed Central

Saffar Moghadam, Ali Akbar; Mahjoubi, Frouzandeh; Reisi, Nahid; Vosough, Parvaneh

2016-01-01

Background & objectives: Fanconi anaemia (FA) is a syndrome with a predisposition to bone marrow failure, congenital anomalies and malignancies. It is characterized by cellular hypersensitivity to cross-linking agents such as mitomycin C (MMC). In the present study, a new approach was selected to investigate FANCA (Fanconi anaemia complementation group A) gene in patients clinically diagnosed with cellular hypersensitivity to DNA cross-linking agent MMC. Methods: Chromosomal breakage analysis was performed to prove the diagnosis of Fanconi anaemia in 318 families. Of these, 70 families had a positive result. Forty families agreed to molecular genetic testing. In total, there were 27 patients with unknown complementary types. Genomic DNA was extracted and total RNA was isolated from fresh whole blood of the patients. The first-strand cDNA was synthesized and the cDNA of each patient was then tested with 21 pairs of overlapping primers. High resolution melting curve analysis was used to screen FANCA, and LinReg software version 1.7 was utilized for analysis of expression. Results: In total, six sequence alterations were identified, which included two stop codons, two frames-shift mutations, one large deletion and one amino acid exchange. FANCA expression was downregulated in patients who had sequence alterations. Interpretation & conclusions: The results of the present study show that high resolution melting (HRM) curve analysis may be useful in the detection of sequence alteration. It is simpler and more costeffective than the multiplex ligation-dependent probe amplification (MLPA) procedure. PMID:27121516

Investigation of FANCA gene in Fanconi anaemia patients in Iran.

PubMed

Moghadam, Ali Akbar Saffar; Mahjoubi, Frouzandeh; Reisi, Nahid; Vosough, Parvaneh

2016-02-01

Fanconi anaemia (FA) is a syndrome with a predisposition to bone marrow failure, congenital anomalies and malignancies. It is characterized by cellular hypersensitivity to cross-linking agents such as mitomycin C (MMC). In the present study, a new approach was selected to investigate FANCA (Fanconi anaemia complementation group A) gene in patients clinically diagnosed with cellular hypersensitivity to DNA cross-linking agent MMC. Chromosomal breakage analysis was performed to prove the diagnosis of Fanconi anaemia in 318 families. Of these, 70 families had a positive result. Forty families agreed to molecular genetic testing. In total, there were 27 patients with unknown complementary types. Genomic DNA was extracted and total RNA was isolated from fresh whole blood of the patients. The first-strand cDNA was synthesized and the cDNA of each patient was then tested with 21 pairs of overlapping primers. High resolution melting curve analysis was used to screen FANCA, and LinReg software version 1.7 was utilized for analysis of expression. In total, six sequence alterations were identified, which included two stop codons, two frames-shift mutations, one large deletion and one amino acid exchange. FANCA expression was downregulated in patients who had sequence alterations. The results of the present study show that high resolution melting (HRM) curve analysis may be useful in the detection of sequence alteration. It is simpler and more cost-effective than the multiplex ligation-dependent probe amplification (MLPA) procedure.

Universal multiplex PCR and CE for quantification of SMN1/SMN2 genes in spinal muscular atrophy.

PubMed

Wang, Chun-Chi; Chang, Jan-Gowth; Jong, Yuh-Jyh; Wu, Shou-Mei

2009-04-01

We established a universal multiplex PCR and CE to calculate the copy number of survival motor neuron (SMN1 and SMN2) genes for clinical screening of spinal muscular atrophy (SMA). In this study, one universal fluorescent primer was designed and applied for multiplex PCR of SMN1, SMN2 and two internal standards (CYBB and KRIT1). These amplicons were separated by conformation sensitive CE. Mixture of hydroxyethyl cellulose and hydroxypropyl cellulose were used in this CE system. Our method provided the potential to separate two 390-bp PCR products that differ in a single nucleotide. Differentiation and quantification of SMN1 and SMN2 are essential for clinical screening of SMA patients and carriers. The DNA samples included 22 SMA patients, 45 parents of SMA patients (obligatory carriers) and 217 controls. For evaluating accuracy, those 284 samples were blind-analyzed by this method and denaturing high pressure liquid chromatography (DHPLC). Eight of the total samples showed different results. Among them, two samples were diagnosed as having only SMN2 gene by DHPLC, however, they contained both SMN1 and SMN2 by our method. They were further confirmed by DNA sequencing. Our method showed good agreement with the DNA sequencing. The multiplex ligation-dependent probe amplification (MLPA) was used for confirming the other five samples, and showed the same results with our CE method. For only one sample, our CE showed different results with MLPA and DNA sequencing. One out of 284 samples (0.35%) belonged to mismatching. Our method provided a better accurate method and convenient method for clinical genotyping of SMA disease.

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.

Prenatal diagnosis for a Chinese family with a de novo DMD gene mutation

PubMed Central

Li, Tao; Zhang, Zhao-jing; Ma, Xin; Lv, Xue; Xiao, Hai; Guo, Qian-nan; Liu, Hong-yan; Wang, Hong-dan; Wu, Dong; Lou, Gui-yu; Wang, Xin; Zhang, Chao-yang; Liao, Shi-xiu

2017-01-01

Abstract Background: Patients with Duchenne muscular dystrophy (DMD) usually have severe and fatal symptoms. At present, there is no effective treatment for DMD, thus it is very important to avoid the birth of children with DMD by effective prenatal diagnosis. We identified a de novo DMD gene mutation in a Chinese family, and make a prenatal diagnosis. Methods: First, multiplex ligation-dependent probe amplification (MLPA) was applied to analyze DMD gene exon deletion/duplication in all family members. The coding sequences of 79 exons in DMD gene were analyzed by Sanger sequencing in the patient; and then according to DMD gene exon mutation in the patient, DMD gene sequencing was performed in the family members. On the basis of results above, the pathogenic mutation in DMD gene was identified. Results: MLPA showed no DMD gene exon deletion/duplication in all family members. Sanger sequencing revealed c.2767_2767delT [p.Ser923LeufsX26] mutation in DMD gene of the patient. Heterozygous deletion mutation (T/-) at this locus was observed in the pregnant woman and her mother and younger sister. The analyses of amniotic fluid samples indicated negative Y chromosome sex-determining gene, no DMD gene exon deletion/duplication, no mutations at c.2767 locus, and the inherited maternal X chromosome different from that of the patient. Conclusion: The pathogenic mutation in DMD gene, c.2767_2767delT [p.Ser923LeufsX26], identified in this family is a de novo mutation. On the basis of specific conditions, it is necessary to select suitable methods to make prenatal diagnosis more effective, accurate, and economic. PMID:29390271

Presymptomatic Diagnosis of Spinal Muscular Atrophy Through Newborn Screening.

PubMed

Chien, Yin-Hsiu; Chiang, Shu-Chuan; Weng, Wen-Chin; Lee, Ni-Chung; Lin, Ching-Jie; Hsieh, Wu-Shiun; Lee, Wang-Tso; Jong, Yuh-Jyh; Ko, Tsang-Ming; Hwu, Wuh-Liang

2017-11-01

To demonstrate the feasibility of presymptomatic diagnosis of spinal muscular atrophy (SMA) through newborn screening (NBS). We performed a screening trial to assess all newborns who underwent routine newborn metabolic screening at the National Taiwan University Hospital newborn screening center between November 2014 and September 2016. A real-time polymerase chain reaction (RT-PCR) genotyping assay for the SMN1/SMN2 intron 7 c.888+100A/G polymorphism was performed to detect homozygous SMN1 deletion using dried blood spot (DBS) samples. Then the exon 7 c.840C>T mutation and SMN2 copy number were determined by both droplet digital PCR (ddPCR) using the original screening DBS and multiplex ligation-dependent probe amplification (MLPA) using a whole blood sample. Of the 120 267 newborns, 15 tested positive according to the RT-PCR assay. The DBS ddPCR assay excluded 8 false-positives, and the other 7 patients were confirmed by the MLPA assay. Inclusion of the second-tier DBS ddPCR screening assay resulted in a positive prediction value of 100%. The incidence of SMA was 1 in 17 181 (95% CI, 1 in 8323 to 1 in 35 468). Two of the 3 patients with 2 copies of SMN2 and all 4 patients with 3 or 4 copies of SMN2 were asymptomatic at the time of diagnosis. Five of the 8 false-positives were caused by intragenic recombination between SMN1 and SMN2. Newborn screening can detect patients affected by SMA before symptom onset and enable early therapeutic intervention. A combination of a RT-PCR and a second-tier ddPCR can accurately diagnose SMA from DBS samples with no false-positives. ClinicalTrials.gov NCT02123186. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of the first recurrent PAR1 deletion in Léri-Weill dyschondrosteosis and idiopathic short stature reveals the presence of a novel SHOX enhancer.

PubMed

Benito-Sanz, Sara; Royo, Jose Luis; Barroso, Eva; Paumard-Hernández, Beatriz; Barreda-Bonis, Ana C; Liu, Pengfei; Gracía, Ricardo; Lupski, James R; Campos-Barros, Ángel; Gómez-Skarmeta, José Luis; Heath, Karen Elise

2012-07-01

SHOX, located in the pseudoautosomal region 1 (PAR1) of the sexual chromosomes, encodes a transcription factor implicated in human growth. Defects in SHOX or its enhancers have been observed in ∼60% of Leri-Weill dyschondrosteosis (LWD) patients, a skeletal dysplasia characterised by short stature and/or the characteristic Madelung deformity, and in 2-5% of idiopathic short stature (ISS). To identify the molecular defect in the remaining genetically undiagnosed LWD and ISS patients, this study screened previously unanalysed PAR1 regions in 124 LWD and 576 ISS probands. PAR1 screening was undertaken by multiplex ligation dependent probe amplification (MLPA). Copy number alterations were subsequently confirmed and delimited by locus-specific custom-designed MLPA, array comparative genomic hybridisation (CGH) and breakpoint junction PCR/sequencing. A recurrent PAR1 deletion downstream of SHOX spanning 47543 bp with identical breakpoints was identified in 19 LWD (15.3%) and 11 ISS (1.9%) probands, from 30 unrelated families. Eight evolutionarily conserved regions (ECRs 1-8) identified within the deleted sequence were evaluated for SHOX regulatory activity by means of chromosome conformation capture (3C) in chicken embryo limbs and luciferase reporter assays in human U2OS osteosarcoma cells. The 3C assay indicated potential SHOX regulatory activity by ECR1, which was subsequently confirmed to act as a SHOX enhancer, operating in an orientation and position independent manner, in human U2OS cells. This study has identified the first recurrent PAR1 deletion in LWD and ISS, which results in the loss of a previously uncharacterised SHOX enhancer. The loss of this enhancer may decrease SHOX transcription, resulting in LWD or ISS due to SHOX haploinsufficiency.

New multiplex real-time PCR approach to detect gene mutations for spinal muscular atrophy.

PubMed

Liu, Zhidai; Zhang, Penghui; He, Xiaoyan; Liu, Shan; Tang, Shi; Zhang, Rong; Wang, Xinbin; Tan, Junjie; Peng, Bin; Jiang, Li; Hong, Siqi; Zou, Lin

2016-08-17

Spinal muscular atrophy (SMA) is the most common autosomal recessive disease in children, and the diagnosis is complicated and difficult, especially at early stage. Early diagnosis of SMA is able to improve the outcome of SMA patients. In our study, Real-time PCR was developed to measure the gene mutation or deletion of key genes for SMA and to further analyse genotype-phenotype correlation. The multiple real-time PCR for detecting the mutations of survival of motor neuron (SMN), apoptosis inhibitory protein (NAIP) and general transcription factor IIH, polypeptide 2 gene (GTF2H2) was established and confirmed by DNA sequencing and multiplex ligation-dependent probe amplification (MLPA). The diagnosis and prognosis of 141 hospitalized children, 100 normal children and further 2000 cases of dry blood spot (DBS) samples were analysed by this multiple real-time PCR. The multiple real-time PCR was established and the accuracy of it to detect the mutations of SMN, NAIP and GTF2H2 was at least 98.8 % comparing with DNA sequencing and MLPA. Among 141 limb movement disorders children, 75 cases were SMA. 71 cases of SMA (94.67 %) were with SMN c.840 mutation, 9 cases (12 %) with NAIP deletion and 3 cases (4 %) with GTF2H2 deletion. The multiple real-time PCR was able to diagnose and predict the prognosis of SMA patients. Simultaneously, the real-time PCR was applied to detect trace DNA from DBS and able to make an early diagnosis of SMA. The clinical and molecular characteristics of SMA in Southwest of China were presented. Our work provides a novel way for detecting SMA in children by using real-time PCR and the potential usage in newborn screening for early diagnosis of SMA.

Unique and atypical deletions in Prader–Willi syndrome reveal distinct phenotypes

PubMed Central

Kim, Soo-Jeong; Miller, Jennifer L; Kuipers, Paul J; German, Jennifer Ruth; Beaudet, Arthur L; Sahoo, Trilochan; Driscoll, Daniel J

2012-01-01

Prader–Willi syndrome (PWS) is a multisystem, contiguous gene disorder caused by an absence of paternally expressed genes within the 15q11.2-q13 region via one of the three main genetic mechanisms: deletion of the paternally inherited 15q11.2-q13 region, maternal uniparental disomy and imprinting defect. The deletion class is typically subdivided into Type 1 and Type 2 based on their proximal breakpoints (BP1–BP3 and BP2–BP3, respectively). Despite PWS being a well-characterized genetic disorder the role of the specific genes contributing to various aspects of the phenotype are not well understood. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) is a recently developed technique that detects copy number changes and aberrant DNA methylation. In this study, we initially applied MS-MLPA to elucidate the deletion subtypes of 88 subjects. In our cohort, 32 had a Type 1 and 49 had a Type 2 deletion. The remaining seven subjects had unique or atypical deletions that were either smaller (n=5) or larger (n=2) than typically described and were further characterized by array-based comparative genome hybridization. In two subjects both the PWS region (15q11.2) and the newly described 15q13.3 microdeletion syndrome region were deleted. The subjects with a unique or an atypical deletion revealed distinct phenotypic features. In conclusion, unique or atypical deletions were found in ∼8% of the deletion subjects with PWS in our cohort. These novel deletions provide further insight into the potential role of several of the genes within the 15q11.2 and the 15q13.3 regions. PMID:22045295

Unique and atypical deletions in Prader-Willi syndrome reveal distinct phenotypes.

PubMed

Kim, Soo-Jeong; Miller, Jennifer L; Kuipers, Paul J; German, Jennifer Ruth; Beaudet, Arthur L; Sahoo, Trilochan; Driscoll, Daniel J

2012-03-01

Prader-Willi syndrome (PWS) is a multisystem, contiguous gene disorder caused by an absence of paternally expressed genes within the 15q11.2-q13 region via one of the three main genetic mechanisms: deletion of the paternally inherited 15q11.2-q13 region, maternal uniparental disomy and imprinting defect. The deletion class is typically subdivided into Type 1 and Type 2 based on their proximal breakpoints (BP1-BP3 and BP2-BP3, respectively). Despite PWS being a well-characterized genetic disorder the role of the specific genes contributing to various aspects of the phenotype are not well understood. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) is a recently developed technique that detects copy number changes and aberrant DNA methylation. In this study, we initially applied MS-MLPA to elucidate the deletion subtypes of 88 subjects. In our cohort, 32 had a Type 1 and 49 had a Type 2 deletion. The remaining seven subjects had unique or atypical deletions that were either smaller (n=5) or larger (n=2) than typically described and were further characterized by array-based comparative genome hybridization. In two subjects both the PWS region (15q11.2) and the newly described 15q13.3 microdeletion syndrome region were deleted. The subjects with a unique or an atypical deletion revealed distinct phenotypic features. In conclusion, unique or atypical deletions were found in ∼8% of the deletion subjects with PWS in our cohort. These novel deletions provide further insight into the potential role of several of the genes within the 15q11.2 and the 15q13.3 regions.

Accurate clinical detection of exon copy number variants in a targeted NGS panel using DECoN.

PubMed

Fowler, Anna; Mahamdallie, Shazia; Ruark, Elise; Seal, Sheila; Ramsay, Emma; Clarke, Matthew; Uddin, Imran; Wylie, Harriet; Strydom, Ann; Lunter, Gerton; Rahman, Nazneen

2016-11-25

Background: Targeted next generation sequencing (NGS) panels are increasingly being used in clinical genomics to increase capacity, throughput and affordability of gene testing. Identifying whole exon deletions or duplications (termed exon copy number variants, 'exon CNVs') in exon-targeted NGS panels has proved challenging, particularly for single exon CNVs.  Methods: We developed a tool for the Detection of Exon Copy Number variants (DECoN), which is optimised for analysis of exon-targeted NGS panels in the clinical setting. We evaluated DECoN performance using 96 samples with independently validated exon CNV data. We performed simulations to evaluate DECoN detection performance of single exon CNVs and to evaluate performance using different coverage levels and sample numbers. Finally, we implemented DECoN in a clinical laboratory that tests BRCA1 and BRCA2 with the TruSight Cancer Panel (TSCP). We used DECoN to analyse 1,919 samples, validating exon CNV detections by multiplex ligation-dependent probe amplification (MLPA).  Results: In the evaluation set, DECoN achieved 100% sensitivity and 99% specificity for BRCA exon CNVs, including identification of 8 single exon CNVs. DECoN also identified 14/15 exon CNVs in 8 other genes. Simulations of all possible BRCA single exon CNVs gave a mean sensitivity of 98% for deletions and 95% for duplications. DECoN performance remained excellent with different levels of coverage and sample numbers; sensitivity and specificity was >98% with the typical NGS run parameters. In the clinical pipeline, DECoN automatically analyses pools of 48 samples at a time, taking 24 minutes per pool, on average. DECoN detected 24 BRCA exon CNVs, of which 23 were confirmed by MLPA, giving a false discovery rate of 4%. Specificity was 99.7%.  Conclusions: DECoN is a fast, accurate, exon CNV detection tool readily implementable in research and clinical NGS pipelines. It has high sensitivity and specificity and acceptable false discovery rate. DECoN is freely available at www.icr.ac.uk/decon.

Update on Novel CCM Gene Mutations in Patients with Cerebral Cavernous Malformations.

PubMed

Scimone, Concetta; Bramanti, Placido; Alafaci, Concetta; Granata, Francesca; Piva, Francesco; Rinaldi, Carmela; Donato, Luigi; Greco, Federica; Sidoti, Antonina; D'Angelo, Rosalia

2017-02-01

Cerebral cavernous malformations (CCMs) are lesions affecting brain microvessels. The pathogenesis is not clearly understood. Conventional classification criterion is based on genetics, and thus, familial and sporadic forms can be distinguished; however, classification of sporadic cases with multiple lesions still remains uncertain. To date, three CCM causative genes have been identified: CCM1/KRIT1, CCM2/MGC4607 and CCM3/PDCD10. In our previous mutation screening, performed in a cohort of 95 Italian patients, with both sporadic and familial cases, we identified several mutations in CCM genes. This study represents further molecular screening in a cohort of 19 Italian patients enrolled by us in the few last years and classified into familial, sporadic and sporadic with multiple lesions cases. Direct sequencing and multiplex ligation-dependent probe amplification (MLPA) analysis were performed to detect point mutations and large genomic rearrangements, respectively. Effects of detected mutations and single-nucleotide polymorphisms (SNPs) were evaluated by an in silico approach and by western blot analysis. A novel nonsense mutation in CCM1 and a novel missense mutation in CCM2 were detected; moreover, several CCM2 gene polymorphisms in sporadic CCM patients were reported. We believe that these data enrich the mutation spectrum of CCM genes, which is useful for genetic counselling to identify both familial and sporadic CCM cases, as early as possible.

Nationwide genetic analysis for molecularly unresolved cystic fibrosis patients in a multiethnic society: implications for preconception carrier screening.

PubMed

Behar, Doron M; Inbar, Ori; Shteinberg, Michal; Gur, Michal; Mussaffi, Huda; Shoseyov, David; Ashkenazi, Moshe; Alkrinawi, Soliman; Bormans, Concetta; Hakim, Fahed; Mei-Zahav, Meir; Cohen-Cymberknoh, Malena; Dagan, Adi; Prais, Dario; Sarouk, Ifat; Stafler, Patrick; Bar Aluma, Bat El; Akler, Gidon; Picard, Elie; Aviram, Micha; Efrati, Ori; Livnat, Galit; Rivlin, Joseph; Bentur, Lea; Blau, Hannah; Kerem, Eitan; Singer, Amihood

2017-05-01

Preconception carrier screening for cystic fibrosis (CF) is usually performed using ethnically targeted panels of selected mutations. This has been recently challenged by the use of expanded, ethnically indifferent, pan-population panels. Israel is characterized by genetically heterogeneous populations carrying a wide range of CFTR mutations. To assess the potential of expanding the current Israeli preconception screening program, we sought the subset of molecularly unresolved CF patients listed in the Israeli CF data registry comprising ~650 patients. An Israeli nationwide genotyping of 152 CF cases, representing 176 patients lacking molecular diagnosis, was conducted. Molecular analysis included Sanger sequencing for all exons and splice sites, multiplex ligation probe amplification (MLPA), and next-generation sequencing of the poly-T/TG tracts. We identified 54 different mutations, of which only 16 overlapped the 22 mutations included in the Israeli preconception screening program. A total of 29/54 (53.7%) mutations were already listed as CF causing by the CFTR2 database, and only 4/54 (7.4%) were novel. Molecular diagnosis was reached in 78/152 (51.3%) cases. Prenatal diagnosis of 24/78 (30.8%) cases could have been achieved by including all CFTR2-causing mutations in the Israeli panel. Our data reveal an overwhelming hidden abundance of CFTR gene mutations suggesting that expanded preconception carrier screening might achieve higher preconception detection rates.

Multiplexed methylation profiles of tumor suppressor genes and clinical outcome in oligodendroglial tumors.

PubMed

Kuo, Lu-Ting; Lu, Hsueh-Yi; Lee, Chien-Chang; Tsai, Jui-Chang; Lai, Hong-Shiee; Tseng, Ham-Min; Kuo, Meng-Fai; Tu, Yong-Kwang

2016-08-01

Aberrant methylation has been associated with transcriptional inactivation of tumor-related genes in a wide spectrum of human neoplasms. The influence of DNA methylation in oligodendroglial tumors is not fully understood. Genomic DNA was isolated from 61 oligodendroglial tumors for analysis of methylation using methylation-specific multiplex ligation-dependent probe amplification assay (MS-MLPA). We correlated methylation status with clinicopathological findings and outcome. The genes found to be most frequently methylated in oligodendroglial tumors were RASSF1A (80.3%), CASP8 (70.5%), and CDKN2A (52.5%). Kaplan-Meier survival curve analysis demonstrated longer duration of progression-free survival in patients with 19q loss, aged less than 38 years, and with a proliferative index of less than 5%. Methylation of the ESR1 promoter is significantly associated with shorter duration of overall survival and progression-free survival, and that methylation of IGSF4 and RASSF1A is significantly associated with shorter duration of progression-free survival. However, none of the methylation status of ESR1, IGSF4, and RASSF1A was of prognostic value for survival in a multivariate Cox model. A number of novel and interesting epigenetic alterations were identified in this study. The findings highlight the importance of methylation profiles in oligodendroglial tumors and their possible involvement in tumorigenesis. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

SHOX gene defects and selected dysmorphic signs in patients of idiopathic short stature and Léri-Weill dyschondrosteosis.

PubMed

Hirschfeldova, K; Solc, R; Baxova, A; Zapletalova, J; Kebrdlova, V; Gaillyova, R; Prasilova, S; Soukalova, J; Mihalova, R; Lnenicka, P; Florianova, M; Stekrova, J

2012-01-10

The aim of the study was to analyze frequency of SHOX gene defects and selected dysmorphic signs in patients of both idiopathic short stature (ISS) and Léri-Weill dyschondrosteosis (LWD), all derived from the Czech population. Overall, 98 subjects were analyzed in the study. Inclusion criteria were the presence of short stature (-2.0 SD), in combination with at least one of the selected dysmorphic signs for the ISS+ group; and the presence of Madelung deformity, without positive karyotyping for the LWD+ group. Each proband was analyzed by use of P018 MLPA kit, which covers SHOX and its regulatory sequences. Additionally, mutational analysis was done of the coding portions of the SHOX. Both extent and breakpoint localizations in the deletions/duplications found were quite variable. Some PAR1 rearrangements were detected, without obvious phenotypic association. In the ISS+ group, MLPA analysis detected four PAR1 deletions associated with a SHOX gene defect, PAR1 duplication with an ambiguous effect, and two SHOX mutations (13.7%). In the LWD+ group, MLPA analysis detected nine deletions in PAR1 region, with a deleterious effect on SHOX, first reported case of isolated SHOX enhancer duplication, and SHOX mutation (68.8%). In both ISS+ and LWD+ groups were positivity associated with a disproportionately short stature; in the ISS+ group, in combination with muscular hypertrophy. It seems that small PAR1 rearrangements might be quite frequent in the population. Our study suggests disproportionateness, especially in combination with muscular hypertrophy, as relevant indicators of ISS to be the effect of SHOX defect. Copyright © 2011 Elsevier B.V. All rights reserved.

High frequency of exon 15 deletion in the FANCA gene in Tunisian patients affected with Fanconi anemia disease: implication for diagnosis.

PubMed

Amouri, Ahlem; Talmoudi, Faten; Messaoud, Olfa; d'Enghien, Catherine D; Rekaya, Mariem B; Allegui, Ines; Azaiez, Héla; Kefi, Rym; Abdelhak, Ahlem; Meseddi, Sondes H; Torjemane, Lamia; Ouederni, Monia; Mellouli, Fethi; Abid, Héla B; Aissaoui, Lamia; Bejaoui, Mohamed; Othmen, Tarek B; Lyonnet, Dominique S; Soulier, Jean; Hachicha, Mongia; Dellagi, Koussay; Abdelhak, Sonia; Fanconi, Tunisian

2014-03-01

Tunisian population is characterized by its heterogeneous ethnic background and high rate of consanguinity. In consequence, there is an increase in the frequency of recessive genetic disorders including Fanconi anemia (FA). The aim of this study was to confirm the existence of a founder haplotype among FA Tunisian patients and to identify the associated mutation in order to develop a simple tool for FA diagnosis. Seventy-four unrelated families with a total of 95 FA patients were investigated. All available family members were genotyped with four microsatellite markers flanking FANCA gene. Haplotype analysis and homozygosity mapping assigned 83 patients belonging to 62 families to the FA-A group. A common haplotype was shared by 42 patients from 26 families at a homozygous state while five patients from five families were heterozygous. Among them, 85% were from southern Tunisia suggesting a founder effect. Using multiplex ligation-dependent probe amplification (MLPA) technique, we have also demonstrated that this haplotype is associated with a total deletion of exon 15 in FANCA gene. Identification of a founder mutation allowed genetic counseling in relatives of these families, better bone marrow graft donor selection and prenatal diagnosis. This mutation should be investigated in priority for patients originating from North Africa and Middle East.

A strategy for molecular diagnostics of Fanconi anemia in Brazilian patients.

PubMed

Pilonetto, Daniela V; Pereira, Noemi F; Bonfim, Carmem M S; Ribeiro, Lisandro L; Bitencourt, Marco A; Kerkhoven, Lianne; Floor, Karijn; Ameziane, Najim; Joenje, Hans; Gille, Johan J P; Pasquini, Ricardo

2017-07-01

Fanconi anemia (FA) is a predominantly autosomal recessive disease with wide genetic heterogeneity resulting from mutations in several DNA repair pathway genes. To date, 21 genetic subtypes have been identified. We aimed to identify the FA genetic subtypes in the Brazilian population and to develop a strategy for molecular diagnosis applicable to routine clinical use. We screened 255 patients from Hospital de Clínicas, Universidade Federal do Paraná for 11 common FA gene mutations. Further analysis by multiplex ligation-dependent probe amplification (MLPA) for FANCA and Sanger sequencing of all coding exons of FANCA , -C , and - G was performed in cases who harbored a single gene mutation. We identified biallelic mutations in 128/255 patients (50.2%): 89, 11, and 28 carried FANCA , FANCC , and FANCG mutations, respectively. Of these, 71 harbored homozygous mutations, whereas 57 had compound heterozygous mutations. In 4/57 heterozygous patients, both mutations were identified by the initial screening, in 51/57 additional analyses was required for classification, and in 2/57 the second mutation remained unidentified. We found 52 different mutations of which 22 were novel. The proposed method allowed genetic subtyping of 126/255 (49.4%) patients at a significantly reduced time and cost, which makes molecular diagnosis of FA Brazilian patients feasible.

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

High frequency of exon 15 deletion in the FANCA gene in Tunisian patients affected with Fanconi anemia disease: implication for diagnosis

PubMed Central

Amouri, Ahlem; Talmoudi, Faten; Messaoud, Olfa; d'Enghien, Catherine D; Rekaya, Mariem B; Allegui, Ines; Azaiez, Héla; Kefi, Rym; Abdelhak, Ahlem; Meseddi, Sondes H; Torjemane, Lamia; Ouederni, Monia; Mellouli, Fethi; Abid, Héla B; Aissaoui, Lamia; Bejaoui, Mohamed; Othmen, Tarek B; Lyonnet, Dominique S; Soulier, Jean; Hachicha, Mongia; Dellagi, Koussay; Abdelhak, Sonia; Fanconi, Tunisian

2014-01-01

Tunisian population is characterized by its heterogeneous ethnic background and high rate of consanguinity. In consequence, there is an increase in the frequency of recessive genetic disorders including Fanconi anemia (FA). The aim of this study was to confirm the existence of a founder haplotype among FA Tunisian patients and to identify the associated mutation in order to develop a simple tool for FA diagnosis. Seventy-four unrelated families with a total of 95 FA patients were investigated. All available family members were genotyped with four microsatellite markers flanking FANCA gene. Haplotype analysis and homozygosity mapping assigned 83 patients belonging to 62 families to the FA-A group. A common haplotype was shared by 42 patients from 26 families at a homozygous state while five patients from five families were heterozygous. Among them, 85% were from southern Tunisia suggesting a founder effect. Using multiplex ligation-dependent probe amplification (MLPA) technique, we have also demonstrated that this haplotype is associated with a total deletion of exon 15 in FANCA gene. Identification of a founder mutation allowed genetic counseling in relatives of these families, better bone marrow graft donor selection and prenatal diagnosis. This mutation should be investigated in priority for patients originating from North Africa and Middle East. PMID:24689079

Identification of rare and novel deletions that cause (δβ)0-thalassaemia and hereditary persistence of foetal haemoglobin in Indian population.

PubMed

Mayuranathan, Thiyagaraj; Rayabaram, Janakiram; Das, Reena; Arora, Neeraj; Edison, Eunice S; Chandy, Mammen; Srivastava, Alok; Velayudhan, Shaji R

2014-06-01

Hereditary persistence of foetal haemoglobin (HPFH) and (δβ)(0) -thalassaemia are conditions caused by large deletions that involve δ- and β-globin genes in the β-globin cluster, and they are characterized by increased haemoglobin (HbF) levels in adults. Significant phenotypic diversity is observed between the different mutations that cause these conditions. Molecular characterization of these deletions is important for accurate molecular diagnosis, and they will also provide the information on the cis-acting genetic regulatory elements present in the β-globin cluster. We performed gap-PCR, multiplex ligation-dependent probe amplification (MLPA), quantitative fluorescent multiplex PCR (QF-MPCR) and DNA sequencing to detect and characterize the deletions in the β-globin cluster. We characterized six different deletions resulting in (δβ)(0) -thalassaemia or HPFH in 51 unrelated families. With the help of multiple genetic tools, we performed comprehensive genetic analysis of HPFH and (δβ)(0) -thalassaemia in Indian population and could define the molecular basis of these conditions in this population. We also identified two novel HPFH mutations, 49.98 kb (HPFH-9) and 86.7 kb (HPFH-10) deletions, in this population. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Exonization of an Intronic LINE-1 Element Causing Becker Muscular Dystrophy as a Novel Mutational Mechanism in Dystrophin Gene.

PubMed

Gonçalves, Ana; Oliveira, Jorge; Coelho, Teresa; Taipa, Ricardo; Melo-Pires, Manuel; Sousa, Mário; Santos, Rosário

2017-10-03

A broad mutational spectrum in the dystrophin ( DMD ) gene, from large deletions/duplications to point mutations, causes Duchenne/Becker muscular dystrophy (D/BMD). Comprehensive genotyping is particularly relevant considering the mutation-centered therapies for dystrophinopathies. We report the genetic characterization of a patient with disease onset at age 13 years, elevated creatine kinase levels and reduced dystrophin labeling, where multiplex-ligation probe amplification (MLPA) and genomic sequencing failed to detect pathogenic variants. Bioinformatic, transcriptomic (real time PCR, RT-PCR), and genomic approaches (Southern blot, long-range PCR, and single molecule real-time sequencing) were used to characterize the mutation. An aberrant transcript was identified, containing a 103-nucleotide insertion between exons 51 and 52, with no similarity with the DMD gene. This corresponded to the partial exonization of a long interspersed nuclear element (LINE-1), disrupting the open reading frame. Further characterization identified a complete LINE-1 (~6 kb with typical hallmarks) deeply inserted in intron 51. Haplotyping and segregation analysis demonstrated that the mutation had a de novo origin. Besides underscoring the importance of mRNA studies in genetically unsolved cases, this is the first report of a disease-causing fully intronic LINE-1 element in DMD , adding to the diversity of mutational events that give rise to D/BMD.

Novel deletions involving the USH2A gene in patients with Usher syndrome and retinitis pigmentosa

PubMed Central

García-García, Gema; Jaijo, Teresa; Aparisi, Maria J.; Larrieu, Lise; Faugère, Valérie; Blanco-Kelly, Fiona; Ayuso, Carmen; Roux, Anne-Francoise; Millán, José M.

2014-01-01

Purpose The aim of the present work was to identify and characterize large rearrangements involving the USH2A gene in patients with Usher syndrome and nonsyndromic retinitis pigmentosa. Methods The multiplex ligation-dependent probe amplification (MLPA) technique combined with a customized array-based comparative genomic hybridization (aCGH) analysis was applied to 40 unrelated patients previously screened for point mutations in the USH2A gene in which none or only one pathologic mutation was identified. Results We detected six large deletions involving USH2A in six out of the 40 cases studied. Three of the patients were homozygous for the deletion, and the remaining three were compound heterozygous with a previously identified USH2A point mutation. In five of these cases, the patients displayed Usher type 2, and the remaining case displayed nonsyndromic retinitis pigmentosa. The exact breakpoint junctions of the deletions found in USH2A in four of these cases were characterized. Conclusions Our study highlights the need to develop improved efficient strategies of mutation screening based upon next generation sequencing (NGS) that reduce cost, time, and complexity and allow simultaneous identification of all types of disease-causing mutations in diagnostic procedures. PMID:25352746

Molecular Diagnostic and Prognostic Subtyping of Gliomas in Tunisian Population.

PubMed

Trabelsi, Saoussen; Chabchoub, Imen; Ksira, Iadh; Karmeni, Nadhir; Mama, Nadia; Kanoun, Samia; Burford, Anna; Jury, Alexa; Mackay, Alan; Popov, Sergey; Bouaouina, Noureddine; Ben Ahmed, Slim; Mokni, Moncef; Tlili, Kalthoum; Krifa, Hedi; Yacoubi, Mohamed Tahar; Jones, Chris; Saad, Ali; H'mida Ben Brahim, Dorra

2017-05-01

It has become increasingly evident that morphologically similar gliomas may have distinct clinical phenotypes arising from diverse genetic signatures. To date, glial tumours from the Tunisian population have not been investigated. To address this, we correlated the clinico-pathology with molecular data of 110 gliomas by a combination of HM450K array, MLPA and TMA-IHC. PTEN loss and EGFR amplification were distributed in different glioma histological groups. However, 1p19q co-deletion and KIAA1549:BRAF fusion were, respectively, restricted to Oligodendroglioma and Pilocytic Astrocytoma. CDKN2A loss and EGFR overexpression were more common within high-grade gliomas. Furthermore, survival statistical correlations led us to identify Glioblastoma (GB) prognosis subtypes. In fact, significant lower overall survival (OS) was detected within GB that overexpressed EGFR and Cox2. In addition, IDH1R132H mutation seemed to provide a markedly survival advantage. Interestingly, the association of IDHR132H mutation and EGFR normal status, as well as the association of differentiation markers, defined GB subtypes with good prognosis. By contrast, poor survival GB subtypes were defined by the combination of PTEN loss with PDGFRa expression and/or EGFR amplification. Additionally, GB presenting p53-negative staining associated with CDKN2A loss or p21 positivity represented a subtype with short survival. Thus, distinct molecular subtypes with individualised prognosis were identified. Interestingly, we found a unique histone mutation in a poor survival young adult GB case. This tumour exceptionally associated the H3F3A G34R mutation and MYCN amplification as well as 1p36 loss and 10q loss. Furthermore, by exhibiting a remarkable methylation profile, it emphasised the oncogenic power of G34R mutation connecting gliomagenesis and chromatin regulation.

Identification of a duplication within the GDF9 gene and novel candidate genes for primary ovarian insufficiency (POI) by a customized high-resolution array comparative genomic hybridization platform.

PubMed

Norling, A; Hirschberg, A L; Rodriguez-Wallberg, K A; Iwarsson, E; Wedell, A; Barbaro, M

2014-08-01

Can high-resolution array comparative genomic hybridization (CGH) analysis of DNA samples from women with primary ovarian insufficiency (POI) improve the diagnosis of the condition and identify novel candidate genes for POI? A mutation affecting the regulatory region of growth differentiation factor 9 (GDF9) was identified for the first time together with several novel candidate genes for POI. Most patients with POI do not receive a molecular diagnosis despite a significant genetic component in the pathogenesis. We performed a case-control study. Twenty-six patients were analyzed by array CGH for identification of copy number variants. Novel changes were investigated in 95 controls and in a separate population of 28 additional patients with POI. The experimental procedures were performed during a 1-year period. DNA samples from 26 patients with POI were analyzed by a customized 1M array-CGH platform with whole genome coverage and probe enrichment targeting 78 genes in sex development. By PCR amplification and sequencing, the breakpoint of an identified partial GDF9 gene duplication was characterized. A multiplex ligation-dependent probe amplification (MLPA) probe set for specific identification of deletions/duplications affecting GDF9 was developed. An MLPA probe set for the identification of additional cases or controls carrying novel candidate regions identified by array-CGH was developed. Sequencing of three candidate genes was performed. Eleven unique copy number changes were identified in a total of 11 patients, including a tandem duplication of 475 bp, containing part of the GDF9 gene promoter region. The duplicated region contains three NOBOX-binding elements and an E-box, important for GDF9 gene regulation. This aberration is likely causative of POI. Fifty-four patients were investigated for copy number changes within GDF9, but no additional cases were found. Ten aberrations constituting novel candidate regions were detected, including a second DNAH6 deletion in a patient with POI. Other identified candidate genes were TSPYL6, SMARCC1, CSPG5 and ZFR2. This is a descriptive study and no functional experiments were performed. The study illustrates the importance of analyzing small copy number changes in addition to sequence alterations in the genetic investigation of patients with POI. Also, promoter regions should be included in the investigation. The study was supported by grants from the Swedish Research council (project no 12198 to A.W. and project no 20324 to A.L.H.), Stockholm County Council (E.I., A.W. and K.R.W.), Foundation Frimurare Barnhuset (A.N., A.W. and M.B.), Karolinska Institutet (A.N., A.L.H., E.I., A.W. and M.B.), Novo Nordic Foundation (A.W.) and Svenska Läkaresällskapet (M.B.). The funding sources had no involvement in the design or analysis of the study. The authors have no competing interests to declare. Not applicable. © The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

FANCA Gene Mutations with 8 Novel Molecular Changes in Indian Fanconi Anemia Patients.

PubMed

Solanki, Avani; Mohanty, Purvi; Shukla, Pallavi; Rao, Anita; Ghosh, Kanjaksha; Vundinti, Babu Rao

2016-01-01

Fanconi anemia (FA), a rare heterogeneous genetic disorder, is known to be associated with 19 genes and a spectrum of clinical features. We studied FANCA molecular changes in 34 unrelated and 2 siblings of Indian patients with FA and have identified 26 different molecular changes of FANCA gene, of which 8 were novel mutations (a small deletion c.2500delC, 4 non-sense mutations c.2182C>T, c.2630C>G, c.3677C>G, c.3189G>A; and 3 missense mutations; c.1273G>C, c.3679 G>C, and c.3992 T>C). Among these only 16 patients could be assigned FA-A complementation group, because we could not confirm single exon deletions detected by MLPA or cDNA amplification by secondary confirmation method and due to presence of heterozygous non-pathogenic variations or heterozygous pathogenic mutations. An effective molecular screening strategy should be developed for confirmation of these mutations and determining the breakpoints for single exon deletions.

FANCA Gene Mutations with 8 Novel Molecular Changes in Indian Fanconi Anemia Patients

PubMed Central

Solanki, Avani; Mohanty, Purvi; Shukla, Pallavi; Rao, Anita; Ghosh, Kanjaksha; Vundinti, Babu Rao

2016-01-01

Fanconi anemia (FA), a rare heterogeneous genetic disorder, is known to be associated with 19 genes and a spectrum of clinical features. We studied FANCA molecular changes in 34 unrelated and 2 siblings of Indian patients with FA and have identified 26 different molecular changes of FANCA gene, of which 8 were novel mutations (a small deletion c.2500delC, 4 non-sense mutations c.2182C>T, c.2630C>G, c.3677C>G, c.3189G>A; and 3 missense mutations; c.1273G>C, c.3679 G>C, and c.3992 T>C). Among these only 16 patients could be assigned FA-A complementation group, because we could not confirm single exon deletions detected by MLPA or cDNA amplification by secondary confirmation method and due to presence of heterozygous non-pathogenic variations or heterozygous pathogenic mutations. An effective molecular screening strategy should be developed for confirmation of these mutations and determining the breakpoints for single exon deletions. PMID:26799702

[An attempt to identify 22q11.2 microdeletions in samples of the Hungarian schizophrenia DNA bank by multiplex ligation-based probe amplification (MLPA): literature review, methodology and results].

PubMed

Klein, Izabella; Szocs, Katalin; Vincze, Katalin; Benkovits, Judit; Somogyi, Szilvia; Herman, Levente; Rethelyi, Janos M

2016-12-01

Schizophrenia is a severe debilitating psychiatric disorder, with a typical onset in adolescence or early adulthood. This condition is characterized by heterogeneous symptoms (hallucinations, delusions, disorganized behaviour, affective flattening, and social isolation) and a life-time prevalence of 0.5-1.2%. In spite of the efforts to uncover the etiology of the disorder, its pathogenesis and neurobiological background are poorly understood. Given the high heritability in schizophrenia, genetic research remains an important area of focus. Besides the common variations of low penetrance - single nucleotid polymorphisms (SNPs) -, rare variants, mainly copy number variations (CNVs) play a role in the genetic architecture of the disorder. The most frequent CNV associated with schizophrenia is the hemizygous deletion of the 22q11.2 region. According to previous research this genetic variant occurs in 1% of the patients and conversely, 25% of the carriers of the 22q11.2 microdeletion will develop schizophrenia. The 22q11.2 deletion syndrome (22Q11DS, velocardiofacial (VCFS) syndrome, DiGeorge-syndrome) is usually a childhood diagnosis. Its prevalence is 1:2000-4000 considering all births. Patients can demonstrate heart developmental disorders, craniofacial (elongated face, hypertelorism), immunological (thymus-hypoplasia), endocrinological (hypocalcaemia) abnormalities, and neurodevelopmental alterations, but only a proportion will have these abnormalities due to incomplete penetrance. The variable symptoms complicate the recognition of the syndrome in the day to day medical practice. 25% of the known 22Q11DS patients develop schizophrenia but the risk of neuropsychiatric problems, like autism, ADHD and childhood conduct disorder is also increased, while early onset Parkinson's disease in also more frequent in adults. The schizophrenia phenotype is not distinguishable at the moment in patients with or without the 22q11 deletion. But emerging evidence suggests that early onset Parkinson's disease is more frequent in 22Q11DS and the effects of clozapine treatment could be different in schizophrenia with 22Q11DS. The question arises what is the incidence rate of the 22q11.2 microdeletion among our Hungarian DNA samples with schizophrenia. To answer the question, we utilized a new method used in routine genetic diagnostics, multiplex ligation-based probe amplification (MLPA). Although we genotyped the DNA of 315 Hungarian schizophrenia patients, we found no 22Q11DS in this cohort. The findings are discussed in terms of basic research and their translation into everyday clinical practice.

Post-mortem cytogenomic investigations in patients with congenital malformations.

PubMed

Dias, Alexandre Torchio; Zanardo, Évelin Aline; Dutra, Roberta Lelis; Piazzon, Flavia Balbo; Novo-Filho, Gil Monteiro; Montenegro, Marilia Moreira; Nascimento, Amom Mendes; Rocha, Mariana; Madia, Fabricia Andreia Rosa; Costa, Thais Virgínia Moura Machado; Milani, Cintia; Schultz, Regina; Gonçalves, Fernanda Toledo; Fridman, Cintia; Yamamoto, Guilherme Lopes; Bertola, Débora Romeo; Kim, Chong Ae; Kulikowski, Leslie Domenici

2016-08-01

Congenital anomalies are the second highest cause of infant deaths, and, in most cases, diagnosis is a challenge. In this study, we characterize patterns of DNA copy number aberrations in different samples of post-mortem tissues from patients with congenital malformations. Twenty-eight patients undergoing autopsy were cytogenomically evaluated using several methods, specifically, Multiplex Ligation-dependent Probe Amplification (MLPA), microsatellite marker analysis with a MiniFiler kit, FISH, a cytogenomic array technique and bidirectional Sanger sequencing, which were performed on samples of different tissues (brain, heart, liver, skin and diaphragm) preserved in RNAlater, in formaldehyde or by paraffin-embedding. The results identified 13 patients with pathogenic copy number variations (CNVs). Of these, eight presented aneuploidies involving chromosomes 13, 18, 21, X and Y (two presented inter- and intra-tissue mosaicism). In addition, other abnormalities were found, including duplication of the TYMS gene (18p11.32); deletion of the CHL1 gene (3p26.3); deletion of the HIC1 gene (17p13.3); and deletion of the TOM1L2 gene (17p11.2). One patient had a pathogenic missense mutation of g.8535C>G (c.746C>G) in exon 7 of the FGFR3 gene consistent with Thanatophoric Dysplasia type I. Cytogenomic techniques were reliable for the analysis of autopsy material and allowed the identification of inter- and intra-tissue mosaicism and a better understanding of the pathogenesis of congenital malformations. Copyright © 2016 Elsevier Inc. All rights reserved.

Clinical and molecular evaluation of SHOX/PAR1 duplications in Leri-Weill dyschondrosteosis (LWD) and idiopathic short stature (ISS).

PubMed

Benito-Sanz, S; Barroso, E; Heine-Suñer, D; Hisado-Oliva, A; Romanelli, V; Rosell, J; Aragones, A; Caimari, M; Argente, J; Ross, J L; Zinn, A R; Gracia, R; Lapunzina, P; Campos-Barros, A; Heath, K E

2011-02-01

Léri-Weill dyschondrosteosis (LWD) is a skeletal dysplasia characterized by disproportionate short stature and the Madelung deformity of the forearm. SHOX mutations and pseudoautosomal region 1 deletions encompassing SHOX or its enhancers have been identified in approximately 60% of LWD and approximately 15% of idiopathic short stature (ISS) individuals. Recently SHOX duplications have been described in LWD/ISS but also in individuals with other clinical manifestations, thus questioning their pathogenicity. The objective of the study was to investigate the pathogenicity of SHOX duplications in LWD and ISS. Multiplex ligation-dependent probe amplification is routinely used in our unit to analyze for SHOX/pseudoautosomal region 1 copy number changes in LWD/ISS referrals. Quantitative PCR, microsatellite marker, and fluorescence in situ hybridization analysis were undertaken to confirm all identified duplications. During the routine analysis of 122 LWD and 613 ISS referrals, a total of four complete and 10 partial SHOX duplications or multiple copy number (n > 3) as well as one duplication of the SHOX 5' flanking region were identified in nine LWD and six ISS cases. Partial SHOX duplications appeared to have a more deleterious effect on skeletal dysplasia and height gain than complete SHOX duplications. Importantly, no increase in SHOX copy number was identified in 340 individuals with normal stature or 104 overgrowth referrals. MLPA analysis of SHOX/PAR1 led to the identification of partial and complete SHOX duplications or multiple copies associated with LWD or ISS, suggesting that they may represent an additional class of mutations implicated in the molecular etiology of these clinical entities.

Metastatic neuroblastoma of the mandible: a cytogenetic and molecular genetic study.

PubMed

Manor, Esther; Kapelushnik, Joseph; Joshua, Ben-Zion; Bodner, Lipa

2012-08-01

Neuroblastoma (NB) jaw metastases are rare. Here, we report on cytogenetic and genetic studies on metastatic NB to the mandible. A 7-year-old boy, with an abdominal neuroblastoma, presented with a mass of the left body of the mandible. Cytogenetic analysis of the original tumor and the mandibular lesion biopsies revealed similar heterogenous subclones with 42 ~ 47,XY,+der(1)(q11 → qter),-2,del(7)(q21.1 → qter),-8,-9,-10,-11,del(11)(q13.3 → qter),-13,-14,-15,-17, + 18-18,der(18)(?),+21,+m1,+m2,+m3,+m4,+m5,+m6,+m7[cp25]. The different markers were identified by SKY analysis. Most of the cells carried 3-6 of these translocations: der(1;21), der(2;9;17), der(2;15;18), der(2;15;Y), der(8;10), der(10;17). Molecular examination using Neuroblastoma MLPA kit (MRC-Holland) revealed gain of 1q25, 1q42, 2q33, 2p23, 2p24 (N-myc), and 21q22, and loss of 11q22, 11q23, 17p13, and 17q11. FISH analysis using N-myc probe showed high amplification levels of N-myc. The cytogenetic and molecular genetic work-ups revealed that the mandibular lesion is a metastasis of the original abdominal tumor and not a second primary caused by the aggressive treatment. Clinical parameters such as : patient's age, site of primary tumor and the mandibular metastasis, together with poor prognosis genetic markers explain the patient's short-term survival.

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

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.

Craniosynostosis in 10q26 deletion patients: A consequence of brain underdevelopment or altered suture biology?

PubMed

Faria, Ágatha Cristhina; Rabbi-Bortolini, Eliete; Rebouças, Maria R G O; de S Thiago Pereira, Andréia L A; Frasson, Milena G Tonini; Atique, Rodrigo; Lourenço, Naila Cristina V; Rosenberg, Carla; Kobayashi, Gerson S; Passos-Bueno, Maria Rita; Errera, Flávia Imbroisi Valle

2016-02-01

Approximately a hundred patients with terminal 10q deletions have been described. They present with a wide range of clinical features always accompanied by delayed development, intellectual disability and craniofacial dysmorphisms. Here, we report a girl and a boy with craniosynostosis, developmental delay and other congenital anomalies. Karyotyping and molecular analysis including Multiplex Ligation dependent probe amplification (MLPA) and Array Comparative Genomic Hybridization (aCGH) were performed in both patients. We detected a 13.1 Mb pure deletion at 10q26.12-q26.3 in the girl and a 10.9 Mb pure deletion at 10q26.13-q26.3 in the boy, both encompassing about 100 genes. The clinical and molecular findings in these patients reinforce the importance of the DOCK1 smallest region of overlap I (SRO I), previously suggested to explain the clinical signs, and together with a review of the literature suggest a second 3.5 Mb region important for the phenotype (SRO II). Genotype-phenotype correlations and literature data suggest that the craniosynostosis is not directly related to dysregulated signaling in suture development, but may be secondary to alterations in brain development instead. Further, genes at 10q26 may be involved in the molecular crosstalk between brain and cranial vault. © 2015 Wiley Periodicals, Inc.

Exonization of an Intronic LINE-1 Element Causing Becker Muscular Dystrophy as a Novel Mutational Mechanism in Dystrophin Gene

PubMed Central

Gonçalves, Ana; Coelho, Teresa; Melo-Pires, Manuel; Sousa, Mário

2017-01-01

A broad mutational spectrum in the dystrophin (DMD) gene, from large deletions/duplications to point mutations, causes Duchenne/Becker muscular dystrophy (D/BMD). Comprehensive genotyping is particularly relevant considering the mutation-centered therapies for dystrophinopathies. We report the genetic characterization of a patient with disease onset at age 13 years, elevated creatine kinase levels and reduced dystrophin labeling, where multiplex-ligation probe amplification (MLPA) and genomic sequencing failed to detect pathogenic variants. Bioinformatic, transcriptomic (real time PCR, RT-PCR), and genomic approaches (Southern blot, long-range PCR, and single molecule real-time sequencing) were used to characterize the mutation. An aberrant transcript was identified, containing a 103-nucleotide insertion between exons 51 and 52, with no similarity with the DMD gene. This corresponded to the partial exonization of a long interspersed nuclear element (LINE-1), disrupting the open reading frame. Further characterization identified a complete LINE-1 (~6 kb with typical hallmarks) deeply inserted in intron 51. Haplotyping and segregation analysis demonstrated that the mutation had a de novo origin. Besides underscoring the importance of mRNA studies in genetically unsolved cases, this is the first report of a disease-causing fully intronic LINE-1 element in DMD, adding to the diversity of mutational events that give rise to D/BMD. PMID:28972564

Heterozygous deletion at the SOX10 gene locus in two patients from a Chinese family with Waardenburg syndrome type II.

PubMed

Wenzhi, He; Ruijin, Wen; Jieliang, Li; Xiaoyan, Ma; Haibo, Liu; Xiaoman, Wang; Jiajia, Xian; Shaoying, Li; Shuanglin, Li; Qing, Li

2015-10-01

Waardenburg syndrome (WS) is a rare disease characterized by sensorineural deafness and pigment disturbance. To date, almost 100 mutations have been reported, but few reports on cases with SOX10 gene deletion. The inheritance pattern of SOX10 gene deletion is still unclear. Our objective was to identify the genetic causes of Waardenburg syndrome type II in a two-generation Chinese family. Clinical evaluations were conducted in both of the patients. Microarray analysis and multiplex ligation-dependent probe amplification (MLPA) were performed to identify disease-related copy number variants (CNVs). DNA sequencing of the SOX10, MITF and SNAI2 genes was performed to identify the pathogenic mutation responsible for WS2. A 280kb heterozygous deletion at the 22q13.1 chromosome region (including SOX10) was detected in both of the patients. No mutation was found in the patients, unaffected family members and 30 unrelated healthy controls. This report is the first to describe SOX10 heterozygous deletions in Chinese WS2 patients. Our result conform the thesis that heterozygous deletions at SOX10 is an important pathogenicity for WS, and present as autosomal dominant inheritance. Nevertheless, heterozygous deletion of the SOX10 gene would be worth investigating to understand their functions and contributions to neurologic phenotypes. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Influence of prophylactic probiotics and selective decontamination on bacterial translocation in patients undergoing pancreatic surgery: a randomized controlled trial.

PubMed

Diepenhorst, Gwendolyn M P; van Ruler, Oddeke; Besselink, Marc G H; van Santvoort, Hjalmar C; Wijnandts, Paul R; Renooij, Willem; Gouma, Dirk J; Gooszen, Hein G; Boermeester, Marja A

2011-01-01

Bacterial translocation (BT) is suspected to play a major role in the development of infections in surgical patients. However, the clinical association between intestinal barrier dysfunction, BT, and septic morbidity has remained unconfirmed. The objective of this study was to study BT in patients undergoing major abdominal surgery and the effects of probiotics, selective decontamination of the digestive tract (SDD), and standard treatment on intestinal barrier function. In a randomized controlled setting, 30 consecutive patients planned for elective pylorus-preserving pancreaticoduodenectomy (PPPD) were allocated to receive perioperatively probiotics, SDD, or standard treatment. To assess intestinal barrier function, intestinal fatty acid-binding protein (mucosal damage) and polyethylene glycol recovery (intestinal permeability) in urine were measured perioperatively. BT was assessed by real-time polymerase chain reaction and multiplex ligation-dependent probe amplification (MLPA) in mesenteric lymph nodes (MLNs) harvested early (baseline control) and at the end of surgery ("end-of-surgery" MLNs, after 3h in PPPD patients). Polymerase chain reaction detected bacterial DNA in 18 of 27 end-of-surgery MLNs and in 13 of 23 control MLNs (P = 0.378). Probiotics and SDD had no significant effect on the number of positive MLNs or the change in bacterial DNA during operation. Multiplex ligation-dependent probe amplification analysis showed significantly increased expression of only 4 of 30 inflammatory mediator-related genes in end-of-surgery compared with early sampled MLN (P < 0.05). Polyethylene glycol recovery was unaffected by operation, probiotics and SDD as compared with standard treatment. Intestinal fatty acid-binding protein levels were increased shortly postoperatively only in patients treated with SDD (P = 0.02). Probiotics and SDD did not influence BT, intestinal permeability, or inflammatory mediator expression. Bacterial translocation after abdominal surgery may be part of normal antigen-sampling processes of the gut.

A novel deletion of SNURF/SNRPN exon 1 in a patient with Prader-Willi-like phenotype.

PubMed

Cao, Yang; AlHumaidi, Susan S; Faqeih, Eissa A; Pitel, Beth A; Lundquist, Patrick; Aypar, Umut

2017-08-01

Here we report the smallest deletion involving SNURF/SNRPN that causes major symptoms of Prader-Willi syndrome (PWS), including hypotonia, dysmorphic features, intellectual disability, and obesity. A female patient with the aforementioned and additional features was referred to the Mayo Clinic Cytogenetics laboratory for genetic testing. Chromosomal microarray analysis and subsequent Sanger sequencing identified a de novo 6.4 kb deletion at 15q11.2, containing exon 1 of the SNURF gene and exon 1 of the shortest isoform of the SNRPN gene. SNURF/SNRPN exon 1, which is methylated on the silent maternal allele, is associated with acetylated histones on the expressed paternal allele. This region also overlaps with the PWS-imprinting center (IC). Subsequent molecular methylation analysis was performed using methylation-specific MLPA (MS-MLPA), which characterized that the deletion of SNURF/SNRPN exon 1 was paternal in origin, consistent with the PWS-like phenotype. Since SNURF/SNRPN gene and the PWS-IC are known to regulate snoRNAs, it is likely that the PWS-like phenotype observed in patients with paternal SNURF/SNRPN deletion is due to the disrupted expression of SNORD116 snoRNAs. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Viral prevalence increases with regional colony abundance in honey bee drones (Apis mellifera L).

PubMed

Forfert, Nadège; Natsopoulou, Myrsini E; Paxton, Robert J; Moritz, Robin F A

2016-10-01

Transmission among colonies is a central feature for the epidemiology of honey bee pathogens. High colony abundance may promote transmission among colonies independently of apiary layout, making colony abundance a potentially important parameter determining pathogen prevalence in populations of honey bees. To test this idea, we sampled male honey bees (drones) from seven distinct drone congregation areas (DCA), and used their genotypes to estimate colony abundance at each site. A multiplex ligation dependent probe amplification assay (MLPA) was used to assess the prevalence of ten viruses, using five common viral targets, in individual drones. There was a significant positive association between colony abundance and number of viral infections. This result highlights the potential importance of high colony abundance for pathogen prevalence, possibly because high population density facilitates pathogen transmission. Pathogen prevalence in drones collected from DCAs may be a useful means of estimating the disease status of a population of honey bees during the mating season, especially for localities with a large number of wild or feral colonies. Copyright © 2016 Elsevier B.V. All rights reserved.

Mutation spectrum of Chinese patients with Bartter syndrome.

PubMed

Han, Yue; Lin, Yi; Sun, Qing; Wang, Shujuan; Gao, Yanxia; Shao, Leping

2017-11-24

Bartter syndrome (BS) has been rarely reported in Chinese population except for a few case reports. This investigation was aimed to analyze the mutations of the causal genes in sixteen Chinese patients with BS, and review their followup and treatment. Identify mutations by the next generation sequencing and the multiplex ligation-dependent probe amplification (MLPA). Clinical characteristics and biochemical findings at the first presentation as well as follow-up were reviewed. 15 different CLCNKB gene mutations were identified in fourteen patients with BS, including 11 novel ones. A novel missense mutation and a novel small deletion were found from SLC12A1 gene. A novel gross deletion was found in CLCNKA gene. A recurrent missense mutation was identified from BSND gene. We found that the whole gene deletion mutation of CLCNKB gene was the most frequent mutation (32%), and the rate of gross deletion was up to 50 percent in this group of Chinese patients. The present study has found 19 mutations, including 14 novel ones, which would enrich the human gene mutation database (HGMD) and provide valuable references to the genetic counseling and diagnosis of the Chinese population.

SHOX gene and conserved noncoding element deletions/duplications in Colombian patients with idiopathic short stature.

PubMed

Sandoval, Gloria Tatiana Vinasco; Jaimes, Giovanna Carola; Barrios, Mauricio Coll; Cespedes, Camila; Velasco, Harvy Mauricio

2014-03-01

SHOX gene mutations or haploinsufficiency cause a wide range of phenotypes such as Leri Weill dyschondrosteosis (LWD), Turner syndrome, and disproportionate short stature (DSS). However, this gene has also been found to be mutated in cases of idiopathic short stature (ISS) with a 3-15% frequency. In this study, the multiplex ligation-dependent probe amplification (MLPA) technique was employed to determine the frequency of SHOX gene mutations and their conserved noncoding elements (CNE) in Colombian patients with ISS. Patients were referred from different centers around the county. From a sample of 62 patients, 8.1% deletions and insertions in the intragenic regions and in the CNE were found. This result is similar to others published in other countries. Moreover, an isolated case of CNE 9 duplication and a new intron 6b deletion in another patient, associated with ISS, are described. This is one of the first studies of a Latin American population in which deletions/duplications of the SHOX gene and its CNE are examined in patients with ISS.

SHOX gene and conserved noncoding element deletions/duplications in Colombian patients with idiopathic short stature

PubMed Central

Sandoval, Gloria Tatiana Vinasco; Jaimes, Giovanna Carola; Barrios, Mauricio Coll; Cespedes, Camila; Velasco, Harvy Mauricio

2014-01-01

SHOX gene mutations or haploinsufficiency cause a wide range of phenotypes such as Leri Weill dyschondrosteosis (LWD), Turner syndrome, and disproportionate short stature (DSS). However, this gene has also been found to be mutated in cases of idiopathic short stature (ISS) with a 3–15% frequency. In this study, the multiplex ligation-dependent probe amplification (MLPA) technique was employed to determine the frequency of SHOX gene mutations and their conserved noncoding elements (CNE) in Colombian patients with ISS. Patients were referred from different centers around the county. From a sample of 62 patients, 8.1% deletions and insertions in the intragenic regions and in the CNE were found. This result is similar to others published in other countries. Moreover, an isolated case of CNE 9 duplication and a new intron 6b deletion in another patient, associated with ISS, are described. This is one of the first studies of a Latin American population in which deletions/duplications of the SHOX gene and its CNE are examined in patients with ISS. PMID:24689071

A unique combination of 17pter trisomy and 21qter monosomy in a boy with developmental delay, severe intellectual disability, growth retardation and dysmorphisms.

PubMed

Zheng, Zhaojing; Yao, Ru-En; Geng, Juan; Jin, Xingming; Shen, Yongnian; Ying, Daming; Fu, Qihua; Yu, Yongguo

2013-03-10

Microduplication at 17p13.3 and microdeletion at 21q22 are both rare chromosomal aberrations. The presence of both genomic imbalances in one patient has not been previously reported in literature. In this study, we performed a molecular diagnostic testing with a whole genome microarray on a 3-year-old boy with developmental delay, mental retardation and multiple malformations. A routine G-banding karyotype analysis was performed using peripheral lymphocytes. Chromosome microarray analysis (CMA) was done using Affymetrix CytoScan™ HD array. Genomic imbalances were further confirmed by multiple ligation-dependent probe amplification (MLPA). The result of karyotyping was normal but CMA detected a 9.8 Mb microduplication at 17p13.3-13.1 (chr17: 1-9,875,545) and a 2.8 Mb microdeletion involving 21q22.3-qter (chr21: 45,239,077-48,097,372). The imbalances were due to a balanced translocation present in patient's mother. The patient was characterized with short stature, profound developmental delay, non-verbal, intellectual disability as well as craniofacial dysmorphism, subtle brain structural anomaly and sparse scalp hair. This is the first patient reported with a combination of a microduplication at 17p13.3-13.1 and a microdeletion at 21q22.3-qter. Both genomic imbalances were undetected by conventional karyotyping but were delineated with CMA test. Synergistic effect from the two rare genomic imbalances is likely responsible for the severe clinical phenotypes observed in this patient. Copyright © 2012 Elsevier B.V. All rights reserved.

Mutational analysis in patients with Autosomal Dominant Polycystic Kidney Disease (ADPKD): Identification of five mutations in the PKD1 gene.

PubMed

Abdelwahed, Mayssa; Hilbert, Pascale; Ahmed, Asma; Mahfoudh, Hichem; Bouomrani, Salem; Dey, Mouna; Hachicha, Jamil; Kamoun, Hassen; Keskes-Ammar, Leila; Belguith, Neïla

2018-05-31

Autosomal Dominant Polycystic Kidney Disease (ADPKD), the most frequent genetic disorder of the kidneys, is characterized by a typical presenting symptoms include cysts development in different organs and a non-cysts manifestations. ADPKD is caused by mutations in PKD1 or PKD2 genes. In this study, we aimed to search for molecular causative defects among PKD1 and PKD2 genes. Eighteen patients were diagnosed based on renal ultrasonography and renal/extra-renal manifestations. Then, Sanger sequencing was performed for PKD1 and PKD2 genes. Multiplex Ligation dependent Probe Amplification method (MLPA) methods was performed for both PKD genes. Mutational analysis of the PKD2 gene revealed the absence of variants and no deletions or duplications of both PKD genes were detected. But three novels mutations i.e. p.S463C exon 7; c. c.11156+2T>C IVS38 and c.8161-1G>A IVS22 and two previously reported c.1522T>C exon 7 and c.412C>T exon 4 mutations in the PKD1 gene were detected. Bioinformatics tools predicted that the novel variants have a pathogenic effects on splicing machinery, pre-mRNA secondary structure and stability and protein stability. Our results highlighted molecular features of Tunisian patients with ADPKD and revealed novel variations that can be utilized in clinical diagnosis and in the evaluation of living kidney donor. To the best of our knowledge, this is the first report of Autosomal Polycystic Kidney Disease in Tunisia. Copyright © 2017. Published by Elsevier B.V.

CNV-ROC: A cost effective, computer-aided analytical performance evaluator of chromosomal microarrays

PubMed Central

Goodman, Corey W.; Major, Heather J.; Walls, William D.; Sheffield, Val C.; Casavant, Thomas L.; Darbro, Benjamin W.

2016-01-01

Chromosomal microarrays (CMAs) are routinely used in both research and clinical laboratories; yet, little attention has been given to the estimation of genome-wide true and false negatives during the assessment of these assays and how such information could be used to calibrate various algorithmic metrics to improve performance. Low-throughput, locus-specific methods such as fluorescence in situ hybridization (FISH), quantitative PCR (qPCR), or multiplex ligation-dependent probe amplification (MLPA) preclude rigorous calibration of various metrics used by copy number variant (CNV) detection algorithms. To aid this task, we have established a comparative methodology, CNV-ROC, which is capable of performing a high throughput, low cost, analysis of CMAs that takes into consideration genome-wide true and false negatives. CNV-ROC uses a higher resolution microarray to confirm calls from a lower resolution microarray and provides for a true measure of genome-wide performance metrics at the resolution offered by microarray testing. CNV-ROC also provides for a very precise comparison of CNV calls between two microarray platforms without the need to establish an arbitrary degree of overlap. Comparison of CNVs across microarrays is done on a per-probe basis and receiver operator characteristic (ROC) analysis is used to calibrate algorithmic metrics, such as log2 ratio threshold, to enhance CNV calling performance. CNV-ROC addresses a critical and consistently overlooked aspect of analytical assessments of genome-wide techniques like CMAs which is the measurement and use of genome-wide true and false negative data for the calculation of performance metrics and comparison of CNV profiles between different microarray experiments. PMID:25595567

Interpretation of acid α-glucosidase activity in creatine kinase elevation: A case of Becker muscular dystrophy.

PubMed

Oitani, Yoshiki; Ishiyama, Akihiko; Kosuga, Motomichi; Iwasawa, Kentaro; Ogata, Ayako; Tanaka, Fumiko; Takeshita, Eri; Shimizu-Motohashi, Yuko; Komaki, Hirofumi; Nishino, Ichizo; Okuyama, Torayuki; Sasaki, Masayuki

2018-05-16

Diagnosis of Pompe disease is sometimes challenging because it exhibits clinical similarities to muscular dystrophy. We describe a case of Becker muscular dystrophy (BMD) with a remarkable reduction in activity of the acid α-glucosidase (GAA) enzyme, caused by a combination of pathogenic mutation and polymorphism variants resulting in pseudodeficiency in GAA. The three-year-old boy demonstrated asymptomatic creatine kinase elevation. Neither exon deletion nor duplication was detected on multiplex ligation-dependent probe amplification (MLPA) of DMD. GAA enzyme activity in both dried blood spots and lymphocytes was low, at 11.7% and 7.7% of normal, respectively. However, genetic analysis of GAA detected only heterozygosity for a nonsense mutation (c.118C > T, p.Arg40 ∗ ). Muscle pathology showed no glycogen deposits and no high acid phosphatase activity. Hematoxylin-eosin staining detected scattered regenerating fibers; the fibers were faint and patchy on immunochemistry staining of dystrophin. The amount of dystrophin protein was reduced to 11.8% of normal, on Western blotting analysis. Direct sequencing analysis of DMD revealed hemizygosity for a nonsense mutation (c.72G > A, p.Trp24 ∗ ). The boy was diagnosed with BMD, despite remarkable reduction in GAA activity; further, he demonstrated heterozygosity for [p.Gly576Ser; p.Glu689Lys] polymorphism variants that indicated pseudodeficiency on another allele in GAA. Pseudodeficiency alleles are detected in approximately 4% of the Asian population; these demonstrate low activity of acid α-glucosidase (GAA), similar to levels found in Pompe disease. Clinicians should be careful in their interpretations of pseudodeficiency alleles that complicate diagnosis in cases of elevated creatine kinase. Copyright © 2018 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Refining the role of PMS2 in Lynch syndrome: germline mutational analysis improved by comprehensive assessment of variants.

PubMed

Borràs, Ester; Pineda, Marta; Cadiñanos, Juan; Del Valle, Jesús; Brieger, Angela; Hinrichsen, Inga; Cabanillas, Ruben; Navarro, Matilde; Brunet, Joan; Sanjuan, Xavier; Musulen, Eva; van der Klift, Helen; Lázaro, Conxi; Plotz, Guido; Blanco, Ignacio; Capellá, Gabriel

2013-08-01

The majority of mismatch repair (MMR) gene mutations causing Lynch syndrome (LS) occur either in MLH1 or MSH2. However, the relative contribution of PMS2 is less well defined. The aim of this study was to evaluate the role of PMS2 in LS by assessing the pathogenicity of variants of unknown significance (VUS) detected in the mutational analysis of PMS2 in a series of Spanish patients. From a cohort of 202 LS suspected patients, 13 patients showing loss of PMS2 expression in tumours were screened for germline mutations in PMS2, using a long range PCR based strategy and multiplex ligation dependent probe amplification (MLPA). Pathogenicity assessment of PMS2 VUS was performed evaluating clinicopathological data, frequency in control population and in silico and in vitro analyses at the RNA and protein level. Overall 25 different PMS2 DNA variants were detected. Fourteen were classified as polymorphisms. Nine variants were classified as pathogenic: seven alterations based on their molecular nature and two after demonstrating a functional defect (c.538-3C>G affected mRNA processing and c.137G>T impaired MMR activity). The c.1569C>G variant was classified as likely neutral while the c.384G>A remained as a VUS. We have also shown that the polymorphic variant c.59G>A is MMR proficient. Pathogenic PMS2 mutations were detected in 69% of patients harbouring LS associated tumours with loss of PMS2 expression. In all, PMS2 mutations account for 6% of the LS cases identified. The comprehensive functional analysis shown here has been useful in the classification of PMS2 VUS and contributes to refining the role of PMS2 in LS.

First description of mutational analysis of MLH1, MSH2 and MSH6 in Algerian families with suspected Lynch syndrome.

PubMed

Ziada-Bouchaar, H; Sifi, K; Filali, T; Hammada, T; Satta, D; Abadi, N

2017-01-01

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant disorder characterized by the early onset of colorectal cancer (CRC) linked to germline defects in Mismatch Repair (MMR) genes. We present here, the first molecular study of the correlation between CRC and mutations occurring in these genes performed in twenty-one unrelated Algerian families. The presence of germline mutations in MMR genes, MLH1, MSH2 and MSH6 genes was tested by sequencing all exons plus adjacent intronic sequences and Multiplex ligand-dependent probe amplification (MLPA) for testing large genomic rearrangements. Pathogenic mutations were identified in 20 % of families with clinical suspicion on HNPCC. Two novel variants described for the first time in Algerian families were identified in MLH1, c.881_884delTCAGinsCATTCCT and a large deletion in MSH6 gene from a young onset of CRC. Moreover, the variants of MSH2 gene: c.942+3A>T, c.1030C>T, the most described ones, were also detected in Algerian families. Furthermore, the families HNPCC caused by MSH6 germline mutation may show an age of onset that is comparable to this of patients with MLH1 and MSH2 mutations. In this study, we confirmed that MSH2, MLH1, and MSH6 contribute to CRC susceptibility. This work represents the implementation of a diagnostic algorithm for the identification of Lynch syndrome patients in Algerian families.

Spectrum of SMARCB1/INI1 Mutations in Familial and Sporadic Rhabdoid Tumors

PubMed Central

Eaton, Katherine W.; Tooke, Laura S.; Wainwright, Luanne M.; Judkins, Alexander R.; Biegel, Jaclyn A.

2011-01-01

Background Germline mutations and deletions of SMARCB1/INI1 in chromosome band 22q11.2 predispose patients to rhabdoid tumor and schwannomatosis. Previous estimates suggested that 15–20% of rhabdoid tumors were caused by an underlying germline abnormality of SMARCB1. However, these studies were limited by case selection and an inability to detect intragenic deletions and duplications. Procedure One hundred matched tumor and blood samples from patients with rhabdoid tumors of the brain, kidney, or soft tissues were analyzed for mutations and deletions of SMARCB1 by FISH, multiplex ligation-dependent probe amplification (MLPA), sequence analysis and high resolution Illumina 610K SNP based oligonucleotide array studies. Results Thirty-five of 100 patients were found to have a germline SMARCB1 abnormality. These abnormalities included point and frameshift mutations, intragenic deletions and duplications, and larger deletions including regions both proximal and distal to SMARCB1. There were 9 cases that demonstrated parent to child transmission of a mutated copy of SMARCB1. In 8 of the 9 cases, one or more family members were also diagnosed with rhabdoid tumor or schwannoma, and 2 of the 8 families presented with multiple affected children in a manner consistent with gonadal mosaicism. Conclusions Approximately one third of newly diagnosed patients with rhabdoid tumor have an underlying genetic predisposition to tumors due to a germline SMARCB1 alteration. Families may demonstrate incomplete penetrance and gonadal mosaicism, which must be considered when counseling families of patients with rhabdoid tumor. PMID:21108436

SHOX duplications found in some cases with type I Mayer-Rokitansky-Kuster-Hauser syndrome.

PubMed

Gervasini, Cristina; Grati, Francesca Romana; Lalatta, Faustina; Tabano, Silvia; Gentilin, Barbara; Colapietro, Patrizia; De Toffol, Simona; Frontino, Giada; Motta, Francesca; Maitz, Silvia; Bernardini, Laura; Dallapiccola, Bruno; Fedele, Luigi; Larizza, Lidia; Miozzo, Monica

2010-10-01

The Mayer-Rokitansky-Küster-Hauser syndrome is defined as congenital aplasia of müllerian ducts derived structures in females with a normal female chromosomal and gonadal sex. Most cases with Mayer-Rokitansky-Küster-Hauser syndrome are sporadic, although familial cases have been reported. The genetic basis of Mayer-Rokitansky-Küster-Hauser syndrome is largely unknown and seems heterogeneous, and a small number of cases were found to have mutations in the WNT4 gene. The aim of this study was to identify possible recurrent submicroscopic imbalances in a cohort of familial and sporadic cases with Mayer-Rokitansky-Küster-Hauser syndrome. Multiplex ligation-dependent probe amplification was used to screen the subtelomeric sequences of all chromosomes in 30 patients with Mayer-Rokitansky-Küster-Hauser syndrome (sporadic, n = 27 and familial, n = 3). Segregation analysis and pyrosequencing were applied to validate the MLPA results in the informative family. Partial duplication of the Xpter pseudoautosomal region 1 containing the short stature homeobox (SHOX) gene was detected in five patients with Mayer-Rokitansky-Küster-Hauser syndrome (familial, n = 3 and sporadic, n = 2) and not in 53 healthy controls. The duplications were not overlapping, and SHOX was never entirely duplicated. Haplotyping in the informative family revealed that SHOX gene duplication was inherited from the unaffected father and was absent in two healthy sisters. Partial duplication of SHOX gene is found in some cases with both familial and sporadic Mayer-Rokitansky-Küster-Hauser type I syndrome.

Spectrum of BRCA1/2 variants in 940 patients from Argentina including novel, deleterious and recurrent germline mutations: impact on healthcare and clinical practice.

PubMed

Solano, Angela Rosaria; Cardoso, Florencia Cecilia; Romano, Vanesa; Perazzo, Florencia; Bas, Carlos; Recondo, Gonzalo; Santillan, Francisco Bernardo; Gonzalez, Eduardo; Abalo, Eduardo; Viniegra, María; Michel, José Davalos; Nuñez, Lina María; Noblia, Cristina Maria; Mc Lean, Ignacio; Canton, Enrique Diaz; Chacon, Reinaldo Daniel; Cortese, Gustavo; Varela, Eduardo Beccar; Greco, Martín; Barrientos, María Laura; Avila, Silvia Adela; Vuotto, Hector Daniel; Lorusso, Antonio; Podesta, Ernesto Jorge; Mando, Oscar Gaspar

2017-09-01

BRCA1/2 mutations in Latin America are scarcely documented and in serious need of knowledge about the spectrum of BRCA pathogenic variants, information which may alter clinical practice and subsequently improve patient outcome. In addition, the search for data on testing policies in different regions constitutes a fundamental strength for the present study, which analyzes BRCA1/2 gene sequences and large rearrangements in 940 probands with familial and/or personal history of breast/ovary cancer (BOC). In non-mutated DNA samples, Multiplex Ligation-dependent Probe Amplification assays (MLPA) were used for the analysis of large rearrangements. Our studies detected 179 deleterious mutations out of 940 (19.04%) probands, including 5 large rearrangements and 22 novel mutations. The recurrent mutations accounted for 15.08% of the total and only 2.87% of the probands analyzed, very different from a Hispanic panel previously described. a) this first comprehensive description of the spectrum in BRCA1/2 sheds light on the low frequency of recurrent mutations; b) this information is key in clinical practice to select adequate sequencing studies in our population, subsequently improve patient outcome and prevent damage associated to false normal reports resulting from the use of invalid population panels; c) panels of mutations from other populations should be cautiously validated before imported, even those of apparently similar origin, a concept to be considered beyond significance in Argentina.

Whole exome sequencing and array-based molecular karyotyping as aids to prenatal diagnosis in fetuses with suspected Simpson-Golabi-Behmel syndrome.

PubMed

Kehrer, Christina; Hoischen, Alexander; Menkhaus, Ralf; Schwab, Eva; Müller, Andreas; Kim, Sarah; Kreiß, Martina; Weitensteiner, Valerie; Hilger, Alina; Berg, Christoph; Geipel, Anne; Reutter, Heiko; Gembruch, Ulrich

2016-10-01

Simpson-Golabi-Behmel (SGBS) syndrome type 1 and type 2 represent rare X-linked prenatal overgrowth disorders. The aim of our study is to describe the prenatal sonographic features as well as the genetic work-up. Retrospective analysis of four cases with a pre- or postnatal diagnosis of SGBS in a single tertiary referral center within a period of 4 years. In the study period, four male fetuses with SGBS were detected. The final diagnosis was made prenatally in three cases. In all cases the second trimester anomaly scan revealed left sided congenital diaphragmatic hernia (CDH) with additional anomalies; three fetuses with SGBS type 1 showed fetal overgrowth. In two of these, whole exome sequencing showed a possible frameshift mutation and a point mutation in the gene GPC3, respectively. In the third case, multiplex ligation-dependent probe amplification (MLPA) revealed a hemizygous duplication of exon 3-7 in the gene GPC3. In the fourth case, SGBS type 2 was confirmed by array comparative genomic hybridization (CGH) of amniotic fluid cells showing a deletion of the gene OFD1. We could demonstrate, that in the presence of a CDH, syndromes of the fetus can be increasingly differentiated by detailed sonography followed by a selective and graded molecular diagnostic using microarray techniques and whole exome sequencing. © 2016 John Wiley & Sons, Ltd. © 2016 John Wiley & Sons, Ltd.

APC alterations are frequently involved in the pathogenesis of acinar cell carcinoma of the pancreas, mainly through gene loss and promoter hypermethylation.

PubMed

Furlan, Daniela; Sahnane, Nora; Bernasconi, Barbara; Frattini, Milo; Tibiletti, Maria Grazia; Molinari, Francesca; Marando, Alessandro; Zhang, Lizhi; Vanoli, Alessandro; Casnedi, Selenia; Adsay, Volkan; Notohara, Kenji; Albarello, Luca; Asioli, Sofia; Sessa, Fausto; Capella, Carlo; La Rosa, Stefano

2014-05-01

Genetic and epigenetic alterations involved in the pathogenesis of pancreatic acinar cell carcinomas (ACCs) are poorly characterized, including the frequency and role of gene-specific hypermethylation, chromosome aberrations, and copy number alterations (CNAs). A subset of ACCs is known to show alterations in the APC/β-catenin pathway which includes mutations of APC gene. However, it is not known whether, in addition to mutation, loss of APC gene function can occur through alternative genetic and epigenetic mechanisms such as gene loss or promoter methylation. We investigated the global methylation profile of 34 tumor suppressor genes, CNAs of 52 chromosomal regions, and APC gene alterations (mutation, methylation, and loss) together with APC mRNA level in 45 ACCs and related peritumoral pancreatic tissues using methylation-specific multiplex ligation probe amplification (MS-MLPA), fluorescence in situ hybridization (FISH), mutation analysis, and reverse transcription-droplet digital PCR. ACCs did not show an extensive global gene hypermethylation profile. RASSF1 and APC were the only two genes frequently methylated. APC mutations were found in only 7 % of cases, while APC loss and methylation were more frequently observed (48 and 56 % of ACCs, respectively). APC mRNA low levels were found in 58 % of cases and correlated with CNAs. In conclusion, ACCs do not show extensive global gene hypermethylation. APC alterations are frequently involved in the pathogenesis of ACCs mainly through gene loss and promoter hypermethylation, along with reduction of APC mRNA levels.

Spectrum of BRCA1/2 variants in 940 patients from Argentina including novel, deleterious and recurrent germline mutations: impact on healthcare and clinical practice

PubMed Central

Solano, Angela Rosaria; Cardoso, Florencia Cecilia; Romano, Vanesa; Perazzo, Florencia; Bas, Carlos; Recondo, Gonzalo; Santillan, Francisco Bernardo; Gonzalez, Eduardo; Abalo, Eduardo; Viniegra, María; Michel, José Davalos; Nuñez, Lina María; Noblia, Cristina Maria; Mc Lean, Ignacio; Canton, Enrique Diaz; Chacon, Reinaldo Daniel; Cortese, Gustavo; Varela, Eduardo Beccar; Greco, Martín; Barrientos, María Laura; Avila, Silvia Adela; Vuotto, Hector Daniel; Lorusso, Antonio; Podesta, Ernesto Jorge; Mando, Oscar Gaspar

2017-01-01

BRCA1/2 mutations in Latin America are scarcely documented and in serious need of knowledge about the spectrum of BRCA pathogenic variants, information which may alter clinical practice and subsequently improve patient outcome. In addition, the search for data on testing policies in different regions constitutes a fundamental strength for the present study, which analyzes BRCA1/2 gene sequences and large rearrangements in 940 probands with familial and/or personal history of breast/ovary cancer (BOC). In non-mutated DNA samples, Multiplex Ligation-dependent Probe Amplification assays (MLPA) were used for the analysis of large rearrangements. Our studies detected 179 deleterious mutations out of 940 (19.04%) probands, including 5 large rearrangements and 22 novel mutations. The recurrent mutations accounted for 15.08% of the total and only 2.87% of the probands analyzed, very different from a Hispanic panel previously described. In conclusion: a) this first comprehensive description of the spectrum in BRCA1/2 sheds light on the low frequency of recurrent mutations; b) this information is key in clinical practice to select adequate sequencing studies in our population, subsequently improve patient outcome and prevent damage associated to false normal reports resulting from the use of invalid population panels; c) panels of mutations from other populations should be cautiously validated before imported, even those of apparently similar origin, a concept to be considered beyond significance in Argentina. PMID:28947987

Maturity Onset Diabetes of the Young (MODY) in Tunisia: Low frequencies of GCK and HNF1A mutations.

PubMed

Ben Khelifa, S; Martinez, R; Dandana, A; Khochtali, I; Ferchichi, S; Castaño, L

2018-04-20

Maturity Onset Diabetes of the Young (MODY) is a monogenic form of diabetes characterized by autosomal dominant inheritance, an early clinical onset and a primary defect in β-cell function. Mutations in the GCK and HNF1A genes are the most common cause of MODY among Caucasians. The etiology of MODY in Tunisia stills a challenge for researchers. The aim of this study was to screen for mutations in GCK, HNF1A, HNF4A and INS genes in North African Tunisians subjects, in whom the clinical profile was very suggestive of MODY. A total of 23 unrelated patients, with clinical presentation of MODY were tested for mutations in GCK, HNF1A, HNF4A and INS genes, using Denaturing High Performance Liquid Chromatography (DHPLC), Multiplex Ligation-depend Probe Amplification (MLPA) and sequencing analysis. We identified the previously reported mutation c-169C > T in one patient as well as a new mutation c-457C > T in two unrelated patients. No mutations were detected in the HNF1A and INS genes. Despite restrictive clinical criteria used for selecting patients in this study, the most common genes known for MODY do not explain the majority of cases in Tunisians. This suggests that there are others candidate or unidentified genes contributing to the etiology of MODY in Tunisians families. Copyright © 2018 Elsevier B.V. All rights reserved.

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.

8p23.1 duplication syndrome differentiated from copy number variation of the defensin cluster at prenatal diagnosis in four new families

PubMed Central

2010-01-01

Background The 8p23.1 duplication syndrome and copy number variation of the 8p23.1 defensin gene cluster are cytogenetically indistinguishable but distinct at the molecular level. To our knowledge, the 8p23.1 duplication syndrome has been described at prenatal diagnosis only once and we report our experience with four further apparent duplications ascertained at prenatal diagnosis. Methods Additional material at band 8p23.1 was detected using conventional G-banded cytogenetics in each case. Multiplex Ligation-dependent Probe Amplification (MLPA) or Fluorescence In Situ Hybridisation (FISH) were used depending on whether only DNA (Cases 1 and 4) or cytogenetic preparations (Cases 2 and 3) were available from the laboratory of origin. The extent of the duplication in Case 1 was retrospectively determined using array Comparative Genomic Hybridisation (array CGH). Results Three cases of 8p23.1 duplication syndrome were found (Cases 1 to 3). Two were de novo and continued to term and the third, a paternally transmitted duplication, was terminated because of a previous child with psychomotor delay and 8p23.1 duplication syndrome. Case 1 was ascertained with a hypoplastic left heart but the ventricular septal and interventricular defects, in Cases 2 and 3 respectively, were found after ascertainment for advanced maternal age. By contrast, case 4 was a maternally transmitted copy number variation of the defensin cluster with normal outcome. Conclusions Our data underline the need to differentiate 8p23.1 duplications from copy number variation of the defensin cluster using FISH, MLPA or array CGH. Cardiac defects were ascertained by ultrasound in only one of the three duplication 8p23.1 pregnancies but were visible in two of the three at 21 to 22 weeks gestation. Our results provide further evidence that both deletion and duplication of the GATA4 transcription factor can give rise to a variety of conotruncal heart defects with variable penetrance and expressivity. PMID:20167067

Waardenburg syndrome: Novel mutations in a large Brazilian sample.

PubMed

Bocángel, Magnolia Astrid Pretell; Melo, Uirá Souto; Alves, Leandro Ucela; Pardono, Eliete; Lourenço, Naila Cristina Vilaça; Marcolino, Humberto Vicente Cezar; Otto, Paulo Alberto; Mingroni-Netto, Regina Célia

2018-06-01

This paper deals with the molecular investigation of Waardenburg syndrome (WS) in a sample of 49 clinically diagnosed probands (most from southeastern Brazil), 24 of them having the type 1 (WS1) variant (10 familial and 14 isolated cases) and 25 being affected by the type 2 (WS2) variant (five familial and 20 isolated cases). Sequential Sanger sequencing of all coding exons of PAX3, MITF, EDN3, EDNRB, SOX10 and SNAI2 genes, followed by CNV detection by MLPA of PAX3, MITF and SOX10 genes in selected cases revealed many novel pathogenic variants. Molecular screening, performed in all patients, revealed 19 causative variants (19/49 = 38.8%), six of them being large whole-exon deletions detected by MLPA, seven (four missense and three nonsense substitutions) resulting from single nucleotide substitutions (SNV), and six representing small indels. A pair of dizygotic affected female twins presented the c.430delC variant in SOX10, but the mutation, imputed to gonadal mosaicism, was not found in their unaffected parents. At least 10 novel causative mutations, described in this paper, were found in this Brazilian sample. Copy-number-variation detected by MLPA identified the causative mutation in 12.2% of our cases, corresponding to 31.6% of all causative mutations. In the majority of cases, the deletions were sporadic, since they were not present in the parents of isolated cases. Our results, as a whole, reinforce the fact that the screening of copy-number-variants by MLPA is a powerful tool to identify the molecular cause in WS patients. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

MYO7A and USH2A gene sequence variants in Italian patients with Usher syndrome.

PubMed

Sodi, Andrea; Mariottini, Alessandro; Passerini, Ilaria; Murro, Vittoria; Tachyla, Iryna; Bianchi, Benedetta; Menchini, Ugo; Torricelli, Francesca

2014-01-01

To analyze the spectrum of sequence variants in the MYO7A and USH2A genes in a group of Italian patients affected by Usher syndrome (USH). Thirty-six Italian patients with a diagnosis of USH were recruited. They received a standard ophthalmologic examination, visual field testing, optical coherence tomography (OCT) scan, and electrophysiological tests. Fluorescein angiography and fundus autofluorescence imaging were performed in selected cases. All the patients underwent an audiologic examination for the 0.25-8,000 Hz frequencies. Vestibular function was evaluated with specific tests. DNA samples were analyzed for sequence variants of the MYO7A gene (for USH1) and the USH2A gene (for USH2) with direct sequencing techniques. A few patients were analyzed for both genes. In the MYO7A gene, ten missense variants were found; three patients were compound heterozygous, and two were homozygous. Thirty-four USH2A gene variants were detected, including eight missense variants, nine nonsense variants, six splicing variants, and 11 duplications/deletions; 19 patients were compound heterozygous, and three were homozygous. Four MYO7A and 17 USH2A variants have already been described in the literature. Among the novel mutations there are four USH2A large deletions, detected with multiplex ligation dependent probe amplification (MLPA) technology. Two potentially pathogenic variants were found in 27 patients (75%). Affected patients showed variable clinical pictures without a clear genotype-phenotype correlation. Ten variants in the MYO7A gene and 34 variants in the USH2A gene were detected in Italian patients with USH at a high detection rate. A selective analysis of these genes may be valuable for molecular analysis, combining diagnostic efficiency with little time wastage and less resource consumption.

Feasibility of nonselective testing for hemoglobinopathies in early pregnancy in The Netherlands.

PubMed

Kaufmann, Judith O; Demirel-Güngör, Gönül; Selles, Anke; Hudig, Cisca; Steen, Gerard; Ponjee, Gabrielle; Holleboom, Cas; Freeman, Liv M; Hendiks, Joris; Wijermans, Pierre; Giordano, Piero C; Kerkhoffs, Jean-Louis

2011-12-01

To examine the feasibility of standardized hemoglobinopathy (HBP) carrier testing for pregnant women in The Netherlands in addition to the standard anemia screening. We assessed the prevalence of HBP in women at the time of the first pregnancy visit using both a prospective cohort (N = 703) and a retrospective series of women selected at random (N = 588). For the purpose of analysis, the population was divided into a high risk and a low risk group for HBP based on maternal ethnicity. Screening for HBP utilized standard screening tests for anemia, with additional high performance liquid chromatography (Variant II); molecular analysis was performed by Gap-polymerase chain reaction (Gap-PCR) and if necessary, direct sequencing and multiplex ligation-dependent probe amplification (MLPA). Family history was reported or collected from the medical records. β-Globin defects were found in 3.9% of the total population (50/1291). The frequency in the high risk population was 5.6% (37/656), compared with 1.2% (6/501) in the low risk group. In the prospective study we found 30 HBP carriers, leading to testing of 16 partners and identification of two couples at risk. One affected child was born. Mean gestational age at the screening was 11.3 weeks with a standard deviation (SD) of 5.8. We found that the prevalence of HBP carriers is high enough in our population to warrant HBP testing for the entire multiethnic population in early pregnancy at the time of anemia screening. This is feasible as most women had their booking early in their first trimester. Copyright © 2011 John Wiley & Sons, Ltd.

CNV-ROC: A cost effective, computer-aided analytical performance evaluator of chromosomal microarrays.

PubMed

Goodman, Corey W; Major, Heather J; Walls, William D; Sheffield, Val C; Casavant, Thomas L; Darbro, Benjamin W

2015-04-01

Chromosomal microarrays (CMAs) are routinely used in both research and clinical laboratories; yet, little attention has been given to the estimation of genome-wide true and false negatives during the assessment of these assays and how such information could be used to calibrate various algorithmic metrics to improve performance. Low-throughput, locus-specific methods such as fluorescence in situ hybridization (FISH), quantitative PCR (qPCR), or multiplex ligation-dependent probe amplification (MLPA) preclude rigorous calibration of various metrics used by copy number variant (CNV) detection algorithms. To aid this task, we have established a comparative methodology, CNV-ROC, which is capable of performing a high throughput, low cost, analysis of CMAs that takes into consideration genome-wide true and false negatives. CNV-ROC uses a higher resolution microarray to confirm calls from a lower resolution microarray and provides for a true measure of genome-wide performance metrics at the resolution offered by microarray testing. CNV-ROC also provides for a very precise comparison of CNV calls between two microarray platforms without the need to establish an arbitrary degree of overlap. Comparison of CNVs across microarrays is done on a per-probe basis and receiver operator characteristic (ROC) analysis is used to calibrate algorithmic metrics, such as log2 ratio threshold, to enhance CNV calling performance. CNV-ROC addresses a critical and consistently overlooked aspect of analytical assessments of genome-wide techniques like CMAs which is the measurement and use of genome-wide true and false negative data for the calculation of performance metrics and comparison of CNV profiles between different microarray experiments. Copyright © 2015 Elsevier Inc. All rights reserved.

Mutation analysis of the APC gene in Taiwanese FAP families: low incidence of APC germline mutation in a distinct subgroup of FAP families.

PubMed

Chiang, J M; Chen, H W; Tang, R P; Chen, J S; Changchien, C R; Hsieh, P S; Wang, J Y

2010-06-01

Familial adenomatous polyposis (FAP) is an autosomal-dominant disease caused by germline mutations in the adenomatous polyposis coli (APC) gene. The affected individuals develop colorectal polyposis and show various extra-colonic manifestations. In this study, we aimed to investigate the genetic and clinical characteristics of FAP in Taiwanese families and analyze the genotype-phenotype correlations. Blood samples were obtained from 66 FAP patients registered in the hereditary colorectal cancer database. Then, germline mutations in the APC genes of these 66 polyposis patients from 47 unrelated FAP families were analyzed. The germline-mutation-negative cases were analyzed by performing multiplex ligation-dependent probe amplification (MLPA) and single-strand conformation polymorphism (SSCP) analysis of the MUTYH gene. Among the analyzed families, 79% (37/47) of the families showed 28 APC mutations, including 19 frameshift mutations, 4 nonsense mutations, 3 genomic deletion mutations, 1 missense mutation, and 1 splice-site mutation. In addition, we identified 15 novel mutations in 32% (15/47) of the families. The cases in which APC mutations were not identified showed significantly lower incidence of profuse polyposis (P = 0.034) and gastroduodenal polyps (P = 0.027). Furthermore, FAP families in which some affected individuals had less than 100 polyps showed significant association with low incidence of APC germline mutations (P = 0.002). We have added the APC germline-mutation data for Taiwanese FAP patients and indicated the presence of an FAP subgroup comprising affected individuals with nonadenomatous polyps or less than 100 adenomatous polyps; this form of FAP is less frequently caused by germline mutations of the APC gene.

Mutation spectrum of Chinese patients with Bartter syndrome

PubMed Central

Han, Yue; Lin, Yi; Sun, Qing; Wang, Shujuan; Gao, Yanxia; Shao, Leping

2017-01-01

Objective Bartter syndrome (BS) has been rarely reported in Chinese population except for a few case reports. This investigation was aimed to analyze the mutations of the causal genes in sixteen Chinese patients with BS, and review their followup and treatment. Methods Identify mutations by the next generation sequencing and the multiplex ligation-dependent probe amplification (MLPA). Clinical characteristics and biochemical findings at the first presentation as well as follow-up were reviewed. Results 15 different CLCNKB gene mutations were identified in fourteen patients with BS, including 11 novel ones. A novel missense mutation and a novel small deletion were found from SLC12A1 gene. A novel gross deletion was found in CLCNKA gene. A recurrent missense mutation was identified from BSND gene. We found that the whole gene deletion mutation of CLCNKB gene was the most frequent mutation (32%), and the rate of gross deletion was up to 50 percent in this group of Chinese patients. Conclusion The present study has found 19 mutations, including 14 novel ones, which would enrich the human gene mutation database (HGMD) and provide valuable references to the genetic counseling and diagnosis of the Chinese population. PMID:29254190

Partial USH2A deletions contribute to Usher syndrome in Denmark.

PubMed

Dad, Shzeena; Rendtorff, Nanna D; Kann, Erik; Albrechtsen, Anders; Mehrjouy, Mana M; Bak, Mads; Tommerup, Niels; Tranebjærg, Lisbeth; Rosenberg, Thomas; Jensen, Hanne; Møller, Lisbeth B

2015-12-01

Usher syndrome is an autosomal recessive disorder characterized by congenital hearing impairment, progressive visual loss owing to retinitis pigmentosa and in some cases vestibular dysfunction. Usher syndrome is divided into three subtypes, USH1, USH2 and USH3. Twelve loci and eleven genes have so far been identified. Duplications and deletions in PCDH15 and USH2A that lead to USH1 and USH2, respectively, have previously been identified in patients from United Kingdom, Spain and Italy. In this study, we investigate the proportion of exon deletions and duplications in PCDH15 and USH2A in 20 USH1 and 30 USH2 patients from Denmark using multiplex ligation-dependent probe amplification (MLPA). Two heterozygous deletions were identified in USH2A, but no deletions or duplications were identified in PCDH15. Next-generation mate-pair sequencing was used to identify the exact breakpoints of the two deletions identified in USH2A. Our results suggest that USH2 is caused by USH2A exon deletions in a small fraction of the patients, whereas deletions or duplications in PCDH15 might be rare in Danish Usher patients.

Ring chromosome 10: report on two patients and review of the literature.

PubMed

Guilherme, Roberta Santos; Kim, Chong Ae; Alonso, Luis Garcia; Honjo, Rachel S; Meloni, Vera Ayres; Christofolini, Denise Maria; Kulikowski, Leslie Domenici; Melaragno, Maria Isabel

2013-02-01

Ring chromosome 10--r(10)--is a rare disorder, with 14 cases reported in the literature, but only two with breakpoint determination by high-resolution techniques. We report here on two patients presenting a ring chromosome 10, studied by G-banding, fluorescent in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA) and SNP-array techniques, in order to investigate ring instability and determine breakpoints. Patient 1 showed a r(10)(p15.3q26.2) with a 7.9 Mb deletion in 10q26.2-q26.2, while patient 2 showed a r(10)(p15.3q26.13) with a 1.0 Mb deletion in 10p15.3 and a 8.8 Mb deletion in 10q26.13-q26.3, both unstable. While patient 1 presented with clinical features usually found in patients with r(10) and terminal 10q deletion, patient 2 presented characteristics so far not described in other patients with r(10), such as Dandy-Walker variant, osteopenia, semi-flexed legs, and dermal pigmentation regions. Our data and the data from literature show that there are no specific clinical findings to define a r(10) syndrome.

Identification of 15 novel partial SHOX deletions and 13 partial duplications, and a review of the literature reveals intron 3 to be a hotspot region.

PubMed

Benito-Sanz, Sara; Belinchon-Martínez, Alberta; Aza-Carmona, Miriam; de la Torre, Carolina; Huber, Celine; González-Casado, Isabel; Ross, Judith L; Thomas, N Simon; Zinn, Andrew R; Cormier-Daire, Valerie; Heath, Karen E

2017-02-01

Short stature homeobox gene (SHOX) is located in the pseudoautosomal region 1 of the sex chromosomes. It encodes a transcription factor implicated in the skeletal growth. Point mutations, deletions or duplications of SHOX or its transcriptional regulatory elements are associated with two skeletal dysplasias, Léri-Weill dyschondrosteosis (LWD) and Langer mesomelic dysplasia (LMD), as well as in a small proportion of idiopathic short stature (ISS) individuals. We have identified a total of 15 partial SHOX deletions and 13 partial SHOX duplications in LWD, LMD and ISS patients referred for routine SHOX diagnostics during a 10 year period (2004-2014). Subsequently, we characterized these alterations using MLPA (multiplex ligation-dependent probe amplification assay), fine-tiling array CGH (comparative genomic hybridation) and breakpoint PCR. Nearly half of the alterations have a distal or proximal breakpoint in intron 3. Evaluation of our data and that in the literature reveals that although partial deletions and duplications only account for a small fraction of SHOX alterations, intron 3 appears to be a breakpoint hotspot, with alterations arising by non-allelic homologous recombination, non-homologous end joining or other complex mechanisms.

Search for copy number variants in chromosomes 15q11-q13 and 22q11.2 in obsessive compulsive disorder

PubMed Central

2010-01-01

Background Obsessive-compulsive disorder (OCD) is a clinically and etiologically heterogeneous syndrome. The high frequency of obsessive-compulsive symptoms reported in subjects with the 22q11.2 deletion syndrome (DiGeorge/velocardiofacial syndrome) or Prader-Willi syndrome (15q11-13 deletion of the paternally derived chromosome), suggests that gene dosage effects in these chromosomal regions could increase risk for OCD. Therefore, the aim of this study was to search for microrearrangements in these two regions in OCD patients. Methods We screened the 15q11-13 and 22q11.2 chromosomal regions for genomic imbalances in 236 patients with OCD using multiplex ligation-dependent probe amplification (MLPA). Results No deletions or duplications involving 15q11-13 or 22q11.2 were identified in our patients. Conclusions Our results suggest that deletions/duplications of chromosomes 15q11-13 and 22q11.2 are rare in OCD. Despite the negative findings in these two regions, the search for copy number variants in OCD using genome-wide array-based methods is a highly promising approach to identify genes of etiologic importance in the development of OCD. PMID:20565924

Search for copy number variants in chromosomes 15q11-q13 and 22q11.2 in obsessive compulsive disorder.

PubMed

Delorme, Richard; Moreno-De-Luca, Daniel; Gennetier, Aurélie; Maier, Wolfgang; Chaste, Pauline; Mössner, Rainald; Grabe, Hans Jörgen; Ruhrmann, Stephan; Falkai, Peter; Mouren, Marie-Christine; Leboyer, Marion; Wagner, Michael; Betancur, Catalina

2010-06-21

Obsessive-compulsive disorder (OCD) is a clinically and etiologically heterogeneous syndrome. The high frequency of obsessive-compulsive symptoms reported in subjects with the 22q11.2 deletion syndrome (DiGeorge/velocardiofacial syndrome) or Prader-Willi syndrome (15q11-13 deletion of the paternally derived chromosome), suggests that gene dosage effects in these chromosomal regions could increase risk for OCD. Therefore, the aim of this study was to search for microrearrangements in these two regions in OCD patients. We screened the 15q11-13 and 22q11.2 chromosomal regions for genomic imbalances in 236 patients with OCD using multiplex ligation-dependent probe amplification (MLPA). No deletions or duplications involving 15q11-13 or 22q11.2 were identified in our patients. Our results suggest that deletions/duplications of chromosomes 15q11-13 and 22q11.2 are rare in OCD. Despite the negative findings in these two regions, the search for copy number variants in OCD using genome-wide array-based methods is a highly promising approach to identify genes of etiologic importance in the development of OCD.

Comprehensive analysis of MGMT promoter methylation: correlation with MGMT expression and clinical response in GBM.

PubMed

Shah, Nameeta; Lin, Biaoyang; Sibenaller, Zita; Ryken, Timothy; Lee, Hwahyung; Yoon, Jae-Geun; Rostad, Steven; Foltz, Greg

2011-01-07

O⁶-methylguanine DNA-methyltransferase (MGMT) promoter methylation has been identified as a potential prognostic marker for glioblastoma patients. The relationship between the exact site of promoter methylation and its effect on gene silencing, and the patient's subsequent response to therapy, is still being defined. The aim of this study was to comprehensively characterize cytosine-guanine (CpG) dinucleotide methylation across the entire MGMT promoter and to correlate individual CpG site methylation patterns to mRNA expression, protein expression, and progression-free survival. To best identify the specific MGMT promoter region most predictive of gene silencing and response to therapy, we determined the methylation status of all 97 CpG sites in the MGMT promoter in tumor samples from 70 GBM patients using quantitative bisulfite sequencing. We next identified the CpG site specific and regional methylation patterns most predictive of gene silencing and improved progression-free survival. Using this data, we propose a new classification scheme utilizing methylation data from across the entire promoter and show that an analysis based on this approach, which we call 3R classification, is predictive of progression-free survival (HR  = 5.23, 95% CI [2.089-13.097], p<0.0001). To adapt this approach to the clinical setting, we used a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) test based on the 3R classification and show that this test is both feasible in the clinical setting and predictive of progression free survival (HR  = 3.076, 95% CI [1.301-7.27], p = 0.007). We discuss the potential advantages of a test based on this promoter-wide analysis and compare it to the commonly used methylation-specific PCR test. Further prospective validation of these two methods in a large independent patient cohort will be needed to confirm the added value of promoter wide analysis of MGMT methylation in the clinical setting.

Comprehensive Analysis of MGMT Promoter Methylation: Correlation with MGMT Expression and Clinical Response in GBM

PubMed Central

Shah, Nameeta; Lin, Biaoyang; Sibenaller, Zita; Ryken, Timothy; Lee, Hwahyung; Yoon, Jae-Geun; Rostad, Steven; Foltz, Greg

2011-01-01

O6-methylguanine DNA-methyltransferase (MGMT) promoter methylation has been identified as a potential prognostic marker for glioblastoma patients. The relationship between the exact site of promoter methylation and its effect on gene silencing, and the patient's subsequent response to therapy, is still being defined. The aim of this study was to comprehensively characterize cytosine-guanine (CpG) dinucleotide methylation across the entire MGMT promoter and to correlate individual CpG site methylation patterns to mRNA expression, protein expression, and progression-free survival. To best identify the specific MGMT promoter region most predictive of gene silencing and response to therapy, we determined the methylation status of all 97 CpG sites in the MGMT promoter in tumor samples from 70 GBM patients using quantitative bisulfite sequencing. We next identified the CpG site specific and regional methylation patterns most predictive of gene silencing and improved progression-free survival. Using this data, we propose a new classification scheme utilizing methylation data from across the entire promoter and show that an analysis based on this approach, which we call 3R classification, is predictive of progression-free survival (HR  = 5.23, 95% CI [2.089–13.097], p<0.0001). To adapt this approach to the clinical setting, we used a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) test based on the 3R classification and show that this test is both feasible in the clinical setting and predictive of progression free survival (HR  = 3.076, 95% CI [1.301–7.27], p = 0.007). We discuss the potential advantages of a test based on this promoter-wide analysis and compare it to the commonly used methylation-specific PCR test. Further prospective validation of these two methods in a large independent patient cohort will be needed to confirm the added value of promoter wide analysis of MGMT methylation in the clinical setting. PMID:21249131

Exon dosage analysis of parkin gene in Chinese sporadic Parkinson's disease.

PubMed

Guo, Ji-Feng; Dong, Xiao-Li; Xu, Qian; Li, Nan; Yan, Xin-Xiang; Xia, Kun; Tang, Bei-Sha

2015-09-14

Parkin gene mutations are by far the most common mutations in both familial Parkinson's disease (PD) and sporadic PD. Approximately, 50% of parkin mutations is exon dosage mutations (i.e., deletions and duplications of entire exons). Here, we first established a MLPA assay for quick detection of parkin exon rearrangements. Then, we studied parkin exon dosage mutations in 755 Chinese sporadic PDdisease patients using the established MLPA assay. We found that there were 25 (3.3%) patients with exon dosage alterations including deletions and duplications, 20 (11.4%) patients with exon rearrangements in 178 early-onset patients, and 5 (0.86%) patients with exon rearrangement mutations in 579 later-onset patients. The percentage of individuals with parkin dosage mutations is more than 33% when the age at onset is less than 30 years old, but less than 7% when the age at onset is more than 30. In these mutations, deletion is the main mutational style, especially in exon 2-5. Our results indicated that exon dosage mutations in parkin gene might be the main cause for sporadic PD, especially in EOP. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Unbalanced translocation 6p/16q (partial monosomy 6p and trisomy 16q): prenatal diagnosis and cytogenetics.

PubMed

Puhl, Alexander G; Zelazny, Julia; Galetzka, Danuta; Skala, Christine; Frey-Mahn, Gabriele; Wellek, Brigitte; Koelbl, Heinz

2010-06-01

Unbalanced translocation 6p/16q in one fetus is a very rare event and the prenatal sonographic findings have never been published before. We will give a short overview of the literature along with a case report focussing on prenatal ultrasound features and molecular cytogenetic analysis. A 21-year-old primigravid woman presented with a singleton pregnancy at 19 weeks' gestation. The fetus revealed a mild hydrocephalus, a ventricular septal defect (VSD), a Dandy-Walker malformation as well as an intrauterine growth retardation (IUGR) and limb anomalities. MLPA analysis from amniotic fluid cells showed an unbalanced translocation from the subtelomeric region of chromosome 6p to the subtelomeric region of chromosome 16q. Karyotype of the fetus was 46, XX.ishder(6)t(6;16)(p2?5;q?13)(pVYS246A+, pVYS228B-, pVYS229A+). Despite the karyotype the mother decided not to interrupt pregnancy. The fetus died in utero within the 39th week of gestation and was delivered vaginally after labour induction, with a birth weight of 1815g. Prenatal FISH and MLPA studies can be very important to help outline the chromosomal area of deletion and duplication and the sonographic findings forebode the cytogenetic region of interest. Subsequent to the processing of the case, a complete Medline search was conducted to review previous cases with similar genetic alterations. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

State of the art and new developments in molecular diagnostics for hemoglobinopathies in multiethnic societies.

PubMed

Harteveld, C L

2014-02-01

For detecting carriers of thalassemia traits, the basic part of diagnostics consists of measurement of the hematological indices followed by mostly automatic separation and measurement of the Hb fractions, while direct Hb separation either on high pressure liquid chromatography or capillary electrophoresis is sufficient to putatively identify carriers of the common Hb variants like HbS, C, E, D, and O-Arab. A putative positive result is reported together with an advice for parents, partner, or family analysis. For couples, presumed at-risk confirmation at the DNA level is essential. In general, this part of diagnostics is done in specialized centers provided with sufficient experience and the technical tools needed to combine hematological and biochemical interpretation with identification of the mutations at the molecular level. State-of-the-art tools are usually available in centers that also provide prenatal diagnosis and should consist of gap-PCR for the common deletions, direct DNA sequencing for all kind of point-mutations and the capacity to uncover novel or rare mutations or disease mechanisms. New developments are MLPA for large and eventually unknown deletion defects and microarray technology for fine mapping and primer design for breakpoint analysis. Gap-PCR primers designed in the region flanking the deletion breakpoints can subsequently be used to facilitate carrier detection of uncommon deletions in family members or isolated populations in laboratories where no microarray technology or MLPA is available. © 2013 John Wiley & Sons Ltd.

A 600 kb triplication in the cat eye syndrome critical region causes anorectal, renal and preauricular anomalies in a three-generation family.

PubMed

Knijnenburg, Jeroen; van Bever, Yolande; Hulsman, Lorette O M; van Kempen, Chantal A P; Bolman, Galhana M; van Loon, Rosa Laura E; Beverloo, H Berna; van Zutven, Laura J C M

2012-09-01

Cat eye syndrome (CES) is caused by a gain of the proximal part of chromosome 22. Usually, a supernumerary marker chromosome is present, containing two extra copies of the chromosome 22q11.1q11.21 region. More sporadically, the gain is present intrachromosomally. The critical region for CES is currently estimated to be about 2.1 Mb and to contain at least 14 RefSeq genes. Gain of this region may cause ocular coloboma, preauricular, anorectal, urogenital and congenital heart malformations. We describe a family in which a 600 kb intrachromosomal triplication is present in at least three generations. The copy number alteration was detected using MLPA and further characterized with interphase and metaphase FISH and SNP-array. The amplified fragment is located in the distal part of the CES region. The family members show anal atresia and preauricular tags or pits, matching part of the phenotype of this syndrome. This finding suggests that amplification of the genes CECR2, SLC25A18 and ATP6V1E1, mapping within the critical region for CES, may be responsible for anorectal, renal and preauricular anomalies in patients with CES.

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.

Major Contribution of Genomic Copy Number Variation in Syndromic Congenital Heart Disease: The Use of MLPA as the First Genetic Test.

PubMed

Monteiro, Rejane A C; de Freitas, Mariana L; Vianna, Gabrielle S; de Oliveira, Valdirene T; Pietra, Rafaella X; Ferreira, Luana C A; Rocha, Patrícia P O; da S Gonçalves, Michele; da C César, Giovana; de S Lima, Joziele; Medeiros, Paula F V; Mazzeu, Juliana F; Jehee, Fernanda S

2017-08-01

Congenital heart disease (CHD) is the most common congenital disorder among live births. When associated with extracardiac abnormalities, it is characterized as a syndromic heart disease (syndromic CHD) and corresponds to 25% of all liveborn infants with a heart defect. The etiology in about 65% of the cases still remains unknown, and in about 35% of the patients, it is associated with genetic factors. In the present study, MLPA and SNP-array techniques were used to investigate a group of 47 patients with syndromic CHD. In total, 16 defects (34%) were identified, of which 12 (25.5%) were classified as pathogenic or probably pathogenic. The most frequent abnormalities were 22q11.2 deletion (22q11.2 deletion syndrome) and 7q11.23 deletion (Williams-Beuren syndrome). We also show that rarer malformations may be associated with syndromic CHD, such as 14q32.33 deletion as well as 17q25.3, 15q11.2 (BP1-BP2), 22q13.31, and 12p13.31 ( SLC2A3 ) duplications. The present study demonstrates that CNVs are important causal factors and should be studied in patients with syndromic CHD. Furthermore, the use of MLPA as a first screening test was appropriate, as this less expensive technology detected 11 of the 12 pathogenic abnormalities (91.6%).

[FANCA gene mutation analysis in Fanconi anemia patients].

PubMed

Chen, Fei; Peng, Guang-Jie; Zhang, Kejian; Hu, Qun; Zhang, Liu-Qing; Liu, Ai-Guo

2005-10-01

To screen the FANCA gene mutation and explore the FANCA protein function in Fanconi anemia (FA) patients. FANCA protein expression and its interaction with FANCF were analyzed using Western blot and immunoprecipitation in 3 cases of FA-A. Genomic DNA was used for MLPA analysis followed by sequencing. FANCA protein was undetectable and FANCA and FANCF protein interaction was impaired in these 3 cases of FA-A. Each case of FA-A contained biallelic pathogenic mutations in FANCA gene. No functional FANCA protein was found in these 3 cases of FA-A, and intragenic deletion, frame shift and splice site mutation were the major pathogenic mutations found in FANCA gene.

Congenital diaphragmatic hernia (CDH) etiology as revealed by pathway genetics.

PubMed

Kantarci, Sibel; Donahoe, Patricia K

2007-05-15

Congenital diaphragmatic hernia (CDH) is a common birth defect with high mortality and morbidity. Two hundred seventy CDH patients were ascertained, carefully phenotyped, and classified as isolated (diaphragm defects alone) or complex (with additional anomalies) cases. We established different strategies to reveal CDH-critical chromosome loci and genes in humans. Candidate genes for sequencing analyses were selected from CDH animal models, genetic intervals of recurrent chromosomal aberration in humans, such as 15q26.1-q26.2 or 1q41-q42.12, as well as genes in the retinoic acid and related pathways and those known to be involved in embryonic lung development. For instance, FOG2, GATA4, and COUP-TFII are all needed for both normal diaphragm and lung development and are likely all in the same genetic and molecular pathway. Linkage analysis was applied first in a large inbred family and then in four multiplex families with Donnai-Barrow syndrome (DBS) associated with CDH. 10K SNP chip and microsatellite markers revealed a DBS locus on chromosome 2q23.3-q31.1. We applied array-based comparative genomic hybridization (aCGH) techniques to over 30, mostly complex, CDH patients and found a de novo microdeletion in a patient with Fryns syndrome related to CDH. Fluorescence in situ hybridization (FISH) and multiplex ligation-dependent probe amplification (MLPA) techniques allowed us to further define the deletion interval. Our aim is to identify genetic intervals and, in those, to prioritize genes that might reveal molecular pathways, mutations in any step of which, might contribute to the same phenotype. More important, the elucidation of pathways may ultimately provide clues to treatment strategies. (c) 2007 Wiley-Liss, Inc.

A novel succinate dehydrogenase subunit B germline variant associated with head and neck paraganglioma in a Dutch kindred: A family-based study.

PubMed

de Vos, B; Rijken, J A; Adank, M A; Hoksbergen, A W J; Bayley, J P; Leemans, C R; Hensen, E F

2018-06-01

In the Netherlands, the majority of hereditary head and neck paragangliomas (HNPGL) are caused by germline variants in the succinate dehydrogenase genes (SDHD, SDHB, SDHAF2). Here, we evaluate a four-generation family linked to a novel SDHB gene variant with the manifestation of a HNPGL. A family-based study. The VU University Medical Center (VUmc) Amsterdam, a tertiary clinic for Otolaryngology and Head and Neck Surgery. The index patients presented with an embryonic rhabdomyosarcoma and a non-Hodgkin lymphoma. Array-based comparative genomic hybridisation (aCGH) analysis and multiplex ligation-dependent probe amplification (MLPA) revealed a novel deletion of exon 1-3 in the SDHB gene, suspected to predispose to paraganglioma (PGL)/pheochromocytoma (PHEO) syndrome type 4. Subsequently, genetic counselling and DNA testing were offered to all family members at risk. Individuals that tested positive for this novel SDHB gene variant were counselled and additional clinical evaluation was offered for the identification of HNPGL and/or PHEO. The DNA of 18 family members was tested, resulting in the identification of 10 carriers of the exon 1-3 deletion in the SDHB gene. One carrier was diagnosed with a carotid body PGL and serum catecholamine excess, which was surgically excised. Negative SDHB immunostaining of the carotid body tumour confirmed that it was caused by the SDHB variant. The remaining 9 carriers showed no evidence of PGL/PHEO. Deletion of exon 1-3 in the SDHB gene is a novel germline variant associated with the formation of hereditary HNPGL. © 2018 The Authors. Clinical Otolaryngology Published by John Wiley & Sons Ltd.

Association of Germline CHEK2 Gene Variants with Risk and Prognosis of Non-Hodgkin Lymphoma

PubMed Central

Havranek, Ondrej; Kleiblova, Petra; Hojny, Jan; Lhota, Filip; Soucek, Pavel; Trneny, Marek; Kleibl, Zdenek

2015-01-01

The checkpoint kinase 2 gene (CHEK2) codes for the CHK2 protein, an important mediator of the DNA damage response pathway. The CHEK2 gene has been recognized as a multi-cancer susceptibility gene; however, its role in non-Hodgkin lymphoma (NHL) remains unclear. We performed mutation analysis of the entire CHEK2 coding sequence in 340 NHL patients using denaturing high-performance liquid chromatography (DHPLC) and multiplex ligation-dependent probe amplification (MLPA). Identified hereditary variants were genotyped in 445 non-cancer controls. The influence of CHEK2 variants on disease risk was statistically evaluated. Identified CHEK2 germline variants included four truncating mutations (found in five patients and no control; P = 0.02) and nine missense variants (found in 21 patients and 12 controls; P = 0.02). Carriers of non-synonymous variants had an increased risk of NHL development [odds ratio (OR) 2.86; 95% confidence interval (CI) 1.42–5.79] and an unfavorable prognosis [hazard ratio (HR) of progression-free survival (PFS) 2.1; 95% CI 1.12–4.05]. In contrast, the most frequent intronic variant c.319+43dupA (identified in 22% of patients and 31% of controls) was associated with a decreased NHL risk (OR = 0.62; 95% CI 0.45–0.86), but its positive prognostic effect was limited to NHL patients with diffuse large B-cell lymphoma (DLBCL) treated by conventional chemotherapy without rituximab (HR-PFS 0.4; 94% CI 0.17–0.74). Our results show that germ-line CHEK2 mutations affecting protein coding sequence confer a moderately-increased risk of NHL, they are associated with an unfavorable NHL prognosis, and they may represent a valuable predictive biomarker for patients with DLBCL. PMID:26506619

Association of Germline CHEK2 Gene Variants with Risk and Prognosis of Non-Hodgkin Lymphoma.

PubMed

Havranek, Ondrej; Kleiblova, Petra; Hojny, Jan; Lhota, Filip; Soucek, Pavel; Trneny, Marek; Kleibl, Zdenek

2015-01-01

The checkpoint kinase 2 gene (CHEK2) codes for the CHK2 protein, an important mediator of the DNA damage response pathway. The CHEK2 gene has been recognized as a multi-cancer susceptibility gene; however, its role in non-Hodgkin lymphoma (NHL) remains unclear. We performed mutation analysis of the entire CHEK2 coding sequence in 340 NHL patients using denaturing high-performance liquid chromatography (DHPLC) and multiplex ligation-dependent probe amplification (MLPA). Identified hereditary variants were genotyped in 445 non-cancer controls. The influence of CHEK2 variants on disease risk was statistically evaluated. Identified CHEK2 germline variants included four truncating mutations (found in five patients and no control; P = 0.02) and nine missense variants (found in 21 patients and 12 controls; P = 0.02). Carriers of non-synonymous variants had an increased risk of NHL development [odds ratio (OR) 2.86; 95% confidence interval (CI) 1.42-5.79] and an unfavorable prognosis [hazard ratio (HR) of progression-free survival (PFS) 2.1; 95% CI 1.12-4.05]. In contrast, the most frequent intronic variant c.319+43dupA (identified in 22% of patients and 31% of controls) was associated with a decreased NHL risk (OR = 0.62; 95% CI 0.45-0.86), but its positive prognostic effect was limited to NHL patients with diffuse large B-cell lymphoma (DLBCL) treated by conventional chemotherapy without rituximab (HR-PFS 0.4; 94% CI 0.17-0.74). Our results show that germ-line CHEK2 mutations affecting protein coding sequence confer a moderately-increased risk of NHL, they are associated with an unfavorable NHL prognosis, and they may represent a valuable predictive biomarker for patients with DLBCL.

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.

First Report of a Single Exon Deletion in TCOF1 Causing Treacher Collins Syndrome

PubMed Central

Beygo, J.; Buiting, K.; Seland, S.; Lüdecke, H.-J.; Hehr, U.; Lich, C.; Prager, B.; Lohmann, D.R.; Wieczorek, D.

2012-01-01

Treacher Collins syndrome (TCS) is a rare craniofacial disorder characterized by facial anomalies and ear defects. TCS is caused by mutations in the TCOF1 gene and follows autosomal dominant inheritance. Recently, mutations in the POLR1D and POLR1C genes have also been identified to cause TCS. However, in a subset of patients no causative mutation could be found yet. Inter- and intrafamilial phenotypic variability is high as is the variety of mainly family-specific mutations identified throughout TCOF1. No obvious correlation between pheno- and genotype could be observed. The majority of described point mutations, small insertions and deletions comprising only a few nucleotides within TCOF1 lead to a premature termination codon. We investigated a cohort of 112 patients with a tentative clinical diagnosis of TCS by multiplex ligation-dependent probe amplification (MLPA) to search for larger deletions not detectable with other methods used. All patients were selected after negative screening for mutations in TCOF1, POLR1D and POLR1C. In 1 patient with an unequivocal clinical diagnosis of TCS, we identified a 3.367 kb deletion. This deletion abolishes exon 3 and is the first described single exon deletion within TCOF1. On RNA level we observed loss of this exon which supposedly leads to haploinsufficiency of TREACLE, the nucleolar phosphoprotein encoded by TCOF1. PMID:22712005

First Report of a Single Exon Deletion in TCOF1 Causing Treacher Collins Syndrome.

PubMed

Beygo, J; Buiting, K; Seland, S; Lüdecke, H-J; Hehr, U; Lich, C; Prager, B; Lohmann, D R; Wieczorek, D

2012-01-01

Treacher Collins syndrome (TCS) is a rare craniofacial disorder characterized by facial anomalies and ear defects. TCS is caused by mutations in the TCOF1 gene and follows autosomal dominant inheritance. Recently, mutations in the POLR1D and POLR1C genes have also been identified to cause TCS. However, in a subset of patients no causative mutation could be found yet. Inter- and intrafamilial phenotypic variability is high as is the variety of mainly family-specific mutations identified throughout TCOF1. No obvious correlation between pheno- and genotype could be observed. The majority of described point mutations, small insertions and deletions comprising only a few nucleotides within TCOF1 lead to a premature termination codon. We investigated a cohort of 112 patients with a tentative clinical diagnosis of TCS by multiplex ligation-dependent probe amplification (MLPA) to search for larger deletions not detectable with other methods used. All patients were selected after negative screening for mutations in TCOF1, POLR1D and POLR1C. In 1 patient with an unequivocal clinical diagnosis of TCS, we identified a 3.367 kb deletion. This deletion abolishes exon 3 and is the first described single exon deletion within TCOF1. On RNA level we observed loss of this exon which supposedly leads to haploinsufficiency of TREACLE, the nucleolar phosphoprotein encoded by TCOF1.

Identical cryptic partial monosomy 20pter and trisomy 20qter in three adult siblings due to a large maternal pericentric inversion: detection by MLPA and breakpoint mapping by SNP array analysis.

PubMed

Stevens, Servi J C; Smeets, Eric E J G L; Blom, Eveline; van Uum, Chris M J; Albrechts, Jozefa C M; Herbergs, Jos; Janssen, Jannie W M; Engelen, John J M

2009-10-01

Genotypic and phenotypic data are presented on three adult siblings with mild to moderate mental retardation and mild dysmorphic features. All three siblings showed a chromosome 20 gain at the q-telomere and loss at the p-telomere in routine subtelomeric MLPA screening. Analysis of GTG-banded chromosomes did not detect any abnormalities, but subtelomeric fluorescent in situ hybridization (FISH) confirmed cryptic partial monosomy of chromosome region 20p13 --> 20pter and cryptic partial trisomy of chromosome region 20q13.33 --> 20qter. Furthermore, FISH analysis in the mother showed a cryptic inv(20)(p13q13.33). This explained the cytogenetic mechanism underlying the chromosomal imbalance in the three children, that is, the meiotic formation of a recombinant chromosome 20 due to crossing-over in the inverted segment. All three children thus carried a rec(20)dup(20q)inv(20)(p13q13.33)mat chromosome. SNP array analysis enabled rapid and detailed imbalance sizing and showed a 1.06 Mb loss in 20p13 and a 2.51 Mb gain in 20q13.33, comprising 21 and 78 genes, respectively. The maternal inversion is the largest described thus far for chromosome 20, comprising 94.4% of its length. Such large inversions result in a particularly high risk for live-born unbalanced offspring because the partial monosomy and trisomy segments are small. Moreover, the inversion size is directly related to the percentage of unbalanced gametes due to high crossing-over change within the inverted segment. The fact that all three children carry an identical chromosomal rearrangement has consequences for genetic counseling for carriers of large pericentric inversions, as the recurrence risk is very high.

Miniaturized isothermal nucleic acid amplification, a review.

PubMed

Asiello, Peter J; Baeumner, Antje J

2011-04-21

Micro-Total Analysis Systems (µTAS) for use in on-site rapid detection of DNA or RNA are increasingly being developed. Here, amplification of the target sequence is key to increasing sensitivity, enabling single-cell and few-copy nucleic acid detection. The several advantages to miniaturizing amplification reactions and coupling them with sample preparation and detection on the same chip are well known and include fewer manual steps, preventing contamination, and significantly reducing the volume of expensive reagents. To-date, the majority of miniaturized systems for nucleic acid analysis have used the polymerase chain reaction (PCR) for amplification and those systems are covered in previous reviews. This review provides a thorough overview of miniaturized analysis systems using alternatives to PCR, specifically isothermal amplification reactions. With no need for thermal cycling, isothermal microsystems can be designed to be simple and low-energy consuming and therefore may outperform PCR in portable, battery-operated detection systems in the future. The main isothermal methods as miniaturized systems reviewed here include nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), rolling circle amplification (RCA), and strand displacement amplification (SDA). Also, important design criteria for the miniaturized devices are discussed. Finally, the potential of miniaturization of some new isothermal methods such as the exponential amplification reaction (EXPAR), isothermal and chimeric primer-initiated amplification of nucleic acids (ICANs), signal-mediated amplification of RNA technology (SMART) and others is presented.

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.

Prospective study of physical activity and sleep in middle-aged and older adults.

PubMed

Tsunoda, Kenji; Kitano, Naruki; Kai, Yuko; Uchida, Ken; Kuchiki, Tsutomu; Okura, Tomohiro; Nagamatsu, Toshiya

2015-06-01

Few prospective cohort studies have examined the association between physical activity (PA) and insomnia prevention, and the effective PA intensity remains unclear. This prospective study explores how PA intensity prevents incident short sleep duration and subjective insufficient sleep in middle-aged and older adults. A self-reported questionnaire gathered data on sleep and PA variables, including moderate low-intensity PA (MLPA); moderate high-intensity PA (MHPA); and vigorous-intensity PA (VPA), during health checkups conducted in Meiji Yasuda Shinjuku Medical Center in Tokyo. This study followed two cohorts from a 2008 baseline survey: (1) participants free of short sleep duration (n=7,061) and (2) participants free of insufficient sleep (n=7,385). They were divided into middle-aged (<60 years; 45.7 [8.8] years for sleep duration and 45.5 [8.8] years for sleep sufficiency) and older adults (both groups aged 65.3 [4.7] years) and followed for a mean 3.4 years until 2013. Data were analyzed in 2014. Engaging in MHPA (hazard ratio [HR]=0.81, 95% CI=0.67, 0.98) and VPA (HR=0.83, 95% CI=0.71, 0.97) had a significant preventive effect on incident subjective insufficient sleep among middle-aged adults. For older adults, only MLPA (HR=0.58, 95% CI=0.42, 0.81) had a significant preventive effect on incident insufficient sleep, and PA did not significantly affect incident short sleep duration. Middle-aged adults engaging in MHPA and VPA and older adults engaging in MLPA can effectively maintain sleep sufficiency. When providing an effective PA program to prevent insomnia, the intensity of PA should correspond to the participant's age. Copyright © 2015 American Journal of Preventive Medicine. Published by Elsevier Inc. All rights reserved.

Non-biased and efficient global amplification of a single-cell cDNA library

PubMed Central

Huang, Huan; Goto, Mari; Tsunoda, Hiroyuki; Sun, Lizhou; Taniguchi, Kiyomi; Matsunaga, Hiroko; Kambara, Hideki

2014-01-01

Analysis of single-cell gene expression promises a more precise understanding of molecular mechanisms of a living system. Most techniques only allow studies of the expressions for limited numbers of gene species. When amplification of cDNA was carried out for analysing more genes, amplification biases were frequently reported. A non-biased and efficient global-amplification method, which uses a single-cell cDNA library immobilized on beads, was developed for analysing entire gene expressions for single cells. Every step in this analysis from reverse transcription to cDNA amplification was optimized. By removing degrading excess primers, the bias due to the digestion of cDNA was prevented. Since the residual reagents, which affect the efficiency of each subsequent reaction, could be removed by washing beads, the conditions for uniform and maximized amplification of cDNAs were achieved. The differences in the amplification rates for randomly selected eight genes were within 1.5-folds, which could be negligible for most of the applications of single-cell analysis. The global amplification gives a large amount of amplified cDNA (>100 μg) from a single cell (2-pg mRNA), and that amount is enough for downstream analysis. The proposed global-amplification method was used to analyse transcript ratios of multiple cDNA targets (from several copies to several thousand copies) quantitatively. PMID:24141095

Isothermal Amplification Methods for the Detection of Nucleic Acids in Microfluidic Devices

PubMed Central

Zanoli, Laura Maria; Spoto, Giuseppe

2012-01-01

Diagnostic tools for biomolecular detection need to fulfill specific requirements in terms of sensitivity, selectivity and high-throughput in order to widen their applicability and to minimize the cost of the assay. The nucleic acid amplification is a key step in DNA detection assays. It contributes to improving the assay sensitivity by enabling the detection of a limited number of target molecules. The use of microfluidic devices to miniaturize amplification protocols reduces the required sample volume and the analysis times and offers new possibilities for the process automation and integration in one single device. The vast majority of miniaturized systems for nucleic acid analysis exploit the polymerase chain reaction (PCR) amplification method, which requires repeated cycles of three or two temperature-dependent steps during the amplification of the nucleic acid target sequence. In contrast, low temperature isothermal amplification methods have no need for thermal cycling thus requiring simplified microfluidic device features. Here, the use of miniaturized analysis systems using isothermal amplification reactions for the nucleic acid amplification will be discussed. PMID:25587397

High-throughput amplification of mature microRNAs in uncharacterized animal models using polyadenylated RNA and stem-loop reverse transcription polymerase chain reaction.

PubMed

Biggar, Kyle K; Wu, Cheng-Wei; Storey, Kenneth B

2014-10-01

This study makes a significant advancement on a microRNA amplification technique previously used for expression analysis and sequencing in animal models without annotated mature microRNA sequences. As research progresses into the post-genomic era of microRNA prediction and analysis, the need for a rapid and cost-effective method for microRNA amplification is critical to facilitate wide-scale analysis of microRNA expression. To facilitate this requirement, we have reoptimized the design of amplification primers and introduced a polyadenylation step to allow amplification of all mature microRNAs from a single RNA sample. Importantly, this method retains the ability to sequence reverse transcription polymerase chain reaction (RT-PCR) products, validating microRNA-specific amplification. Copyright © 2014 Elsevier Inc. All rights reserved.

[Numeric alterations in the dys gene and their association with clinical features].

PubMed

Mampel, Alejandra; Echeverría, María Inés; Vargas, Ana Lía; Roque, María

2011-01-01

The Duchenne/Becker muscular dystrophy is a hereditary miopathy with a recessive sex-linked pattern. The related gene is called DYS and the coded protein plays a crucial role in the anchorage between the cytoskeleton and the cellular membrane in muscle cells. Different clinical manifestations are observed depending on the impact of the genetic alteration on the protein. The global register of mutations reveals an enhanced frequency for deletions/duplications of one or more exons affecting the DYS gene. In the present work, numeric alterations have been studied in the 79 exons of the DYS gene. The study has been performed on 59 individuals, including 31 independent cases and 28 cases with a familial link. The applied methodology was Multiplex Ligation Dependent Probe Amplification (MLPA). In the 31 independent cases clinical data were established: i.e. the clinical score, the Raven test percentiles, and the creatininphosphokinase (CPK) blood values. Our results reveal a 61.3% frequency of numeric alterations affecting the DYS gene in our population, provoking all of them a reading frame shift. The rate for de novo mutations was identified as 35.2%. Alterations involving a specific region of one exon were observed with high frequency, affecting a specific region. A significant association was found between numeric alterations and a low percentile for the Raven test. These data contribute to the local knowledge of genetic alterations and their phenotypic impact for the Duchenne/Becker disease.

Copy number profiling of adult relapsed B-cell precursor acute lymphoblastic leukemia reveals potential leukemia progression mechanisms.

PubMed

Ribera, Jordi; Zamora, Lurdes; Morgades, Mireia; Mallo, Mar; Solanes, Neus; Batlle, Montserrat; Vives, Susana; Granada, Isabel; Juncà, Jordi; Malinverni, Roberto; Genescà, Eulàlia; Guàrdia, Ramon; Mercadal, Santiago; Escoda, Lourdes; Martinez-Lopez, Joaquín; Tormo, Mar; Esteve, Jordi; Pratcorona, Marta; Martinez-Losada, Carmen; Solé, Francesc; Feliu, Evarist; Ribera, Josep-Maria

2017-11-01

The outcome of relapsed adult acute lymphoblastic leukemia (ALL) remains dismal despite new therapeutic approaches. Previous studies analyzing relapse samples have shown a high degree of heterogeneity regarding gene alterations without an evident relapse signature. Bone marrow or peripheral blood samples from 31 adult B-cell precursor ALL patients at first relapse, and 21 paired diagnostic samples were analyzed by multiplex ligation probe-dependent amplification (MLPA). Nineteen paired diagnostic and relapse samples of these 21 patients were also analyzed by SNP arrays. A trend to acquire homozygous CDKN2A/B deletions and a significant increase in the number of copy number alterations (CNA) was observed from diagnosis to first relapse. Evolution from an ancestral clone was the main pattern of clonal evolution. Relapse samples were extremely heterogeneous regarding CNA frequencies. However, CDKN2A/B, PAX5, ETV6, ATM, IKZF1, VPREB1, and TP53 deletions and duplications of 1q, 8q, 17q, 21, X/Y PAR1, and Xp were frequently detected at relapse. Duplications of genes involved in cell proliferation, drug resistance and stem cell homeostasis regulation, as well as deletions of KDM6A and STAG2 genes emerged as specific alterations at relapse. Genomics of relapsed adult B-cell precursor ALL is highly heterogeneous, although some recurrent lesions involved in essential pathways deregulation were frequently observed. Selective and simultaneous targeting of these deregulated pathways may improve the results of current salvage therapies. © 2017 Wiley Periodicals, Inc.

Methylation status as a predictor of intravesical Bacillus Calmette-Guérin (BCG) immunotherapy response of high grade non-muscle invasive bladder tumor.

PubMed

Husek, Petr; Pacovsky, Jaroslav; Chmelarova, Marcela; Podhola, Miroslav; Brodak, Milos

2017-06-01

Genetic and epigenetic alterations play an important role in urothelial cancer pathogenesis. Deeper understanding of these processes could help us achieve better diagnosis and management of this life-threatening disease. The aim of this research was to evaluate the methylation status of selected tumor suppressor genes for predicting BCG response in patients with high grade non-muscle-invasive bladder tumor (NMIBC). We retrospectively evaluated 82 patients with high grade non-muscle-invasive bladder tumor (stage Ta, T1, CIS) who had undergone BCG instillation therapy. We compared epigenetic methylation status in BCG-responsive and BCG-failure groups. We used the MS-MLPA (Methylation-Specific Multiplex Ligation-Dependent Probe Amplification probe sets ME001 and ME004. The control group was 13 specimens of normal urotel (bladder tissue)). Newly identified methylations in high grade NMIBC were found in MUS81a, NTRK1 and PCCA. The methylation status of CDKN2B (P=0.00312 ** ) and MUS81a (P=0.0191 * ) is associated with clinical outcomes of BCG instillation therapy response. CDKN2B and MUS81a unmethylation was found in BCG failure patients. The results show that the methylation status of selected tumor suppressor genes (TSGs) has the potential for predicting BCG response in patients with NMIBC high grade tumors. Tumor suppressor genes such as CDKN2b, MUS81a, PFM-1, MSH6 and THBS1 are very promising for future research.

[The prevalence of SHOX gene deletion in children with idiopathic short stature. A multicentric study].

PubMed

Dávid, Anna; Butz, Henriett; Halász, Zita; Török, Dóra; Nyirő, Gábor; Muzsnai, Ágota; Csákváry, Violetta; Luczay, Andrea; Sallai, Ágnes; Hosszú, Éva; Felszeghy, Enikő; Tar, Attila; Szántó, Zsuzsanna; Fekete, Gy László; Kun, Imre; Patócs, Attila; Bertalan, Rita

2017-08-01

The isolated haploinsufficiency of the SHOX gene is one of the most common cause of short stature determined by monogenic mutations. The heterozygous deviation of the gene can be detected in 2-15% of patients with idiopathic short stature (ISS), in 50-90% of patients with Leri-Weill dyschondrosteosis syndrome (LWS), and in almost 100% of patients with Turner syndrome. The aim of our study was to evaluate the frequency of SHOX gene haploinsufficiency in children with ISS, LWS and in patients having Turner syndrome phenotype (TF), but normal karyotype, and to identify the dysmorphic signs characteristic for SHOX gene deficiency. A total of 144 patients were included in the study. Multiplex Ligation-dependent Probe Amplification (MLPA) method was used to identify the SHOX gene haploinsufficiency. The relationships between clinical data (axiological parameters, skeletal disorders, dysmorphic signs) and genotype were analyzed by statistical methods. 11 (7.6%) of the 144 patients showed SHOX gene deficiency with female dominance (8/11, 81% female). The SHOX positive patients had a significantly higher BMI (in 5/11 vs. 20/133 cases, p<0.02) and presented more frequent dysmorphic signs (9/11vs 62/133, p = 0.02). Madelung deformity of the upper limbs was also significantly more frequent among the SHOX positive patients (4/11, i.e. 36%, vs. 14/133, i.e. 10%, p = 0.0066). There were no statistically significant differences between the mean age, mean height and auxological measurements (sitting height/height, arm span/height) between the two groups of patients. The occurrence of SHOX gene haploinsufficiency observed in our population corresponds to the literature data. In SHOX positive patients, in addition to short stature, the dysmorphic signs have a positive predictive value for SHOX gene alterations. However, the SHOX deletion detected in a patient with idiopathic short stature without dysmorphic signs suggest that SHOX deletion analysis can be recommended in patients with ISS. Orv Hetil. 2017; 158(34): 1351-1356.

Identification of diagnostic markers in colorectal cancer via integrative epigenomics and genomics data

PubMed Central

KOK-SIN, TEOW; MOKHTAR, NORFILZA MOHD; HASSAN, NUR ZARINA ALI; SAGAP, ISMAIL; ROSE, ISA MOHAMED; HARUN, ROSLAN; JAMAL, RAHMAN

2015-01-01

Apart from genetic mutations, epigenetic alteration is a common phenomenon that contributes to neoplastic transformation in colorectal cancer. Transcriptional silencing of tumor-suppressor genes without changes in the DNA sequence is explained by the existence of promoter hypermethylation. To test this hypothesis, we integrated the epigenome and transcriptome data from a similar set of colorectal tissue samples. Methylation profiling was performed using the Illumina InfiniumHumanMethylation27 BeadChip on 55 paired cancer and adjacent normal epithelial cells. Fifteen of the 55 paired tissues were used for gene expression profiling using the Affymetrix GeneChip Human Gene 1.0 ST array. Validation was carried out on 150 colorectal tissues using the methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) technique. PCA and supervised hierarchical clustering in the two microarray datasets showed good separation between cancer and normal samples. Significant genes from the two analyses were obtained based on a ≥2-fold change and a false discovery rate (FDR) P-value of <0.05. We identified 1,081 differentially hypermethylated CpG sites and 36 hypomethylated CpG sites. We also found 709 upregulated and 699 downregulated genes from the gene expression profiling. A comparison of the two datasets revealed 32 overlapping genes with 27 being hypermethylated with downregulated expression and 4 hypermethylated with upregulated expression. One gene was found to be hypomethylated and downregulated. The most enriched molecular pathway identified was cell adhesion molecules that involved 4 overlapped genes, JAM2, NCAM1, ITGA8 and CNTN1. In the present study, we successfully identified a group of genes that showed methylation and gene expression changes in well-defined colorectal cancer tissues with high purity. The integrated analysis gives additional insight regarding the regulation of colorectal cancer-associated genes and their underlying mechanisms that contribute to colorectal carcinogenesis. PMID:25997610

Parkin dosage mutations have greater pathogenicity in familial PD than simple sequence mutations

PubMed Central

Pankratz, N; Kissell, D K.; Pauciulo, M W.; Halter, C A.; Rudolph, A; Pfeiffer, R F.; Marder, K S.; Foroud, T; Nichols, W C.

2009-01-01

Objective: Mutations in both alleles of parkin have been shown to result in Parkinson disease (PD). However, it is unclear whether haploinsufficiency (presence of a mutation in only 1 of the 2 parkin alleles) increases the risk for PD. Methods: We performed comprehensive dosage and sequence analysis of all 12 exons of parkin in a sample of 520 independent patients with familial PD and 263 controls. We evaluated whether presence of a single parkin mutation, either a sequence (point mutation or small insertion/deletion) or dosage (whole exon deletion or duplication) mutation, was found at increased frequency in cases as compared with controls. We then compared the clinical characteristics of cases with 0, 1, or 2 parkin mutations. Results: We identified 55 independent patients with PD with at least 1 parkin mutation and 9 controls with a single sequence mutation. Cases and controls had a similar frequency of single sequence mutations (3.1% vs 3.4%, p = 0.83); however, the cases had a significantly higher rate of dosage mutations (2.6% vs 0%, p = 0.009). Cases with a single dosage mutation were more likely to have an earlier age at onset (50% with onset at ≤45 years) compared with those with no parkin mutations (10%, p = 0.00002); this was not true for cases with only a single sequence mutation (25% with onset at ≤45 years, p = 0.06). Conclusions: Parkin haploinsufficiency, specifically for a dosage mutation rather than a point mutation or small insertion/deletion, is a risk factor for familial PD and may be associated with earlier age at onset. GLOSSARY ADL = Activities of Daily Living; GDS = Geriatric Depression Scale; MLPA = multiplex ligation-dependent probe amplification; MMSE = Mini-Mental State Examination; PD = Parkinson disease; UPDRS = Unified Parkinson’s Disease Rating Scale. PMID:19636047

Comparison of CpG island methylator phenotype (CIMP) frequency in colon cancer using different probe- and gene-specific scoring alternatives on recommended multi-gene panels.

PubMed

Berg, Marianne; Hagland, Hanne R; Søreide, Kjetil

2014-01-01

In colorectal cancer a distinct subgroup of tumours demonstrate the CpG island methylator phenotype (CIMP). However, a consensus of how to score CIMP is not reached, and variation in definition may influence the reported CIMP prevalence in tumours. Thus, we sought to compare currently suggested definitions and cut-offs for methylation markers and how they influence CIMP classification in colon cancer. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), with subsequent fragment analysis, was used to investigate methylation of tumour samples. In total, 31 CpG sites, located in 8 different genes (RUNX3, MLH1, NEUROG1, CDKN2A, IGF2, CRABP1, SOCS1 and CACNA1G) were investigated in 64 distinct colon cancers and 2 colon cancer cell lines. The Ogino gene panel includes all 8 genes, in addition to the Weisenberger panel of which only 5 of the 8 genes included were investigated. In total, 18 alternative combinations of scoring of CIMP positivity on probe-, gene-, and panel-level were analysed and compared. For 47 samples (71%), the CIMP status was constant and independent of criteria used for scoring; 34 samples were constantly scored as CIMP negative, and 13 (20%) consistently scored as CIMP positive. Only four of 31 probes (13%) investigated showed no difference in the numbers of positive samples using the different cut-offs. Within the panels a trend was observed that increasing the gene-level stringency resulted in a larger difference in CIMP positive samples than increasing the probe-level stringency. A significant difference between positive samples using 'the most stringent' as compared to 'the least stringent' criteria (20% vs 46%, respectively; p<0.005) was demonstrated. A statistical significant variation in the frequency of CIMP depending on the cut-offs and genes included in a panel was found, with twice as many positives samples by least compared to most stringent definition used.

Mutation spectrum of RB1 mutations in retinoblastoma cases from Singapore with implications for genetic management and counselling

PubMed Central

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. PMID:28575107

Gain of 1q As a Prognostic Biomarker in Wilms Tumors (WTs) Treated With Preoperative Chemotherapy in the International Society of Paediatric Oncology (SIOP) WT 2001 Trial: A SIOP Renal Tumours Biology Consortium Study

PubMed Central

Chagtai, Tasnim; Zill, Christina; Dainese, Linda; Wegert, Jenny; Savola, Suvi; Popov, Sergey; Mifsud, William; Vujanić, Gordan; Sebire, Neil; Le Bouc, Yves; Ambros, Peter F.; Kager, Leo; O'Sullivan, Maureen J.; Blaise, Annick; Bergeron, Christophe; Mengelbier, Linda Holmquist; Gisselsson, David; Kool, Marcel; Tytgat, Godelieve A.M.; van den Heuvel-Eibrink, Marry M.; Graf, Norbert; van Tinteren, Harm; Coulomb, Aurore; Gessler, Manfred; Williams, Richard Dafydd

2016-01-01

Purpose Wilms tumor (WT) is the most common pediatric renal tumor. Treatment planning under International Society of Paediatric Oncology (SIOP) protocols is based on staging and histologic assessment of response to preoperative chemotherapy. Despite high overall survival (OS), many relapses occur in patients without specific risk factors, and many successfully treated patients are exposed to treatments with significant risks of late effects. To investigate whether molecular biomarkers could improve risk stratification, we assessed 1q status and other potential copy number biomarkers in a large WT series. Materials and Methods WT nephrectomy samples from 586 SIOP WT 2001 patients were analyzed using a multiplex ligation-dependent probe amplification (MLPA) assay that measured the copy number of 1q and other regions of interest. Results One hundred sixty-seven (28%) of 586 WTs had 1q gain. Five-year event-free survival (EFS) was 75.0% in patients with 1q gain (95% CI, 68.5% to 82.0%) and 88.2% in patients without gain (95% CI, 85.0% to 91.4%). OS was 88.4% with gain (95% CI, 83.5% to 93.6%) and 94.4% without gain (95% CI, 92.1% to 96.7%). In univariable analysis, 1q gain was associated with poorer EFS (P < .001; hazard ratio, 2.33) and OS (P = .01; hazard ratio, 2.16). The association of 1q gain with poorer EFS retained significance in multivariable analysis adjusted for 1p and 16q loss, sex, stage, age, and histologic risk group. Gain of 1q remained associated with poorer EFS in tumor subsets limited to either intermediate-risk localized disease or nonanaplastic localized disease. Other notable aberrations associated with poorer EFS included MYCN gain and TP53 loss. Conclusion Gain of 1q is a potentially valuable prognostic biomarker in WT, in addition to histologic response to preoperative chemotherapy and tumor stage. PMID:27432915

MET amplification as a potential therapeutic target in gastric cancer

PubMed Central

Kawakami, Hisato; Okamoto, Isamu; Arao, Tokuzo; Okamoto, Wataru; Matsumoto, Kazuko; Taniguchi, Hirokazu; Kuwata, Kiyoko; Yamaguchi, Haruka; Nishio, Kazuto; Nakagawa, Kazuhiko; Yamada, Yasuhide

2013-01-01

Our aim was to investigate both the prevalence of MET amplification in gastric cancer as well as the potential of this genetic alteration to serve as a therapeutic target in gastric cancer. MET amplification was assessed by initial screening with a PCR-based copy number assay followed by confirmatory FISH analysis in formalin-fixed, paraffin-embedded specimens of gastric cancer obtained at surgery. The effects of MET tyrosine kinase inhibitors (MET-TKIs) in gastric cancer cells with or without MET amplification were also examined. The median MET copy number in 266 cases of gastric cancer was 1.7, with a range of 0.41 to 21.3. We performed FISH analysis for the 15 cases with the highest MET copy numbers. MET amplification was confirmed in the four assessable cases with a MET copy number of at least 4, whereas MET amplification was not detected in those with a gene copy number of <4. The prevalence of MET amplification was thus 1.5% (4 out of 266 cases). Inhibition of MET by MET-TKIs resulted in the induction of apoptosis accompanied by attenuation of downstream MET signaling in gastric cancer cell lines with MET amplification but not in those without this genetic change. MET amplification identifies a small but clinically important subgroup of gastric cancer patients who are likely to respond to MET-TKIs. Furthermore, screening with a PCR-based copy number assay is an efficient way to reduce the number of patients requiring confirmation of MET amplification by FISH analysis. PMID:23327903

Clinicopathological and prognostic significance of cyclin D1 amplification in patients with breast cancer: a meta-analysis.

PubMed

He, Qi; Wu, Jingxun; Liu, Xin-Li; Ma, Yi-Han; Wu, Xiao-Ting; Wang, Wen-Yi; An, Han-Xiang

2017-01-01

Cyclin D1 plays a critical role in tumorigenesis and the regulation of the G1/S transition in the cell cycle. The relationship between cyclin D1 amplification and clinicopathological parameters in patients with breast cancer remains controversial and its impact on survival outcome is not completely clear. We conducted a meta-analysis to investigate the associations between cyclin D1 gene amplification and certain clinicopathological characteristics and the prognosis in breast cancer. Literature search of PubMed (up to August 3, 2016) was performed. We used Stata 12.0 (Stata Corporation, Texas, US) to analyze the correlations between cyclin D1 amplification and clinicopathological features and the prognostic indicator relapse free survival (RFS) and overall survival (OS) in patients with breast cancer. Publication bias analysis and sensitivity analysis were performed. A total of 9,238 breast cancer patients from 21 studies were included. The pooled odds ratios (ORs) indicated that cyclin D1 amplification was significantly associated with estrogen receptor (ER), progesterone receptor (PR), histological grade and lymph node status, but not associated with human epidermal growth factor receptor-2 (HER2) and tumor size. The combined hazard ratios (HRs) for RFS and OS showed that patients with cyclin D1 amplification displayed a 1.31-fold higher risk of recurrence (HR =1.31, 95% confidence interval (95% CI):1.02-1.60, p<0.01), and a risk of mortality 1.22-fold higher times greater than those without cyclin D1 amplification (HR=1.22, 95% CI:0.99- 1.44, p<0.01), respectively. Our meta-analysis indicated that cyclin D1 amplification is significantly associated with established clinicopathological variables and can be used as a poor prognostic indicator for patients with breast cancer.

A Polyphasic and Taxogenomic Evaluation Uncovers Arcobacter cryaerophilus as a Species Complex That Embraces Four Genomovars

PubMed Central

Pérez-Cataluña, Alba; Collado, Luis; Salgado, Oscar; Lefiñanco, Violeta; Figueras, María J.

2018-01-01

The species Arcobacter cryaerophilus is found in many food products of animal origin and is the dominating species in wastewater. In addition, it is associated with cases of farm animal and human infectious diseases,. The species embraces two subgroups i.e., 1A (LMG 24291T = LMG 9904T) and 1B (LMG 10829) that can be differentiated by their 16S rRNA-RFLP pattern. However, some authors, on the basis of the shared intermediate levels of DNA-DNA hybridization, have suggested abandoning the subgroup classification. This contradiction indicates that the taxonomy of this species is not yet resolved. The objective of the present study was to perform a taxonomic evaluation of the diversity of A. cryaerophilus. Genomic information was used along with a Multilocus Phylogenetic Analysis (MLPA) and phenotypic characterization on a group of 52 temporally and geographically dispersed strains, coming from different types of samples and hosts from nine countries. The MLPA analysis showed that those strains formed four clusters (I–IV). Values of Average Nucleotide Identity (ANI) and in silico DNA-DNA Hybridization (isDDH) obtained between 13 genomes representing strains of the four clusters were below the proposed cut-offs of 96 and 70%, respectively, confirming that each of the clusters represented a different genomic species. However, none of the evaluated phenotypic tests enabled their unequivocal differentiation into species. Therefore, the genomic delimited clusters should be considered genomovars of the species A. cryaerophilus. These genomovars could have different clinical importance, since only the cluster I included strains isolated from human specimens. The discovery of at least one stable distinctive phenotypic character would be needed to define each cluster or genomovar as a different species. Until then, we propose naming them “A. cryaerophilus gv. pseudocryaerophilus” (Cluster I = LMG 10229T), “A. cryaerophilus gv. crypticus” (Cluster II = LMG 9065T), “A. cryaerophilus gv. cryaerophilus” (Cluster III = LMG 24291T) and “A. cryaerophilus gv. occultus” (Cluster IV = LMG 29976T).

Genome amplification of single sperm using multiple displacement amplification.

PubMed

Jiang, Zhengwen; Zhang, Xingqi; Deka, Ranjan; Jin, Li

2005-06-07

Sperm typing is an effective way to study recombination rate on a fine scale in regions of interest. There are two strategies for the amplification of single meiotic recombinants: repulsion-phase allele-specific PCR and whole genome amplification (WGA). The former can selectively amplify single recombinant molecules from a batch of sperm but is not scalable for high-throughput operation. Currently, primer extension pre-amplification is the only method used in WGA of single sperm, whereas it has limited capacity to produce high-coverage products enough for the analysis of local recombination rate in multiple large regions. Here, we applied for the first time a recently developed WGA method, multiple displacement amplification (MDA), to amplify single sperm DNA, and demonstrated its great potential for producing high-yield and high-coverage products. In a 50 mul reaction, 76 or 93% of loci can be amplified at least 2500- or 250-fold, respectively, from single sperm DNA, and second-round MDA can further offer >200-fold amplification. The MDA products are usable for a variety of genetic applications, including sequencing and microsatellite marker and single nucleotide polymorphism (SNP) analysis. The use of MDA in single sperm amplification may open a new era for studies on local recombination rates.

Molecular diagnosis of α-thalassemia in a multiethnic population.

PubMed

Gilad, Oded; Shemer, Orna Steinberg; Dgany, Orly; Krasnov, Tanya; Nevo, Michal; Noy-Lotan, Sharon; Rabinowicz, Ron; Amitai, Nofar; Ben-Dor, Shifra; Yaniv, Isaac; Yacobovich, Joanne; Tamary, Hannah

2017-06-01

α-Thalassemia, one of the most common genetic diseases, is caused by deletions or point mutations affecting one to four α-globin genes. Molecular diagnosis is important to prevent the most severe forms of the disease. However, the diagnosis of α-thalassemia is complex due to a high variability of the genetic defects involved, with over 250 described mutations. We summarize herein the findings of genetic analyses of DNA samples referred to our laboratory for the molecular diagnosis of α-thalassemia, along with a detailed clinical description. We utilized a diagnostic algorithm including Gap-PCR, to detect known deletions, followed by sequencing of the α-globin gene, to identify known and novel point mutations, and multiplex ligation-dependent probe amplification (MLPA) for the diagnosis of rare or novel deletions. α-Thalassemia was diagnosed in 662 of 975 samples referred to our laboratory. Most commonly found were deletions (75.3%, including two novel deletions previously described by us); point mutations comprised 25.4% of the cases, including five novel mutations. Our population included mostly Jews (of Ashkenazi and Sephardic origin) and Muslim Arabs, who presented with a higher rate of point mutations and hemoglobin H disease. Overall, we detected 53 different genotype combinations causing a spectrum of clinical phenotypes, from asymptomatic to severe anemia. Our work constitutes the largest group of patients with α-thalassemia originating in the Mediterranean whose clinical characteristics and molecular basis have been determined. We suggest a diagnostic algorithm that leads to an accurate molecular diagnosis in multiethnic populations. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Effect of non-tuberculous Mycobacteria on host biomarkers potentially relevant for tuberculosis management.

PubMed

Dhanasekaran, S; Jenum, Synne; Stavrum, Ruth; Wiker, Harald G; Kenneth, John; Vaz, Mario; Doherty, T Mark; Grewal, Harleen M S

2014-10-01

Non-tuberculous mycobacteria (NTM) are different from Mycobacterium tuberculosis (MTB) both in their ubiquitous environmental distribution and in their reduced capacity to cause disease. While often neglected in favour of other infectious diseases, NTM may interfere with important aspects of TB control and management, namely the efficacy of new anti-tuberculosis (TB) vaccines; the immuno-diagnostic Tuberculin skin test (TST) and QuantiFERON TB Gold In Tube assay (QFTGIT); and immune biomarkers explored for their diagnostic and/or predictive potential. Our objective was therefore to explore host immune biomarkers in children who had NTM isolated from respiratory and/or gastric specimens. The present study was nested within a prospective cohort study of BCG-vaccinated neonates in Southern India. In this setting, immune biomarkers from peripheral blood were analyzed in 210 children aged <3 years evaluated for TB using dual-colour-Reverse-Transcriptase-Multiple-Ligation-dependent-Probe-Amplification (dcRT-MLPA) and Bio-Plex assays. The children were classified based on clinical examination, chest X-rays and mycobacterial culture reports as either: 1) TB disease, 2) NTM present and 3) controls. The study shows a down-regulation of RAB33A (p<0.001) and up-regulation of TGFβ1, IL-2 and IL-6 (all p<0.05) in children with TB disease, and that RAB33A, TGFBR2 and IL-10 (all p<0.05) were differentially expressed in children with NTM present when compared to children that were culture negative for MTB and NTM (controls). Carriage of NTM may reduce the specificity of future diagnostic and predictive immune biomarkers relevant to TB management.

Spectrum of mutations in homozygous familial hypercholesterolemia in India, with four novel mutations.

PubMed

Setia, Nitika; Saxena, Renu; Arora, Anjali; Verma, Ishwar C

2016-12-01

Homozygous familial hypercholesterolemia (FH) is a rare but serious, inherited disorder of lipid metabolism characterized by very high total and LDL cholesterol levels from birth. It presents as cutaneous and tendon xanthomas since childhood, with or without cardiac involvement. FH is commonly caused by mutations in three genes, i.e. LDL receptor (LDLR), apolipoprotein B (ApoB) and PCSK9. We aimed to determine the spectrum of mutations in cases of homozygous FH in Asian Indians and evaluate if there was any similarity to the mutations observed in Caucasians. Sixteen homozygous FH subjects from eleven families were analyzed for mutations by Sanger sequencing. Large rearrangements in LDLR gene were evaluated by multiplex ligation probe dependent amplification (MLPA) technique. Ten mutations were observed in LDLR gene, of which four mutations were novel. No mutation was detected in ApoB gene and common PCSK9 mutation (p.D374Y). Fourteen cases had homozygous mutations; one had compound heterozygous mutation, while no mutation was detected in one clinically homozygous case. We report an interesting "Triple hit" case with features of homozygous FH. The spectrum of mutations in the Asian Indian population is quite heterogeneous. Of the mutations identified, 40% were novel. No mutation was observed in exons 3, 9 and 14 of LDLR gene, which are considered to be hot spots in studies done on Asian Indians in South Africa. Early detection followed by aggressive therapy, and cascade screening of extended families has been initiated to reduce the morbidity and mortality in these patients. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The ICR96 exon CNV validation series: a resource for orthogonal assessment of exon CNV calling in NGS data.

PubMed

Mahamdallie, Shazia; Ruark, Elise; Yost, Shawn; Ramsay, Emma; Uddin, Imran; Wylie, Harriett; Elliott, Anna; Strydom, Ann; Renwick, Anthony; Seal, Sheila; Rahman, Nazneen

2017-01-01

Detection of deletions and duplications of whole exons (exon CNVs) is a key requirement of genetic testing. Accurate detection of this variant type has proved very challenging in targeted next-generation sequencing (NGS) data, particularly if only a single exon is involved. Many different NGS exon CNV calling methods have been developed over the last five years. Such methods are usually evaluated using simulated and/or in-house data due to a lack of publicly-available datasets with orthogonally generated results. This hinders tool comparisons, transparency and reproducibility. To provide a community resource for assessment of exon CNV calling methods in targeted NGS data, we here present the ICR96 exon CNV validation series. The dataset includes high-quality sequencing data from a targeted NGS assay (the TruSight Cancer Panel) together with Multiplex Ligation-dependent Probe Amplification (MLPA) results for 96 independent samples. 66 samples contain at least one validated exon CNV and 30 samples have validated negative results for exon CNVs in 26 genes. The dataset includes 46 exon CNVs in BRCA1 , BRCA2 , TP53 , MLH1 , MSH2 , MSH6 , PMS2 , EPCAM or PTEN , giving excellent representation of the cancer predisposition genes most frequently tested in clinical practice. Moreover, the validated exon CNVs include 25 single exon CNVs, the most difficult type of exon CNV to detect. The FASTQ files for the ICR96 exon CNV validation series can be accessed through the European-Genome phenome Archive (EGA) under the accession number EGAS00001002428.

Expansion of genetic testing in the division of functional genomics, research center for bioscience and technology, tottori university from 2000 to 2013.

PubMed

Adachi, Kaori

2014-03-01

At the Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University, we have been making an effort to establish a genetic testing facility that can provide the same screening procedures conducted worldwide. Direct Sequencing of PCR products is the main method to detect point mutations, small deletions and insertions. Multiplex Ligation-dependent Probe Amplification (MLPA) was used to detect large deletions or insertions. Expansion of the repeat was analyzed for triplet repeat diseases. Original primers were constructed for 41 diseases when the reported primers failed to amplify the gene. Prediction of functional effects of human nsSNPs (PolyPhen) was used for evaluation of novel mutations. From January 2000 to September 2013, a total of 1,006 DNA samples were subjected to genetic testing in the Division of Functional Genomics, Research Center for Bioscience and Technology, Tottori University. The hospitals that requested genetic testing were located in 43 prefectures in Japan and in 11 foreign countries. The genetic testing covered 62 diseases, and mutations were detected in 287 out of 1,006 with an average mutation detection rate of 24.7%. There were 77 samples for prenatal diagnosis. The number of samples has rapidly increased since 2010. In 2013, the next-generation sequencers were introduced in our facility and are expected to provide more comprehensive genetic testing in the near future. Nowadays, genetic testing is a popular and powerful tool for diagnosis of many genetic diseases. Our genetic testing should be further expanded in the future.

Ankle-Foot Orthosis in Duchenne Muscular Dystrophy: A 4 year Experience in a Multidisciplinary Neuromuscular Disorders Clinic.

PubMed

Gupta, Anupam; Nalini, Atchayaram; Arya, Shanti Prakash; Vengalil, Seena; Khanna, Meeka; Krishnan, Rashmi; Taly, Arun B

2017-03-01

To assess Ankle-Foot-Orthosis (AFO) requirement and ambulation in Duchenne Muscular Dystrophy (DMD) patients seen over a period of 4 y at a multi-disciplinary Neuromuscular disorders clinic (NMD). A study was conducted in university quaternary research hospital with DMD patients confirmed by MLPA (multiplex ligation - dependent probe amplification) method and were evaluated between January 2012 and December 2015. Their ambulatory status, detailed neurological and functional status were recorded. Requirement of AFOs was determined and provided. In total 126 DMD children reported to the NMD clinic. Mean age at presentation was 7.6 y (range 2 to12 y, SD 2.1). Mean duration of illness at first evaluation was 3.4 y (range 0.5 to 10 y, SD 2.0). AFO's were advised at a mean age of 8.5 y (range 7 to 12 y, SD 1.8). Fifty-nine patients were advised AFO as resting or walking splint. At last follow-up 113 patients were still ambulatory whereas 13 had become wheel chair bound. Out of 59 patients, 48 were still wearing AFOs and the remaining discontinued AFOs for various reasons. Children with DMD require wearing of AFOs as resting or walking splint, mostly in first or early second decade of life. As there is some gap between onset of clinical signs and requirement of orthosis, follow-up preferably at a multidisciplinary clinic at regular intervals is desirable for timely intervention in the form of AFOs or other splints to prolong ambulatory status in these patients.

[Two cases of Duchenne muscular dystrophy over 40 years after onset].

PubMed

Ishizaki, Masatoshi; Ueyama, Hidetsugu; Masuda, Teruaki; Nishida, Yasuto; Imamura, Shigehiro; Ando, Yukio

2013-01-01

We report two 45 year old men with Duchenne muscular dystrophy. Case 1 showed a deleted exon 50 of the dystrophin gene by MLPA analysis, and Case 2 showed deleted exons 46-52. Both patients presented with severe weakness of the skeletal muscles and respiratory dysfunction, while cardiac involvement was mild and cognitive function was almost normal. The patients are able to shop at a mall, participate in activities, and attend hobbies, although they are bedridden with artificial respiration through tracheotomy. With the progress of the respiratory care and cardiac protective therapy, the prognosis of Duchenne muscular dystrophy has improved remarkably. At present, it is possible to survive over 40 years with maintenance of quality of life, if cardiac damage is not severe.

Assessment of ERBB2 and EGFR gene amplification and protein expression in gastric carcinoma by immunohistochemistry and fluorescence in situ hybridization

PubMed Central

2011-01-01

Background The goal of this study was to investigate ERBB2(HER2) and EGFR gene amplification and protein expression in gastric cancer. Fluorescence in situ hybridization (FISH) and immunohistochemistry were used to analyze ERBB2 and EGFR gene amplification and protein expression in 69 cases of gastric cancer. Results FISH analysis revealed that 20.3% of the cases exhibited ERBB2 gene amplification. Increases in ERBB2 copy number and gene amplification were present in 52.2% of the samples. Expression of the ERBB2 protein was observed in 42.0% of cases. FISH analysis detected EGFR gene amplification in 29.0% of samples. Increases in EGFR copy number and gene amplification occurred in 57.9% of samples, and EGFR protein expression was present in 52.2% of samples. Both ERBB2 and EGFR gene amplification were 3 cases (4.3%), but abnormalities in both ERBB2 and EGFR gene copy number were present 36.2% of samples. ERBB2 and EGFR gene amplification were significantly associated with the depth of tumor invasion (P < 0.05) and lymph node metastasis (P < 0.05), but not with sex, age, or histological type (P > 0.05). Conclusions Our data indicated that ERBB2 and EGFR genetic abnormalities were associated with the prognosis of gastric cancer. Clinical assessment of ERBB2 and EGFR amplification may represent an important factor for the development of personalized treatment programs for gastic cancer. PMID:21689422

Identification of a novel 15.5 kb SHOX deletion associated with marked intrafamilial phenotypic variability and analysis of its molecular origin.

PubMed

Alexandrou, Angelos; Papaevripidou, Ioannis; Tsangaras, Kyriakos; Alexandrou, Ioanna; Tryfonidis, Marios; Christophidou-Anastasiadou, Violetta; Zamba-Papanicolaou, Eleni; Koumbaris, George; Neocleous, Vassos; Phylactou, Leonidas A; Skordis, Nicos; Tanteles, George A; Sismani, Carolina

2016-12-01

Haploinsufficiency of the short stature homeobox contaning SHOX gene has been shown to result in a spectrum of phenotypes ranging from Leri-Weill dyschondrosteosis (LWD) at the more severe end to SHOX-related short stature at the milder end of the spectrum. Most alterations are whole gene deletions, point mutations within the coding region, or microdeletions in its flanking sequences. Here, we present the clinical and molecular data as well as the potential molecular mechanism underlying a novel microdeletion, causing a variable SHOX-related haploinsufficiency disorder in a three-generation family. The phenotype resembles that of LWD in females, in males, however, the phenotypic expression is milder. The 15523-bp SHOX intragenic deletion, encompassing exons 3-6, was initially detected by array-CGH, followed by MLPA analysis. Sequencing of the breakpoints indicated an Alu recombination-mediated deletion (ARMD) as the potential causative mechanism.

Nucleic acid tool enzymes-aided signal amplification strategy for biochemical analysis: status and challenges.

PubMed

Qing, Taiping; He, Dinggeng; He, Xiaoxiao; Wang, Kemin; Xu, Fengzhou; Wen, Li; Shangguan, Jingfang; Mao, Zhengui; Lei, Yanli

2016-04-01

Owing to their highly efficient catalytic effects and substrate specificity, the nucleic acid tool enzymes are applied as 'nano-tools' for manipulating different nucleic acid substrates both in the test-tube and in living organisms. In addition to the function as molecular scissors and molecular glue in genetic engineering, the application of nucleic acid tool enzymes in biochemical analysis has also been extensively developed in the past few decades. Used as amplifying labels for biorecognition events, the nucleic acid tool enzymes are mainly applied in nucleic acids amplification sensing, as well as the amplification sensing of biorelated variations of nucleic acids. With the introduction of aptamers, which can bind different target molecules, the nucleic acid tool enzymes-aided signal amplification strategies can also be used to sense non-nucleic targets (e.g., ions, small molecules, proteins, and cells). This review describes and discusses the amplification strategies of nucleic acid tool enzymes-aided biosensors for biochemical analysis applications. Various analytes, including nucleic acids, ions, small molecules, proteins, and cells, are reviewed briefly. This work also addresses the future trends and outlooks for signal amplification in nucleic acid tool enzymes-aided biosensors.

Calibrating genomic and allelic coverage bias in single-cell sequencing.

PubMed

Zhang, Cheng-Zhong; Adalsteinsson, Viktor A; Francis, Joshua; Cornils, Hauke; Jung, Joonil; Maire, Cecile; Ligon, Keith L; Meyerson, Matthew; Love, J Christopher

2015-04-16

Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1-10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (∼0.1 × ) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples.

Calibrating genomic and allelic coverage bias in single-cell sequencing

PubMed Central

Francis, Joshua; Cornils, Hauke; Jung, Joonil; Maire, Cecile; Ligon, Keith L.; Meyerson, Matthew; Love, J. Christopher

2016-01-01

Artifacts introduced in whole-genome amplification (WGA) make it difficult to derive accurate genomic information from single-cell genomes and require different analytical strategies from bulk genome analysis. Here, we describe statistical methods to quantitatively assess the amplification bias resulting from whole-genome amplification of single-cell genomic DNA. Analysis of single-cell DNA libraries generated by different technologies revealed universal features of the genome coverage bias predominantly generated at the amplicon level (1–10 kb). The magnitude of coverage bias can be accurately calibrated from low-pass sequencing (~0.1 ×) to predict the depth-of-coverage yield of single-cell DNA libraries sequenced at arbitrary depths. We further provide a benchmark comparison of single-cell libraries generated by multi-strand displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC). Finally, we develop statistical models to calibrate allelic bias in single-cell whole-genome amplification and demonstrate a census-based strategy for efficient and accurate variant detection from low-input biopsy samples. PMID:25879913

Use of Sequence-Independent, Single-Primer-Amplification (SISPA) for rapid detection, identification, and characterization of avian RNA viruses

USDA-ARS?s Scientific Manuscript database

Current technologies with next generation sequencing have revolutionized metagenomics analysis of clinical samples. To achieve the non-selective amplification and recovery of low abundance genetic sequences, a simplified Sequence-Independent, Single-Primer Amplification (SISPA) technique in combinat...

Frequent amplification of receptor tyrosine kinase genes in welldifferentiated/ dedifferentiated liposarcoma.

PubMed

Asano, Naofumi; Yoshida, Akihiko; Mitani, Sachiyo; Kobayashi, Eisuke; Shiotani, Bunsyo; Komiyama, Motokiyo; Fujimoto, Hiroyuki; Chuman, Hirokazu; Morioka, Hideo; Matsumoto, Morio; Nakamura, Masaya; Kubo, Takashi; Kato, Mamoru; Kohno, Takashi; Kawai, Akira; Kondo, Tadashi; Ichikawa, Hitoshi

2017-02-21

Well-differentiated liposarcoma (WDLPS) and dedifferentiated liposarcoma (DDLPS) are closely related tumors commonly characterized by MDM2/CDK4 gene amplification, and lack clinically effective treatment options when inoperable. To identify novel therapeutic targets, we performed targeted genomic sequencing analysis of 19 WDLPS and 37 DDLPS tumor samples using a panel of 104 cancer-related genes (NCC oncopanel v3) developed specifically for genomic testing to select suitable molecular targeted therapies. The results of this analysis indicated that these sarcomas had very few gene mutations and a high frequency of amplifications of not only MDM2 and CDK4 but also other genes. Potential driver mutations were found in only six (11%) samples; however, gene amplification events (other than MDM2 and CDK4 amplification) were identified in 30 (54%) samples. Receptor tyrosine kinase (RTK) genes in particular were amplified in 18 (32%) samples. In addition, growth of a WDLPS cell line with IGF1R amplification was suppressed by simultaneous inhibition of CDK4 and IGF1R, using palbociclib and NVP-AEW541, respectively. Combination therapy with CDK4 and RTK inhibitors may be an effective therapeutic option for WDLPS/DDLPS patients with RTK gene amplification.

Single-molecule dilution and multiple displacement amplification for molecular haplotyping.

PubMed

Paul, Philip; Apgar, Josh

2005-04-01

Separate haploid analysis is frequently required for heterozygous genotyping to resolve phase ambiguity or confirm allelic sequence. We demonstrate a technique of single-molecule dilution followed by multiple strand displacement amplification to haplotype polymorphic alleles. Dilution of DNA to haploid equivalency, or a single molecule, is a simple method for separating di-allelic DNA. Strand displacement amplification is a robust method for non-specific DNA expansion that employs random hexamers and phage polymerase Phi29 for double-stranded DNA displacement and primer extension, resulting in high processivity and exceptional product length. Single-molecule dilution was followed by strand displacement amplification to expand separated alleles to microgram quantities of DNA for more efficient haplotype analysis of heterozygous genes.

Molecular analysis of single oocyst of Eimeria by whole genome amplification (WGA) based nested PCR.

PubMed

Wang, Yunzhou; Tao, Geru; Cui, Yujuan; Lv, Qiyao; Xie, Li; Li, Yuan; Suo, Xun; Qin, Yinghe; Xiao, Lihua; Liu, Xianyong

2014-09-01

PCR-based molecular tools are widely used for the identification and characterization of protozoa. Here we report the molecular analysis of Eimeria species using combined methods of whole genome amplification (WGA) and nested PCR. Single oocyst of Eimeria stiedai or Eimeriamedia was directly used for random amplification of the genomic DNA with either primer extension preamplification (PEP) or multiple displacement amplification (MDA), and then the WGA product was used as template in nested PCR with species-specific primers for ITS-1, 18S rDNA and 23S rDNA of E. stiedai and E. media. WGA-based PCR was successful for the amplification of these genes from single oocyst. For the species identification of single oocyst isolated from mixed E. stiedai or E. media, the results from WGA-based PCR were exactly in accordance with those from morphological identification, suggesting the availability of this method in molecular analysis of eimerian parasites at the single oocyst level. WGA-based PCR method can also be applied for the identification and genetic characterization of other protists. Copyright © 2014 Elsevier Inc. All rights reserved.

Amplification of Genomic DNA for Decoy Receptor 3 Predicts Post-Resection Disease Recurrence in Breast Cancer Patients.

PubMed

Kanbayashi, Chizuko; Koyama, Yu; Ichikawa, Hiroshi; Sakata, Eiko; Hasegawa, Miki; Toshikawa, Chie; Manba, Naoko; Ikarashi, Mayuko; Kobayashi, Takashi; Minagawa, Masahiro; Kosugi, Shin-Ichi; Wakai, Toshifumi

2014-02-01

Decoy receptor 3 (DcR3), a member of the tumor necrosis factor receptor (TNFR) superfamily, shows inhibitory effects on Fas-mediated apoptosis. Currently, data are lacking on the correlation between DcR3 and the recurrence of breast cancer. The authors examined DcR3 mRNA expression and genomic amplification in breast cancer, and investigated the effect of DcR3 gene amplification on prognosis of patients. A total of 95 patients formed the basis of the current retrospective study. DcR3 mRNA expression in breast cancer tissues was examined by RNase protection assay and in situ hybridization. DcR3 gene amplification was examined by quantitative polymerase chain reaction. The correlation between DcR3 gene amplification status and clinicopathological factors was examined and also the relationship between DcR3-Amp and relapse and survival. The relative copy numbers of DcR3 genomic DNA correlated significantly with the levels of DcR3 mRNA expression (ρ = 0.755, P = 0.0067). In addition, lymphatic invasion correlated significantly with DcR3 gene amplification (P = 0.012). However, there was no correlation between the remaining clinicopathological factors and DcR3 gene amplification. In the univariate analysis, the recurrence-free survival (RFS) rate of patients who were positive for DcR3 gene amplification was significantly lower than that of patients who were negative for DcR3 gene amplification (P = 0.0271). Multivariate analysis showed that DcR3 gene amplification (P = 0.028) and disease stage (P < 0.001) remained significant independent predictors of RFS. DcR3 gene amplification was significantly correlated with lymphatic invasion, and also DcR3 gene amplification predicts recurrence after resection, which may be an important prognostic factor in breast cancer patients.

Clinical and genetic characteristics of Pseudohypoparathyroidism in the Chinese population.

PubMed

Chu, Xueying; Zhu, Yan; Wang, Ou; Nie, Min; Quan, Tingting; Xue, Yu; Wang, Wenbo; Jiang, Yan; Li, Mei; Xia, Weibo; Xing, Xiaoping

2018-02-01

Pseudohypoparathyroidism (PHP) is caused by mutations and epimutations in the GNAS locus, and characterized by the possibility of resistance to multiple hormones and Albright's hereditary osteodystrophy. PHP can be classified into the forms 1A/C, sporadic 1B and familial 1B. To obtain an overall view of the clinical and genetic characteristics of the Chinese PHP patient population. From 2000 to 2016, 120 patients were recruited and studied using Sanger sequencing, methylation-specific multiple ligation-dependent probe amplification (MS-MLPA) and combined bisulfite restriction analysis (COBRA). Of these patients, 104 had positive molecular alterations indicative of certain forms of PHP and were included in data analysis. Clinical and laboratory features were compared between PHP1A/C and PHP1B patients. Ten PHP1A/C, 21 familial PHP1B and 73 sporadic PHP1B patients were identified. Four novel GNAS mutations were discovered in these patients, including c.1038+1G>T, c.530+2T>C, c.880_883delCAAG and c.311_312delAAG, insT. The most common symptoms in this series were recurrent tetany (89.4%) and epilepsy (47.1%). The prevalence of weight excess increased with age for PHP1B (10%-35%) and PHP1A/C (50%-75%). Intracranial calcification had a prevalence of 94.6% and correlated with seizures (r = .227, P = .029). Cataracts occurred in 56.2% PHP patients, and there was a trend towards longer disease duration in patients with cataracts (P = .051). Statistically significant differences (P < .05) were observed when comparing certain clinical characteristics between PHP1B and PHP1A/C patients, including age of onset (10 vs 7 year), short stature (21.3% vs 70%), rounded face (60.6% vs 100%), brachydactyly (25.5% vs 100%), ectopic ossification (1.1% vs 40%) and TSH resistance (44.6% vs 90%), respectively. This study is the largest single-centre series of PHP patients and summarizes the clinical and genetic features of the Chinese PHP population. While there was substantial clinical overlap between PHP1A/C and PHP1B, differences in disease progression were observed. © 2017 John Wiley & Sons Ltd.

FGFR1 Amplification Is Often Homogeneous and Strongly Linked to the Squamous Cell Carcinoma Subtype in Esophageal Carcinoma

PubMed Central

Burkhardt, Lia; Simon, Ronald; Steurer, Stefan; Burdak-Rothkamm, Susanne; Jacobsen, Frank; Sauter, Guido; Krech, Till

2015-01-01

Background and Aims Amplification of the fibroblast growth factor receptor 1 (FGFR1) is believed to predict response to multi-kinase inhibitors targeting FGFR1. Esophageal cancer is an aggressive disease, for which novel targeted therapies are highly warranted. Methods This study was designed to investigate the prevalence and clinical significance of FGFR1 amplification in a tissue microarray containing 346 adenocarcinomas and 254 squamous cell carcinomas of the esophagus, using dual-labeling fluorescence in situ hybridization (FISH) analysis. Results FGFR1 amplification, defined as a ratio of FGFR1:centromere 8 copy numbers ≥ 2.0, was more frequently seen in squamous cell carcinoma (8.9% of 202 interpretable cases) than in adenocarcinoma (1.6% of 308; p<0.0001). There was no association between FGFR1 amplification and tumor phenotype or clinical outcome. To study potential heterogeneity of FGFR1 amplification, all available tumor blocks from 23 FGFR1 amplified tumors were analyzed on conventional large sections. This analysis revealed complete homogeneity of FGFR1 amplification in 20 (86.9%) primary tumors and in all available lymph node metastases. Remarkably, FGFR1 amplification was also seen in dysplasia adjacent to tumor in 6 of 9 patients with FGFR1 amplified primary cancers. Conclusions In conclusion, FGFR1 amplification occurs in a relevant subgroup of carcinomas of the esophagus and may play a particular role for development of squamous cell cancers. The high homogeneity of FGFR1 amplification suggests that patients with FGFR1 amplified esophageal cancers may particularly benefit from anti-FGFR1 therapies and prompt for clinical studies in this tumor type. PMID:26555375

Somatic Mutation Allelic Ratio Test Using ddPCR (SMART-ddPCR): An Accurate Method for Assessment of Preferential Allelic Imbalance in Tumor DNA.

PubMed

de Smith, Adam J; Walsh, Kyle M; Hansen, Helen M; Endicott, Alyson A; Wiencke, John K; Metayer, Catherine; Wiemels, Joseph L

2015-01-01

The extent to which heritable genetic variants can affect tumor development has yet to be fully elucidated. Tumor selection of single nucleotide polymorphism (SNP) risk alleles, a phenomenon called preferential allelic imbalance (PAI), has been demonstrated in some cancer types. We developed a novel application of digital PCR termed Somatic Mutation Allelic Ratio Test using Droplet Digital PCR (SMART-ddPCR) for accurate assessment of tumor PAI, and have applied this method to test the hypothesis that heritable SNPs associated with childhood acute lymphoblastic leukemia (ALL) may demonstrate tumor PAI. These SNPs are located at CDKN2A (rs3731217) and IKZF1 (rs4132601), genes frequently lost in ALL, and at CEBPE (rs2239633), ARID5B (rs7089424), PIP4K2A (rs10764338), and GATA3 (rs3824662), genes located on chromosomes gained in high-hyperdiploid ALL. We established thresholds of AI using constitutional DNA from SNP heterozygotes, and subsequently measured allelic copy number in tumor DNA from 19-142 heterozygote samples per SNP locus. We did not find significant tumor PAI at these loci, though CDKN2A and IKZF1 SNPs showed a trend towards preferential selection of the risk allele (p = 0.17 and p = 0.23, respectively). Using a genomic copy number control ddPCR assay, we investigated somatic copy number alterations (SCNA) underlying AI at CDKN2A and IKZF1, revealing a complex range of alterations including homozygous and hemizygous deletions and copy-neutral loss of heterozygosity, with varying degrees of clonality. Copy number estimates from ddPCR showed high agreement with those from multiplex ligation-dependent probe amplification (MLPA) assays. We demonstrate that SMART-ddPCR is a highly accurate method for investigation of tumor PAI and for assessment of the somatic alterations underlying AI. Furthermore, analysis of publicly available data from The Cancer Genome Atlas identified 16 recurrent SCNA loci that contain heritable cancer risk SNPs associated with a matching tumor type, and which represent candidate PAI regions warranting further investigation.

HER-2 amplification in tubular carcinoma of the breast.

PubMed

Oakley, Gerard J; Tubbs, Raymond R; Crowe, Joseph; Sebek, Bruce; Budd, G Thomas; Patrick, Rebecca J; Procop, Gary W

2006-07-01

The prognostic and therapeutic implications of HER-2 gene amplification and estrogen and progesterone receptor status in breast cancer are well described. To address the relative paucity of information concerning HER-2 amplification for tubular carcinomas, we assessed the frequency of gene amplification in 55 tubular carcinomas of the breast from 54 patients, 5 of which had axillary node metastases. The HER-2 gene copy number was assessed by fluorescence in situ hybridization for the majority of tumors analyzed, whereas estrogen and progesterone receptor status was achieved by immunohistochemical analysis. HER-2 gene amplification was not observed in any of the tumors examined, and most were estrogen receptor-positive. This HER-2 gene amplification frequency was significantly lower than the frequency of gene amplification previously reported for all invasive ductal carcinoma of no special type (P < .01). HER-2 gene amplification likely occurs infrequently, or not at all, in tubular carcinomas of the breast, whereas most express estrogen receptors.

Evidence of high-elevation amplification versus Arctic amplification

NASA Astrophysics Data System (ADS)

Wang, Qixiang; Fan, Xiaohui; Wang, Mengben

2016-01-01

Elevation-dependent warming in high-elevation regions and Arctic amplification are of tremendous interest to many scientists who are engaged in studies in climate change. Here, using annual mean temperatures from 2781 global stations for the 1961-2010 period, we find that the warming for the world’s high-elevation stations (>500 m above sea level) is clearly stronger than their low-elevation counterparts; and the high-elevation amplification consists of not only an altitudinal amplification but also a latitudinal amplification. The warming for the high-elevation stations is linearly proportional to the temperature lapse rates along altitudinal and latitudinal gradients, as a result of the functional shape of Stefan-Boltzmann law in both vertical and latitudinal directions. In contrast, neither altitudinal amplification nor latitudinal amplification is found within the Arctic region despite its greater warming than lower latitudes. Further analysis shows that the Arctic amplification is an integrated part of the latitudinal amplification trend for the low-elevation stations (≤500 m above sea level) across the entire low- to high-latitude Northern Hemisphere, also a result of the mathematical shape of Stefan-Boltzmann law but only in latitudinal direction.

Evidence of high-elevation amplification versus Arctic amplification

PubMed Central

Wang, Qixiang; Fan, Xiaohui; Wang, Mengben

2016-01-01

Elevation-dependent warming in high-elevation regions and Arctic amplification are of tremendous interest to many scientists who are engaged in studies in climate change. Here, using annual mean temperatures from 2781 global stations for the 1961–2010 period, we find that the warming for the world’s high-elevation stations (>500 m above sea level) is clearly stronger than their low-elevation counterparts; and the high-elevation amplification consists of not only an altitudinal amplification but also a latitudinal amplification. The warming for the high-elevation stations is linearly proportional to the temperature lapse rates along altitudinal and latitudinal gradients, as a result of the functional shape of Stefan-Boltzmann law in both vertical and latitudinal directions. In contrast, neither altitudinal amplification nor latitudinal amplification is found within the Arctic region despite its greater warming than lower latitudes. Further analysis shows that the Arctic amplification is an integrated part of the latitudinal amplification trend for the low-elevation stations (≤500 m above sea level) across the entire low- to high-latitude Northern Hemisphere, also a result of the mathematical shape of Stefan-Boltzmann law but only in latitudinal direction. PMID:26753547

Contribution of MLH1 constitutional methylation for Lynch syndrome diagnosis in patients with tumor MLH1 downregulation.

PubMed

Pinto, Diana; Pinto, Carla; Guerra, Joana; Pinheiro, Manuela; Santos, Rui; Vedeld, Hege Marie; Yohannes, Zeremariam; Peixoto, Ana; Santos, Catarina; Pinto, Pedro; Lopes, Paula; Lothe, Ragnhild; Lind, Guro Elisabeth; Henrique, Rui; Teixeira, Manuel R

2018-02-01

Constitutional epimutation of the two major mismatch repair genes, MLH1 and MSH2, has been identified as an alternative mechanism that predisposes to the development of Lynch syndrome. In the present work, we aimed to investigate the prevalence of MLH1 constitutional methylation in colorectal cancer (CRC) patients with abnormal expression of the MLH1 protein in their tumors. In a series of 38 patients who met clinical criteria for Lynch syndrome genetic testing, with loss of MLH1 expression in the tumor and with no germline mutations in the MLH1 gene (35/38) or with tumors presenting the BRAF p.Val600Glu mutation (3/38), we screened for constitutional methylation of the MLH1 gene promoter using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) in various biological samples. We found four (4/38; 10.5%) patients with constitutional methylation in the MLH1 gene promoter. RNA studies demonstrated decreased MLH1 expression in the cases with constitutional methylation when compared with controls. We could infer the mosaic nature of MLH1 constitutional hypermethylation in tissues originated from different embryonic germ layers, and in one family we could show that it occurred de novo. We conclude that constitutional MLH1 methylation occurs in a significant proportion of patients who have loss of MLH1 protein expression in their tumors and no MLH1 pathogenic germline mutation. Furthermore, we provide evidence that MLH1 constitutional hypermethylation is the molecular mechanism behind about 3% of Lynch syndrome families diagnosed in our institution, especially in patients with early onset or multiple primary tumors without significant family history. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Combination of gene expression patterns in whole blood discriminate between tuberculosis infection states

PubMed Central

2014-01-01

Background Genetic factors are involved in susceptibility or protection to tuberculosis (TB). Apart from gene polymorphisms and mutations, changes in levels of gene expression, induced by non-genetic factors, may also determine whether individuals progress to active TB. Methods We analysed the expression level of 45 genes in a total of 47 individuals (23 healthy household contacts and 24 new smear-positive pulmonary TB patients) in Addis Ababa using a dual colour multiplex ligation-dependent probe amplification (dcRT-MLPA) technique to assess gene expression profiles that may be used to distinguish TB cases and their contacts and also latently infected (LTBI) and uninfected household contacts. Results The gene expression level of BLR1, Bcl2, IL4d2, IL7R, FCGR1A, MARCO, MMP9, CCL19, and LTF had significant discriminatory power between sputum smear-positive TB cases and household contacts, with AUCs of 0.84, 0.81, 0.79, 0.79, 0.78, 0.76, 0.75, 0.75 and 0.68 respectively. The combination of Bcl2, BLR1, FCGR1A, IL4d2 and MARCO identified 91.66% of active TB cases and 95.65% of household contacts without active TB. The expression of CCL19, TGFB1, and Foxp3 showed significant difference between LTBI and uninfected contacts, with AUCs of 0.85, 0.82, and 0.75, respectively, whereas the combination of BPI, CCL19, FoxP3, FPR1 and TGFB1 identified 90.9% of QFT- and 91.6% of QFT+ household contacts. Conclusions Expression of single and especially combinations of host genes can accurately differentiate between active TB cases and healthy individuals as well as between LTBI and uninfected contacts. PMID:24885723

Effect of Non-tuberculous Mycobacteria on Host Biomarkers Potentially Relevant for Tuberculosis Management

PubMed Central

Dhanasekaran, S.; Jenum, Synne; Stavrum, Ruth; Wiker, Harald G.; Kenneth, John; Vaz, Mario; Doherty, T. Mark; Grewal, Harleen M. S.

2014-01-01

Background Non-tuberculous mycobacteria (NTM) are different from Mycobacterium tuberculosis (MTB) both in their ubiquitous environmental distribution and in their reduced capacity to cause disease. While often neglected in favour of other infectious diseases, NTM may interfere with important aspects of TB control and management, namely the efficacy of new anti-tuberculosis (TB) vaccines; the immuno-diagnostic Tuberculin skin test (TST) and QuantiFERON TB Gold In Tube assay (QFTGIT); and immune biomarkers explored for their diagnostic and/or predictive potential. Our objective was therefore to explore host immune biomarkers in children who had NTM isolated from respiratory and/or gastric specimens. Methodology and Principle Findings The present study was nested within a prospective cohort study of BCG-vaccinated neonates in Southern India. In this setting, immune biomarkers from peripheral blood were analyzed in 210 children aged <3 years evaluated for TB using dual-colour-Reverse-Transcriptase-Multiple-Ligation-dependent-Probe-Amplification (dcRT-MLPA) and Bio-Plex assays. The children were classified based on clinical examination, chest X-rays and mycobacterial culture reports as either: 1) TB disease, 2) NTM present and 3) controls. The study shows a down-regulation of RAB33A (p<0.001) and up-regulation of TGFβ1, IL-2 and IL-6 (all p<0.05) in children with TB disease, and that RAB33A, TGFBR2 and IL-10 (all p<0.05) were differentially expressed in children with NTM present when compared to children that were culture negative for MTB and NTM (controls). Conclusions and Significance Carriage of NTM may reduce the specificity of future diagnostic and predictive immune biomarkers relevant to TB management. PMID:25329719

Sonic Hedgehog mutations are not a common cause of congenital hypopituitarism in the absence of complex midline cerebral defects.

PubMed

Paulo, Sabrina Soares; Fernandes-Rosa, Fábio L; Turatti, Wendy; Coeli-Lacchini, Fernanda Borchers; Martinelli, Carlos E; Nakiri, Guilherme S; Moreira, Ayrton C; Santos, Antônio C; de Castro, Margaret; Antonini, Sonir R

2015-04-01

Sonic Hedgehog (SHH) and GLI2, an obligatory mediator of SHH signal transduction, are holoprosencephaly (HPE)-associated genes essential in pituitary formation. GLI2 variants have been found in patients with congenital hypopituitarism without complex midline cerebral defects (MCD). However, data on the occurrence of SHH mutations in these patients are limited. We screened for SHH and GLI2 mutations or copy number variations (CNV) in patients with congenital hypopituitarism without MCD or with variable degrees of MCD. Detailed data on clinical, laboratory and neuroimaging findings of 115 patients presenting with congenital hypopituitarism without MCD, septo-optic dysplasia or HPE were analysed. The SHH and GLI2 genes were directly sequenced, and the presence of gene CNV was analysed by multiplex ligation-dependent probe amplification (MLPA). Anterior pituitary deficiency was found in 74% and 53% of patients with SOD or HPE, respectively. Diabetes insipidus was common in patients with HPE (47%) but infrequent in patients with congenital hypopituitarism or SOD (7% and 8%, respectively). A single heterozygous nonsense SHH mutation (p.Tyr175Ter) was found in a patient presenting with hypopituitarism and alobar HPE. No other SHH mutations or CNV were found. Nine GLI2 variations (8 missense and 1 frameshift) including a homozygous and a compound heterozygous variation were found in patients with congenital hypopituitarism or SOD, but not in HPE patients. No GLI2 CNV were found. SHH mutations or copy number variations are not a common cause of congenital hypopituitarism in patients without complex midline cerebral defects. GLI2 variants are found in some patients with congenital hypopituitarism without complex midline cerebral defects or septo-optic dysplasia. However, functional analyses of these variants are needed to strengthen genotype-phenotype relationship. © 2014 John Wiley & Sons Ltd.

Spectrum of PAH gene variants among a population of Han Chinese patients with phenylketonuria from northern China.

PubMed

Liu, Ning; Huang, Qiuying; Li, Qingge; Zhao, Dehua; Li, Xiaole; Cui, Lixia; Bai, Ying; Feng, Yin; Kong, Xiangdong

2017-10-05

Phenylketonuria (PKU), which primarily results from a deficiency of phenylalanine hydroxylase (PAH), is one of the most common inherited inborn errors of metabolism that impairs postnatal cognitive development. The incidence of various PAH variations differs by race and ethnicity. The aim of the present study was to characterize the PAH gene variants of a Han population from Northern China. In total, 655 PKU patients and their families were recruited for this study; each proband was diagnosed both clinically and biochemically with phenylketonuria. Subjects were sequentially screened for single-base variants and exon deletions or duplications within PAH via direct Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). A spectrum of 174 distinct PAH variants was identified: 152 previously documented variants and 22 novel variants. While single-base variants were distributed throughout the 13 exons, they were particularly concentrated in exons 7 (33.3%), 11 (14.2%), 6 (13.2%), 12 (11.0%), 3 (10.4%), and 5 (4.4%). The predominant variant was p.Arg243Gln (17.7%), followed by Ex6-96A > G (8.3%), p.Val399 = (6.4%), p.Arg53His (4.7%), p.Tyr356* (4.7%), p.Arg241Cys (4.6%), p.Arg413Pro (4.6%), p.Arg111* (4.4%), and c.442-1G > A (3.4%). Notably, two patients were also identified as carrying de novo variants. The composition of PAH gene variants in this Han population from Northern China was distinct from those of other ethnic groups. As such, the construction of a PAH gene variant database for Northern China is necessary to lay a foundation for genetic-based diagnoses, prenatal diagnoses, and population screening.

Multilocus genetics to reconstruct aeromonad evolution

PubMed Central

2012-01-01

Background Aeromonas spp. are versatile bacteria that exhibit a wide variety of lifestyles. In an attempt to improve the understanding of human aeromonosis, we investigated whether clinical isolates displayed specific characteristics in terms of genetic diversity, population structure and mode of evolution among Aeromonas spp. A collection of 195 Aeromonas isolates from human, animal and environmental sources was therefore genotyped using multilocus sequence analysis (MLSA) based on the dnaK, gltA, gyrB, radA, rpoB, tsf and zipA genes. Results The MLSA showed a high level of genetic diversity among the population, and multilocus-based phylogenetic analysis (MLPA) revealed 3 major clades: the A. veronii, A. hydrophila and A. caviae clades, among the eleven clades detected. Lower genetic diversity was observed within the A. caviae clade as well as among clinical isolates compared to environmental isolates. Clonal complexes, each of which included a limited number of strains, mainly corresponded to host-associated subsclusters of strains, i.e., a fish-associated subset within A. salmonicida and 11 human-associated subsets, 9 of which included only disease-associated strains. The population structure was shown to be clonal, with modes of evolution that involved mutations in general and recombination events locally. Recombination was detected in 5 genes in the MLSA scheme and concerned approximately 50% of the STs. Therefore, these recombination events could explain the observed phylogenetic incongruities and low robustness. However, the MLPA globally confirmed the current systematics of the genus Aeromonas. Conclusions Evolution in the genus Aeromonas has resulted in exceptionally high genetic diversity. Emerging from this diversity, subsets of strains appeared to be host adapted and/or “disease specialized” while the A. caviae clade displayed an atypical tempo of evolution among aeromonads. Considering that A. salmonicida has been described as a genetically uniform pathogen that has adapted to fish through evolution from a variable ancestral population, we hypothesize that the population structure of aeromonads described herein suggested an ongoing process of adaptation to specialized niches associated with different degrees of advancement according to clades and clusters. PMID:22545815

Clinical analysis by next-generation sequencing for NSCLC patients with MET amplification resistant to osimertinib.

PubMed

Wang, Yubo; Li, Li; Han, Rui; Jiao, Lin; Zheng, Jie; He, Yong

2018-04-01

The efficacy of osimertinib was compromised by the development of resistance mechanisms, such as MET amplification. However, cohort studies of osimertinib resistance mechanism, and the correlation of MET and progression-free survival (PFS) after osimertinib resistance have been poorly investigated. This study was carried out to study the acquired MET amplification after osimertinib resistance in advanced lung adenocarcinoma patients, and interrogate the correlation of clinical prognosis and MET amplification. We performed capture-based sequencing on longitudinal plasma and tissue samples obtained before osimertinib treatment and after resistance development from lung adenocarcinoma patients to investigate the underlying resistance mechanism. We also investigated the correlation of MET amplification and patient prognosis after osimertinib resistance using Kaplan-Meier analysis. Paired biopsies before osimertinib treatment and after the resistance development revealed underlying resistance mechanisms. In addition, a cohort of 13 patients who developed disease progression after osimertinib resistance was investigated. Patients with MET amplification after osimertinib resistance commonly had inferior median progression-free survival (mPFS) than patients without MET amplification appearance or increase (3.5 months vs. 9.9 months, p = .117). Patients in MET amplification group also displayed poor median overall survival (mOS) compared to MET amplification negative group (15.6 months vs. 30.7 months, p = .885). Furthermore, combinatorial treatment of first/third-generation EGFR-TKI and crizotinib was efficaciously administrated into two patients with newly acquired MET amplification after osimertinib resistance. Partial responses were achieved by them, both clinically and radiographically. We investigated the osimertinib resistance mechanism in a small cohort of lung adenocarcinoma patients, and demonstrated MET amplification was correlated with inferior PFS/OS after osimertinib treatment. Moreover, we reported the first clinical evidence of efficacy generated by combination of first-generation EGFR-TKI icotinib and crizotinib after the resistance to osimertinib. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

PCR amplification on microarrays of gel immobilized oligonucleotides

DOEpatents

Strizhkov, Boris; Tillib, Sergei; Mikhailovich, Vladimir; Mirzabekov, Andrei

2003-11-04

The invention relates two general methods for performing PCR amplification, combined with the detection and analysis of the PCR products on a microchip. In the first method, the amplification occurs both outside and within a plurality of gel pads on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. In the second method, PCR amplification also takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid such as that which separates the individual gel pads into environments which resemble micro-miniaturized test tubes.

Expanding the clinical spectrum of COL1A1 mutations in different forms of glaucoma.

PubMed

Mauri, Lucia; Uebe, Steffen; Sticht, Heinrich; Vossmerbaeumer, Urs; Weisschuh, Nicole; Manfredini, Emanuela; Maselli, Edoardo; Patrosso, Mariacristina; Weinreb, Robert N; Penco, Silvana; Reis, André; Pasutto, Francesca

2016-08-02

Primary congenital glaucoma (PCG) and early onset glaucomas are one of the major causes of children and young adult blindness worldwide. Both autosomal recessive and dominant inheritance have been described with involvement of several genes including CYP1B1, FOXC1, PITX2, MYOC and PAX6. However, mutations in these genes explain only a small fraction of cases suggesting the presence of further candidate genes. To elucidate further genetic causes of these conditions whole exome sequencing (WES) was performed in an Italian patient, diagnosed with PCG and retinal detachment, and his unaffected parents. Sanger sequencing of the complete coding region of COL1A1 was performed in a total of 26 further patients diagnosed with PCG or early onset glaucoma. Exclusion of pathogenic variations in known glaucoma genes as CYP1B1, MYOC, FOXC1, PITX2 and PAX6 was additionally done per Sanger sequencing and Multiple Ligation-dependent Probe Amplification (MLPA) analysis. In the patient diagnosed with PCG and retinal detachment, analysis of WES data identified compound heterozygous variants in COL1A1 (p.Met264Leu; p.Ala1083Thr). Targeted COL1A1 screening of 26 additional patients detected three further heterozygous variants (p.Arg253*, p.Gly767Ser and p.Gly154Val) in three distinct subjects: two of them diagnosed with early onset glaucoma and mild form of osteogenesis imperfecta (OI), one patient with a diagnosis of PCG at age 4 years. All five variants affected evolutionary, highly conserved amino acids indicating important functional restrictions. Molecular modeling predicted that the heterozygous variants are dominant in effect and affect protein stability and thus the amount of available protein, while the compound heterozygous variants act as recessive alleles and impair binding affinity to two main COL1A1 binding proteins: Hsp47 and fibronectin. Dominant inherited mutations in COL1A1 are known causes of connective tissues disorders such as OI. These disorders are also associated with different ocular abnormalities, although recognition of the common pathology for both features is seldom being recognized. Our results expand the role of COL1A1 mutations in different forms of early-onset glaucoma with and without signs of OI. Thus, we suggest including COL1A1 mutation screening in the genetic work-up of glaucoma cases and detailed ophthalmic examinations with fundus analysis in patients with OI.

Estrogen receptor alpha gene amplification in breast cancer: 25 years of debate

PubMed Central

Holst, Frederik

2016-01-01

Twenty-five years ago, Nembrot and colleagues reported amplification of the estrogen receptor alpha gene (ESR1) in breast cancer, initiating a broad and still ongoing scientific debate on the prevalence and clinical significance of this genetic aberration, which affects one of the most important genes in breast cancer. Since then, a multitude of studies on this topic has been published, covering a wide range of divergent results and arguments. The reported prevalence of this alteration in breast cancer ranges from 0% to 75%, suggesting that ESR1 copy number analysis is hampered by technical and interpreter issues. To date, two major issues related to ESR1 amplification remain to be conclusively addressed: (1) The extent to which abundant amounts of messenger RNA can mimic amplification in standard fluorescence in situ hybridization assays in the analysis of strongly expressed genes like ESR1, and (2) the clinical relevance of ESR1 amplification: Such relevance is strongly disputed, with data showing predictive value for response as well as for resistance of the cancer to anti-estrogen therapies, or for subsequent development of cancers in the case of precursor lesions that display amplification of ESR1. This review provides a comprehensive summary of the various views on ESR1 amplification, and highlights explanations for the contradictions and conflicting data that could inform future ESR1 research. PMID:27081639

Carboxylesterase gene amplifications associated with insecticide resistance in Aedes albopictus: Geographical distribution and evolutionary origin

PubMed Central

Grigoraki, Linda; Pipini, Dimitra; Labbé, Pierrick; Chaskopoulou, Alexandra; Weill, Mylene; Vontas, John

2017-01-01

Background Aedes albopictus is one of the most invasive human disease vectors. Its control has been largely based on insecticides, such as the larvicide temephos. Temephos resistance has been associated with the up-regulation, through gene amplification, of two carboxylesterase (CCE) genes closely linked on the genome, capable of sequestering and metabolizing temephos oxon, the activated form of temephos. Principal findings Here, we investigated the occurrence, geographical distribution and origin of the CCE amplicon in Ae. albopictus populations from several geographical regions worldwide. The haplotypic diversity at the CCEae3a locus revealed high polymorphism, while phylogenetic analysis showed an absence of correlation between haplotype similarity and geographic origin. Two types of esterase amplifications were found, in two locations only (Athens and Florida): one, previously described, results in the amplification of both CCEae3a and CCEae6a; the second is being described for the first time and results in the amplification of CCEae3a only. The two amplification events are independent, as confirmed by sequence analysis. All individuals from Athens and Florida carrying the CCEae3a-CCEae6a co-amplicon share a common haplotype, indicating a single amplification event, which spread between the two countries. Significance The importance of passive transportation of disease vectors, including individuals carrying resistance mechanisms, is discussed in the light of efficient and sustainable vector control strategies. PMID:28394886

Topoisomerase expression and amplification in solid tumours: Analysis of 24,262 patients

PubMed Central

Heestand, Gregory M.; Schwaederle, Maria; Gatalica, Zoran; Arguello, David; Kurzrock, Razelle

2017-01-01

Background Topoisomerase I (TOPO1) and topoisomerase IIα (TOP2A) are specific targets of multiple chemotherapy drugs. Increased expression of TOPO1 protein and amplification of the TOP2A gene have been associated with treatment response in colorectal and breast cancers, respectively. TOPO1 and TOP2A may be potential therapeutic targets in other malignancies as well. Summary of methods We analysed TOPO1 protein expression and TOP2A gene amplification in patients (n = 24,262 specimens) with diverse cancers. Since HER2 and TOP2A co-amplification have been investigated for predictive value regarding anthracycline benefit, we analysed specimens for HER2 amplification as well. Results Overexpressed TOPO1 protein was present in 51% of the tumours. Four percent of the tumours had TOP2A amplification, with gallbladder tumours and gastroesophageal/oesophageal tumours having rates over 10%. Overall, 4903 specimens were assessed for both TOP2A and HER2 amplification; 129 (2.6%) had co-amplification. High rates (>40%) of HER2 amplification were seen in patients with TOP2A amplification in breast, ovarian, gastroesophageal/oesophageal and pancreatic cancer. Conclusion Our data indicate that increased TOPO1 expression and TOP2A amplification, as well as HER2 co-alterations, are present in multiple malignancies. The implications of these observations regarding sensitivity to chemotherapy not traditionally administered to these tumour types merits investigation. PMID:28728050

Breast cancers with EGFR and HER2 co-amplification favor distant metastasis and poor clinical outcome

PubMed Central

Guo, Peng; Pu, Tianjie; Chen, Shinan; Qiu, Yan; Zhong, Xiaorong; Zheng, Hong; Chen, Lina; Bu, Hong; Ye, Feng

2017-01-01

ErbB signaling serves essential roles in invasive ductal carcinoma (IDC). The aim of the present study was to assess gene amplification in ErbB family members in IDC with clinical implications. Quantitative polymerase chain reaction and fluorescence in situ hybridization were performed on formalin-fixed paraffin-embedded tumor samples for gene amplification detection. The clinical and histopathological characteristics, as well as the prognostic significance, were analyzed. Among the 119 IDC patients evaluated, epidermal growth factor receptor [EGFR; also known as human epidermal growth factor receptor (HER)1], HER2, HER3 and HER4 gene amplification was observed in 30 (25.2%), 44 (36.9%), 0 (0.0%) and 1 (0.8%) patients, respectively. EGFR amplification was associated with estrogen receptor status (P=0.028) and higher possibilities of recurrence (P=0.015) and distant metastasis (following initial surgery) (P=0.011). In survival analysis, EGFR amplification was also associated with disease-free survival (DFS) (P=0.001) and overall survival (OS) (P=0.003). HER2 amplification was associated with larger tumor size (P=0.006), later clinical stage (P=0.003) and distant metastasis (following initial surgery) (P=0.006). In survival analysis, HER2 amplification was also associated with DFS (P=0.011). Notably, the present study identified a group of patients in whom EGFR and HER2 were co-amplified. This group of patients appeared to have a higher possibility of metastasis (when diagnosed) (P=0.014) and distant metastasis (following initial surgery) (P<0.001). In survival analysis, these patients were noticed to be associated with DFS (P<0.001) and OS (P=0.002). With respect to treatment regimen, this was also true for the DFS association with chemotherapy (P<0.001), radiotherapy (P<0.001) and hormonal therapy (P=0.001). The present results suggest that EGFR and HER2 amplification favor distant metastasis following initial surgery and are significantly associated with poor clinical outcome in breast cancer patients. PMID:29181099

chipPCR: an R package to pre-process raw data of amplification curves.

PubMed

Rödiger, Stefan; Burdukiewicz, Michał; Schierack, Peter

2015-09-01

Both the quantitative real-time polymerase chain reaction (qPCR) and quantitative isothermal amplification (qIA) are standard methods for nucleic acid quantification. Numerous real-time read-out technologies have been developed. Despite the continuous interest in amplification-based techniques, there are only few tools for pre-processing of amplification data. However, a transparent tool for precise control of raw data is indispensable in several scenarios, for example, during the development of new instruments. chipPCR is an R: package for the pre-processing and quality analysis of raw data of amplification curves. The package takes advantage of R: 's S4 object model and offers an extensible environment. chipPCR contains tools for raw data exploration: normalization, baselining, imputation of missing values, a powerful wrapper for amplification curve smoothing and a function to detect the start and end of an amplification curve. The capabilities of the software are enhanced by the implementation of algorithms unavailable in R: , such as a 5-point stencil for derivative interpolation. Simulation tools, statistical tests, plots for data quality management, amplification efficiency/quantification cycle calculation, and datasets from qPCR and qIA experiments are part of the package. Core functionalities are integrated in GUIs (web-based and standalone shiny applications), thus streamlining analysis and report generation. http://cran.r-project.org/web/packages/chipPCR. Source code: https://github.com/michbur/chipPCR. stefan.roediger@b-tu.de Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Loop-Mediated Isothermal Amplification (LAMP) Signature Identification Software

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

Torres, C.

2009-03-17

This is an extendable open-source Loop-mediated isothermal AMPlification (LAMP) signature design program called LAVA (LAMP Assay Versatile Analysis). LAVA was created in response to limitations of existing LAMP signature programs.

A deletion and a duplication in distal 22q11.2 deletion syndrome region. Clinical implications and review

PubMed Central

Fernández, Luis; Nevado, Julián; Santos, Fernando; Heine-Suñer, Damià; Martinez-Glez, Victor; García-Miñaur, Sixto; Palomo, Rebeca; Delicado, Alicia; Pajares, Isidora López; Palomares, María; García-Guereta, Luis; Valverde, Eva; Hawkins, Federico; Lapunzina, Pablo

2009-01-01

Background Individuals affected with DiGeorge and Velocardiofacial syndromes present with both phenotypic diversity and variable expressivity. The most frequent clinical features include conotruncal congenital heart defects, velopharyngeal insufficiency, hypocalcemia and a characteristic craniofacial dysmorphism. The etiology in most patients is a 3 Mb recurrent deletion in region 22q11.2. However, cases of infrequent deletions and duplications with different sizes and locations have also been reported, generally with a milder, slightly different phenotype for duplications but with no clear genotype-phenotype correlation to date. Methods We present a 7 month-old male patient with surgically corrected ASD and multiple VSDs, and dysmorphic facial features not clearly suggestive of 22q11.2 deletion syndrome, and a newborn male infant with cleft lip and palate and upslanting palpebral fissures. Karyotype, FISH, MLPA, microsatellite markers segregation studies and SNP genotyping by array-CGH were performed in both patients and parents. Results Karyotype and FISH with probe N25 were normal for both patients. MLPA analysis detected a partial de novo 1.1 Mb deletion in one patient and a novel partial familial 0.4 Mb duplication in the other. Both of these alterations were located at a distal position within the commonly deleted region in 22q11.2. These rearrangements were confirmed and accurately characterized by microsatellite marker segregation studies and SNP array genotyping. Conclusion The phenotypic diversity found for deletions and duplications supports a lack of genotype-phenotype correlation in the vicinity of the LCRC-LCRD interval of the 22q11.2 chromosomal region, whereas the high presence of duplications in normal individuals supports their role as polymorphisms. We suggest that any hypothetical correlation between the clinical phenotype and the size and location of these alterations may be masked by other genetic and/or epigenetic modifying factors. PMID:19490635

Integrated microfluidic systems for cell lysis, mixing/pumping and DNA amplification

NASA Astrophysics Data System (ADS)

Lee, Chia-Yen; Lee, Gwo-Bin; Lin, Jr-Lung; Huang, Fu-Chun; Liao, Chia-Sheng

2005-06-01

The present paper reports a fully automated microfluidic system for the DNA amplification process by integrating an electroosmotic pump, an active micromixer and an on-chip temperature control system. In this DNA amplification process, the cell lysis is initially performed in a micro cell lysis reactor. Extracted DNA samples, primers and reagents are then driven electroosmotically into a mixing region where they are mixed by the active micromixer. The homogeneous mixture is then thermally cycled in a micro-PCR (polymerase chain reaction) chamber to perform DNA amplification. Experimental results show that the proposed device can successfully automate the sample pretreatment operation for DNA amplification, thereby delivering significant time and effort savings. The new microfluidic system, which facilitates cell lysis, sample driving/mixing and DNA amplification, could provide a significant contribution to ongoing efforts to miniaturize bio-analysis systems by utilizing a simple fabrication process and cheap materials.

Models and methods to characterize site amplification from a pair of records

USGS Publications Warehouse

Safak, E.

1997-01-01

The paper presents a tutorial review of the models and methods that are used to characterize site amplification from the pairs of rock- and soil-site records, and introduces some new techniques with better theoretical foundations. The models and methods discussed include spectral and cross-spectral ratios, spectral ratios for downhole records, response spectral ratios, constant amplification factors, parametric models, physical models, and time-varying filters. An extensive analytical and numerical error analysis of spectral and cross-spectral ratios shows that probabilistically cross-spectral ratios give more reliable estimates of site amplification. Spectral ratios should not be used to determine site amplification from downhole-surface recording pairs because of the feedback in the downhole sensor. Response spectral ratios are appropriate for low frequencies, but overestimate the amplification at high frequencies. The best method to be used depends on how much precision is required in the estimates.

RUNX1 Amplification Increases the Risk for Thrombosis in Children With B-cell Acute Lymphoblastic Leukemia.

PubMed

Boucher, Maria O; Smitherman, Andrew B; Pahl, Kristy S; Rao, Kathleen W; Deal, Allison M; Blatt, Julie

2016-04-01

RUNX1 (AML1) amplification in patients with B-cell acute lymphoblastic leukemia (B-ALL) has been associated with poor survival for unclear reasons. Our anecdotal experience suggests that children with B-ALL and RUNX1 amplification might be predisposed to thrombosis. We performed a retrospective cohort study of children with B-ALL treated from 2008 to 2014 at the North Carolina Children's Hospital. Patient demographics, cytogenetics, and diagnosis of thrombosis were extracted by blinded chart review. Analysis was performed examining the relationship between RUNX1 amplification and thrombosis. We identified 119 patients with B-ALL and a median age of 4.9 years (interquartile range, 2.9 to 8.6 y) at diagnosis. Four patients (3%) had RUNX1 amplification. The average number of RUNX1 copies among those with amplification was 5 (SD 0.81 [range, 4 to 6]). Eighteen thromboses were diagnosed within 6 months of starting treatment. These events were more likely among patients with RUNX1 amplification than in patients without amplification (75% vs. 13%; RR 5.75, 95% confidence interval, 2.75-12.01). RUNX1 amplification may predispose to early thrombotic events in children with B-ALL which could, in part, contribute to their poorer outcomes. Treatment implications, including possible prophylactic anticoagulation of patients with of RUNX1 amplification, justify larger studies to confirm these findings.

CDK4 Amplification Predicts Recurrence of Well-Differentiated Liposarcoma of the Abdomen

PubMed Central

Ha, Sang Yun; Paik, Kwang Yeol; Lee, Seung Eun; Kim, Jong Man; Park, Jae Berm; Kwon, Choon Hyuck David; Joh, Jae-Won; Choi, Yoon-La; Kim, Sung Joo

2014-01-01

Background The absence of CDK4 amplification in liposarcomas is associated with favorable prognosis. We aimed to identify the factors associated with tumor recurrence in patients with well-differentiated (WD) and dedifferentiated (DD) liposarcomas. Methods From 2000 to 2010, surgical resections for 101 WD and DD liposarcomas were performed. Cases in which complete surgical resections with curative intent were carried out were selected. MDM2 and CDK4 gene amplification were analyzed by quantitative real-time polymerase chain reaction (Q-PCR). Results There were 31 WD and 17 DD liposarcomas. Locoregional recurrence was observed in 11 WD and 3 DD liposarcomas. WD liposarcomas showed better patient survival compared to DD liposarcomas (P<0.05). Q-PCR analysis of the liposarcomas revealed the presence of CDK4 amplification in 44 cases (91.7%) and MDM2 amplification in 46 cases (95.8%). WD liposarcomas with recurrence after surgical resection had significantly higher levels of CDK4 amplification compared to those without recurrence (P = 0.041). High level of CDK4 amplification (cases with CDK4 amplification higher than the median 7.54) was associated with poor recurrence-free survival compared to low CDK4 amplification in both univariate (P = 0.012) and multivariate analyses (P = 0.020). Conclusions Level of CDK4 amplification determined by Q-PCR was associated with the recurrence of WD liposarcomas after surgical resection. PMID:25121597

Amplification of seismic waves by the Seattle basin, Washington State

USGS Publications Warehouse

Pratt, T.L.; Brocher, T.M.; Weaver, C.S.; Creager, K.C.; Snelson, C.M.; Crosson, R.S.; Miller, K.C.; Trehu, A.M.

2003-01-01

Recordings of the 1999 Mw 7.6 Chi-Chi (Taiwan) earthquake, two local earthquakes, and five blasts show seismic-wave amplification over a large sedimentary basin in the U.S. Pacific Northwest. For weak ground motions from the Chi-Chi earthquake, the Seattle basin amplified 0.2- to 0.8-Hz waves by factors of 8 to 16 relative to bedrock sites west of the basin. The amplification and peak frequency change during the Chi-Chi coda: the initial S-wave arrivals (0-30 sec) had maximum amplifications of 12 at 0.5-0.8 Hz, whereas later arrivals (35-65 sec) reached amplifications of 16 at 0.3-0.5 Hz. Analysis of local events in the 1.0- to 10.0-Hz frequency range show fourfold amplifications for 1.0-Hz weak ground motion over the Seattle basin. Amplifications decrease as frequencies increase above 1.0 Hz, with frequencies above 7 Hz showing lower amplitudes over the basin than at bedrock sites. Modeling shows that resonance in low-impedance deposits forming the upper 550 m of the basin beneath our profile could cause most of the observed amplification, and the larger amplification at later arrival times suggests surface waves also play a substantial role. These results emphasize the importance of shallow deposits in determining ground motions over large basins.

Prognostic value of HMGA2, CDK4, and JUN amplification in well-differentiated and dedifferentiated liposarcomas.

PubMed

Saâda-Bouzid, Esma; Burel-Vandenbos, Fanny; Ranchère-Vince, Dominique; Birtwisle-Peyrottes, Isabelle; Chetaille, Bruno; Bouvier, Corinne; Château, Marie-Christine; Peoc'h, Michel; Battistella, Maxime; Bazin, Audrey; Gal, Jocelyn; Michiels, Jean-François; Coindre, Jean-Michel; Pedeutour, Florence; Bianchini, Laurence

2015-11-01

HMGA2, CDK4, and JUN genes have been described as frequently coamplified with MDM2 in atypical lipomatous tumor, well-differentiated liposarcoma, and dedifferentiated liposarcoma. We studied the frequency of amplification of these genes in a series of 48 dedifferentiated liposarcomas and 68 atypical lipomatous tumors/well-differentiated liposarcomas. We correlated their amplification status with clinicopathological features and outcomes. Histologically, both CDK4 (P=0.007) and JUN (P=0.005) amplifications were associated with dedifferentiated liposarcoma, whereas amplification of the proximal parts of HMGA2 (5'-untranslated region (UTR) and exons 1-3) was associated with atypical lipomatous tumor/well-differentiated liposarcoma (P=0.01). CDK4 amplification was associated with axial tumors. Amplification of 5'-UTR and exons 1-3 of HMGA2 was associated with primary status and grade 1. Shorter overall survival was correlated with: age >64 years (P=0.03), chemotherapy used in first intent (P<0.001), no surgery (P=0.003), grade 3 (P<0.001), distant metastasis (P<0.001), node involvement (P=0.006), and CDK4 amplification (P=0.07). In multivariate analysis, distant metastasis (HR=8.8) and grade 3 (HR=18.2) were associated with shorter overall survival. A shorter recurrence-free survival was associated with dedifferentiated liposarcoma (P<0.001), grade 3 (P<0.001), node involvement (P<0.001), distant metastasis (P=0.02), recurrent status (P=0.009), axial location (P=0.001), and with molecular features such as CDK4 (P=0.05) and JUN amplification (P=0.07). Amplification of 5'-UTR and exons 1-3 (P=0.08) and 3'-UTR (P=0.01) of HMGA2 were associated with longer recurrence-free survival. Distant metastasis was associated with shorter recurrence-free survival (HR=5.8) in multivariate analysis. Dedifferentiated liposarcoma type was associated with axial location, grade 3 and recurrent status. In conclusion, we showed that the amplification of HMGA2 was associated with the atypical lipomatous tumor/well-differentiated liposarcoma histological type and a good prognosis, whereas CDK4 and JUN amplifications were associated with dedifferentiated liposarcoma histology and a bad prognosis. In addition, we also provided the first description of the molecular evolution of a well-differentiated liposarcoma into four successive dedifferentiated liposarcoma relapses, which was consistent with our general observations.

A novel method, digital genome scanning detects KRAS gene amplification in gastric cancers: involvement of overexpressed wild-type KRAS in downstream signaling and cancer cell growth

PubMed Central

2009-01-01

Background Gastric cancer is the third most common malignancy affecting the general population worldwide. Aberrant activation of KRAS is a key factor in the development of many types of tumor, however, oncogenic mutations of KRAS are infrequent in gastric cancer. We have developed a novel quantitative method of analysis of DNA copy number, termed digital genome scanning (DGS), which is based on the enumeration of short restriction fragments, and does not involve PCR or hybridization. In the current study, we used DGS to survey copy-number alterations in gastric cancer cells. Methods DGS of gastric cancer cell lines was performed using the sequences of 5000 to 15000 restriction fragments. We screened 20 gastric cancer cell lines and 86 primary gastric tumors for KRAS amplification by quantitative PCR, and investigated KRAS amplification at the DNA, mRNA and protein levels by mutational analysis, real-time PCR, immunoblot analysis, GTP-RAS pull-down assay and immunohistochemical analysis. The effect of KRAS knock-down on the activation of p44/42 MAP kinase and AKT and on cell growth were examined by immunoblot and colorimetric assay, respectively. Results DGS analysis of the HSC45 gastric cancer cell line revealed the amplification of a 500-kb region on chromosome 12p12.1, which contains the KRAS gene locus. Amplification of the KRAS locus was detected in 15% (3/20) of gastric cancer cell lines (8–18-fold amplification) and 4.7% (4/86) of primary gastric tumors (8–50-fold amplification). KRAS mutations were identified in two of the three cell lines in which KRAS was amplified, but were not detected in any of the primary tumors. Overexpression of KRAS protein correlated directly with increased KRAS copy number. The level of GTP-bound KRAS was elevated following serum stimulation in cells with amplified wild-type KRAS, but not in cells with amplified mutant KRAS. Knock-down of KRAS in gastric cancer cells that carried amplified wild-type KRAS resulted in the inhibition of cell growth and suppression of p44/42 MAP kinase and AKT activity. Conclusion Our study highlights the utility of DGS for identification of copy-number alterations. Using DGS, we identified KRAS as a gene that is amplified in human gastric cancer. We demonstrated that gene amplification likely forms the molecular basis of overactivation of KRAS in gastric cancer. Additional studies using a larger cohort of gastric cancer specimens are required to determine the diagnostic and therapeutic implications of KRAS amplification and overexpression. PMID:19545448

Novel Bioluminescent Quantitative Detection of Nucleic Acid Amplification in Real-Time

PubMed Central

Gandelman, Olga A.; Church, Vicki L.; Moore, Cathy A.; Kiddle, Guy; Carne, Christopher A.; Parmar, Surendra; Jalal, Hamid; Tisi, Laurence C.; Murray, James A. H.

2010-01-01

Background The real-time monitoring of polynucleotide amplification is at the core of most molecular assays. This conventionally relies on fluorescent detection of the amplicon produced, requiring complex and costly hardware, often restricting it to specialised laboratories. Principal Findings Here we report the first real-time, closed-tube luminescent reporter system for nucleic acid amplification technologies (NAATs) enabling the progress of amplification to be continuously monitored using simple light measuring equipment. The Bioluminescent Assay in Real-Time (BART) continuously reports through bioluminescent output the exponential increase of inorganic pyrophosphate (PPi) produced during the isothermal amplification of a specific nucleic acid target. BART relies on the coupled conversion of inorganic pyrophosphate (PPi) produced stoichiometrically during nucleic acid synthesis to ATP by the enzyme ATP sulfurylase, and can therefore be coupled to a wide range of isothermal NAATs. During nucleic acid amplification, enzymatic conversion of PPi released during DNA synthesis into ATP is continuously monitored through the bioluminescence generated by thermostable firefly luciferase. The assay shows a unique kinetic signature for nucleic acid amplifications with a readily identifiable light output peak, whose timing is proportional to the concentration of original target nucleic acid. This allows qualitative and quantitative analysis of specific targets, and readily differentiates between negative and positive samples. Since quantitation in BART is based on determination of time-to-peak rather than absolute intensity of light emission, complex or highly sensitive light detectors are not required. Conclusions The combined chemistries of the BART reporter and amplification require only a constant temperature maintained by a heating block and are shown to be robust in the analysis of clinical samples. Since monitoring the BART reaction requires only a simple light detector, the iNAAT-BART combination is ideal for molecular diagnostic assays in both laboratory and low resource settings. PMID:21152399

Four photon parametric amplification. [in unbiased Josephson junction

NASA Technical Reports Server (NTRS)

Parrish, P. T.; Feldman, M. J.; Ohta, H.; Chiao, R. Y.

1974-01-01

An analysis is presented describing four-photon parametric amplification in an unbiased Josephson junction. Central to the theory is the model of the Josephson effect as a nonlinear inductance. Linear, small signal analysis is applied to the two-fluid model of the Josephson junction. The gain, gain-bandwidth product, high frequency limit, and effective noise temperature are calculated for a cavity reflection amplifier. The analysis is extended to multiple (series-connected) junctions and subharmonic pumping.

[Principle of LAMP method--a simple and rapid gene amplification method].

PubMed

Ushikubo, Hiroshi

2004-06-01

So far nucleic acid test (NAT) has been employed in various fields, including infectious disease diagnoses. However, due to its complicated procedures and relatively high cost, it has not been widely utilized in many actual diagnostic applications. We have therefore developed a simple and rapid gene amplification technology, Loop-mediated Isothermal Amplification (LAMP) method, which has shown prominent results of surpassing the performance of the conventional gene amplification methods. LAMP method acquires three main features: (1) all reaction can be carried out under isothermal conditions; (2) the amplification efficiency is extremely high and tremendous amount of amplification products can be obtained; and (3) the reaction is highly specific. Furthermore, developed from the standard LAMP method, a rapid LAMP method, by adding in the loop primers, can reduce the amplification time from the previous 1 hour to less than 30 minutes. Enormous amount of white precipitate of magnesium pyrophosphate is produced as a by-product of the amplification, therefore, direct visual detection is possible without using any reaction indicators and detection equipments. We believe LAMP technology, with the integration of these features, can rightly apply to clinical genetic testing, food and environmental analysis, as well as NAT in different fields.

Recent advances in signal amplification strategy based on oligonucleotide and nanomaterials for microRNA detection-a review.

PubMed

Chen, Ying-Xu; Huang, Ke-Jing; Niu, Ke-Xin

2018-01-15

MicroRNAs (MiRNAs) play multiple crucial regulating roles in cell which can regulate one third of protein-coding genes. MiRNAs participate in the developmental and physiological processes of human body, while their aberrant adjustment will be more likely to trigger diseases such as cancers, kidney disease, central nervous system diseases, cardiovascular diseases, diabetes, viral infections and so on. What's worse, for the detection of miRNAs, their small size, high sequence similarity, low abundance and difficult extraction from cells impose great challenges in the analysis. Hence, it's necessary to fabricate accurate and sensitive biosensing platform for miRNAs detection. Up to now, researchers have developed many signal-amplification strategies for miRNAs detection, including hybridization chain reaction, nuclease amplification, rolling circle amplification, catalyzed hairpin assembly amplification and nanomaterials based amplification. These methods are typical, feasible and frequently used. In this review, we retrospect recent advances in signal amplification strategies for detecting miRNAs and point out the pros and cons of them. Furthermore, further prospects and promising developments of the signal-amplification strategies for detecting miRNAs are proposed. Copyright © 2017 Elsevier B.V. All rights reserved.

Estimation of empirical site amplification factors in Taiwan

NASA Astrophysics Data System (ADS)

Chung, Chi-Hsuan; Wen, Kuo-Liang; Kuo, Chun-Hsiang

2017-04-01

Lots of infrastructures are under construction in metropolises in Taiwan in recent years and thus leads to increasement of population density and urbanization in those area. Taiwan island is located in plate boundaries in which the high seismicity is caused by active tectonic plates. The Chi-Chi earthquake (Mw 7.6) in 1999 caused a fatality of more than 2000, and the Meinong earthquake (Mw 6.5) in 2016 caused a fatality of 117 in Tainan city as well as damages on hundreds of buildings. The cases imply seismic vulnerability of urban area. During the improvements for seismic hazard analysis and seismic design, consideration of seismic site amplifications in different site conditions is one of important issues. This study used selected and processed strong motion records observed by the TSMIP network. The site conditions considered as Vs30 used in this study were investigated at most stations (Kuo et al. 2012; Kuo et al. 2016). Since strong motion records and site conditions are both available, we are able to use the data to analyze site amplifications of seismic waves at different periods. The result may be a reference for future modification of seismic design codes to decrease potential seismic hazards and losses. We adopted the strong motion and site database of the SSHAC (Senior Seismic Hazard Analysis Committee) Level 3 project in Taiwan. The selected significant crustal and subduction events of magnitude larger than six for analysis. The amplification factors of PGA, PGV, PGD, and spectra acceleration at 0.3, 1.0, and 3.0 seconds were evaluated using the processed strong motions. According to the recommendation of SSHAC Level 3 project, the site condition of Vs30 = 760 m/s is considered as the reference rock site in this study. The stations with Vs30 between 600 m/s and 900 m/s and used as the reference rock sites in reality. For each event, we find a reference rock site and other site within a certain distance (region dependent) to calculate site amplifications of ground motions. Relationships of site amplification factors and Vs30 are therefore derived for strong motions by regression analysis. Soil nonlinearity (decrease of amplifications) has to be considered at soft soil sites during a strong shaking. We also discuss amplification factors in terms of different intensities if data is available.

Genomic Analysis Reveals a Common Breakpoint in Amplifications of the Plasmodium vivax Multidrug Resistance 1 Locus in Thailand

PubMed Central

Auburn, Sarah; Serre, David; Pearson, Richard D.; Amato, Roberto; Sriprawat, Kanlaya; To, Sheren; Handayuni, Irene; Suwanarusk, Rossarin; Russell, Bruce; Drury, Eleanor; Stalker, Jim; Miotto, Olivo; Kwiatkowski, Dominic P.; Nosten, Francois; Price, Ric N.

2016-01-01

In regions of coendemicity for Plasmodium falciparum and Plasmodium vivax where mefloquine is used to treat P. falciparum infection, drug pressure mediated by increased copy numbers of the multidrug resistance 1 gene (pvmdr1) may select for mefloquine-resistant P. vivax. Surveillance is not undertaken routinely owing in part to methodological challenges in detection of gene amplification. Using genomic data on 88 P. vivax samples from western Thailand, we identified pvmdr1 amplification in 17 isolates, all exhibiting tandem copies of a 37.6–kilobase pair region with identical breakpoints. A novel breakpoint-specific polymerase chain reaction assay was designed to detect the amplification. The assay demonstrated high sensitivity, identifying amplifications in 13 additional, polyclonal infections. Application to 132 further samples identified the common breakpoint in all years tested (2003–2015), with a decline in prevalence after 2012 corresponding to local discontinuation of mefloquine regimens. Assessment of the structure of pvmdr1 amplification in other geographic regions will yield information about the population-specificity of the breakpoints and underlying amplification mechanisms. PMID:27456706

Evaluation of HER-2/neu status in breast cancer specimens using immunohistochemistry (IHC) & fluorescence in-situ hybridization (FISH) assay.

PubMed

Goud, Kalal Iravathy; Dayakar, Seetha; Vijayalaxmi, Kolanupaka; Babu, Saidam Jangu; Reddy, P Vijay Anand

2012-03-01

Fluorescence in situ hybridization (FISH) is increasingly being recognized as the most accurate and predictive test for HER 2/neu gene amplification and response to therapy in breast cancer. In the present study we investigated HER-2/neu gene amplification by FISH in breast carcinoma tissue specimens and compared the results with that of immunohistochemical (IHC) analysis. A total of 90 breast carcinoma tissue samples were used for immunohistochemical (IHC) and FISH analysis. IHC was performed by using mouse monoclonal antibody to the intracellular domain of HER-2/neu protein. Each slide was scored in a blinded fashion by two pathologists according to the manufacturer's recommended criteria. FISH analysis was performed on paraffin embedded breast tumour tissue sections. The polysomy for centromere 17 (Spec green signal) was read as green signals less than 4 as moderate polysomy, and more than 4 as highly polysomy. Thirty of the 90 patients had negative results by IHC and FISH. Of the 28 patients with the score of 2+ by IHC, 20 were FISH positive for HER-2/neu gene amplification, three were FISH negative and five patients showed equivocal (1.8-2.2) results by FISH. These five cases were retested for IHC and FISH on different paraffin embedded tissue blocks, and all five were found positive for HER-2/neu gene amplification. Twenty five patients with the score of 3+ by IHC were FISH positive for HER-2/neu gene amplification (>2.2). Seven cases with the score of 3+ by IHC were FISH negative for HER-2/neu gene amplification (>2.2), and showed polysomy of chromosome number 17 high polysomy > 4. Our results indicated that HER-2/neu status by FISH should be performed in all cases of breast tumour with a 2+ score by IHC. Cases demonstrating a 3+ score by IHC may be subjected to FISH to rule out polysomy of chromosome 17 which could be falsely interpreted as HER-2/neu overexpression by IHC analysis. There is also a need for establishing a clinically validated cut-off value for HER-2/neu FISH amplification against IHC which may be further compared and calibrated.

Comparison of PCR primer-based strategies for characterization of ammonia oxidizer communities in environmental samples.

PubMed

Mahmood, Shahid; Freitag, Thomas E; Prosser, James I

2006-06-01

PCR-based techniques are commonly used to characterize microbial communities, but are subject to bias that is difficult to assess. This study aimed to evaluate bias of several PCR primer-based strategies used to study diversity of autotrophic ammonia oxidizers. 16S rRNA genes from soil- or sediment-DNA were amplified using primers considered either selective or specific for betaproteobacterial ammonia oxidizers. Five approaches were assessed: (a) amplification with primers betaAMO143f-betaAMO1315r; (b) amplification with primers CTO189f-CTO654r; (c) nested amplification with betaAMO143f-betaAMO1315r followed by CTO189f-CTO654r primers; (d) nested amplification with betaAMO143f-betaAMO1315r and CTO189f-Pf1053r primers; (e) nested amplification with 27f-1492r and CTO189f-CTO654r primers. Amplification products were characterized by denaturing gradient gel electrophoresis (DGGE) analysis after further amplification with 357f-GC-518r primers. DGGE profiles of soil communities were heterogeneous and depended on the approach followed. Ammonia oxidizer diversity was higher using approaches (b), (c) and (e) than using (a) and (d), where sequences of the most prominent bands showed similarities to nonammonia oxidizers. Profiles from marine sediments were more consistent, regardless of the approach adopted, and sequence analysis of excised bands indicated that these consisted of ammonia oxidizers only. The study demonstrates the importance of choice of primer, of screening for sequences of nontarget organisms and use of several approaches when characterizing microbial communities in natural environments.

Improved Method for Direct Detection of Environmental Microorganisms Using an Amplification of 16S rDNA Region

NASA Astrophysics Data System (ADS)

Tsujimura, M.; Akutsu, J.; Zhang, Z.; Sasaki, M.; Tajima, H.; Kawarabayasi, Y.

2004-12-01

The thermostable proteins or enzymes were expected to be capable to be utilized in many areas of industries. Many thermophilic microorganisms, which possess the thermostable proteins or enzymes, were identified from the extreme environment. However, many unidentified and uncultivable microorganisms are still remaining in the environment on the earth. It is generally said that the cultivable microorganisms are less than 1% of entire microorganisms living in the earth, remaining over 99% are still uncultivable. As an approach to the uncultivable microorganisms, the PCR amplification of 16S rDNA region using primer sets designed from the conserved region has been generally utilized for detection and community analysis of microorganism in the environment. However, the facts, that PCR amplification introduces the mutation in the amplified DNA fragment and efficiency of PCR amplification is depend on the sequences of primer sets, indicated that the improving of PCR analysis was necessary for more correct detection of microorganisms. As the result of evaluation for the quality of DNA polymerases, sequences of primers used for amplification and conditions of PCR amplification, the DNA polymerase, the primer set and the conditions for amplification, which did not amplify the DNA fragment from the DNA contaminated within the DNA polymerase itself, were successfully selected. Also the rate of mutation in the DNA fragment amplified was evaluated using this conditions and the genomic DNA from cultivable microbes as a template. The result indicated the rate of mutation introduced by PCR was approximately 0.1% to 0.125%. The improved method using these conditions and error rate calculated was applied for the analysis of microorganisms in the geothermal environment. The result indicated that four kinds of dominant microorganisms, including both of bacteria and archaea, were alive within soil in the hot spring in Tohoku Area. We would like to apply this improved method to detection of microorganisms with important genes from more other environments.

Towards the analysis of the genomes of single cells: further characterisation of the multiple displacement amplification.

PubMed

Panelli, Simona; Damiani, Giuseppe; Espen, Luca; Micheli, Gioacchino; Sgaramella, Vittorio

2006-05-10

The development of methods for the analysis and comparison of the nucleic acids contained in single cells is an ambitious and challenging goal that may provide useful insights in many physiopathological processes. We review here some of the published protocols for the amplification of whole genomes (WGA). We focus on the reaction known as Multiple Displacement Amplification (MDA), which probably represents the most reliable and efficient WGA protocol developed to date. We discuss some recent advances and applications, as well as some modifications to the reaction, which should improve its use and enlarge its range of applicability possibly to degraded genomes, and also to RNA via complementary DNA.

ESR1 gene amplification in endometrial carcinomas: a clinicopathological analysis.

PubMed

Rahman, Mohammed Tanjimur; Nakayama, Kentaro; Rahman, Munmun; Ishikawa, Masako; Katagiri, Hiroshi; Katagiri, Atsuko; Ishibashi, Tomoka; Sato, Emi; Iida, Kouji; Ishikawa, Noriyuki; Nakayama, Naomi; Miyazaki, Kohji

2013-09-01

This study investigated the clinicopathological significance of estrogen receptor 1 (ESR1) gene amplification and its relationship to phosphatase and tensin homolog (PTEN), human epidermal growth factor receptor 2 (HER2), MutL homolog 1 (MLH1), p53, and AT rich interactive domain 1A (ARID1A) expression in endometrial carcinomas. ESR1 amplification and expression were assessed by fluorescence in situ hybridization and immunohistochemistry. Clinical data were collected by retrospective chart review. ESR1 amplification was identified in 13 out of 111 (11.7%) endometrial carcinomas. No significant association was observed between ESR1 amplification and International Federation of Gynecology and Obstetrics (FIGO) stage (p=0.17), histological grade (p=0.35), lymph node metastasis (p=0.51), or deep myometrial invasion (p=0.46). ESR1 amplification was independent of PTEN, p53, HER2, MLH1, and ARID1A protein expression. Patients without estrogen receptor (ER) or progesterone receptor (PR) expression had shorter progression-free and overall survival than those with ER or PR expression (p<0.01). ESR1 amplification is independent of known clinicopathological factors related to poor prognosis and PTEN, p53, HER2, MLH1, and ARID1A protein expression, suggesting ESR1 amplification may be an early event in endometrial carcinoma development.

MYC and MYCN amplification can be reliably assessed by aCGH in medulloblastoma.

PubMed

Bourdeaut, Franck; Grison, Camille; Maurage, Claude-Alain; Laquerriere, Annie; Vasiljevic, Alexandre; Delisle, Marie-Bernadette; Michalak, Sophie; Figarella-Branger, Dominique; Doz, François; Richer, Wilfrid; Pierron, Gaelle; Miquel, Catherine; Delattre, Olivier; Couturier, Jérôme

2013-04-01

As prognostic factors, MYC and MYCN amplifications are routinely assessed in medulloblastomas. Fluorescence in situ hybridization (FISH) is currently considered as the technique of reference. Recently, array comparative genomic hybridization (aCGH) has been developed as an alternative technique to evaluate genomic abnormalities in other tumor types; however, this technique has not been widely adopted as a replacement for FISH in medulloblastoma. In this study, 34 tumors were screened by both FISH and aCGH. In all cases showing amplification by FISH, aCGH also unambiguously revealed the abnormality. The aCGH technique was also performed on tumors showing no amplification by FISH, and the absence of amplification was confirmed in all cases. Interestingly, one tumor showed a subclonal MYC amplification by FISH. This subclonal amplification was observed in approximately 20% of tumor cells and was clearly evident on aCGH. In conclusion, our analysis confirms that aCGH is as safe as FISH for the detection of MYC/MYCN gene amplification. Given its cost efficiency in comparison to two FISH tests and the global genomic information additionally provided by an aCGH experiment, this reproducible technique can be safely retained as an alternative to FISH for routine investigation of medulloblastoma. Copyright © 2013 Elsevier Inc. All rights reserved.

High amplification levels of MDM2 and CDK4 correlate with poor outcome in patients with dedifferentiated liposarcoma: A cytogenomic microarray analysis of 47 cases.

PubMed

Ricciotti, Robert W; Baraff, Aaron J; Jour, George; Kyriss, McKenna; Wu, Yu; Liu, Yuhua; Li, Shao-Chun; Hoch, Benjamin; Liu, Yajuan J

2017-12-01

Dedifferentiated liposarcoma (DDLS) is characterized at the molecular level by amplification of genes within 12q13-15 including MDM2 and CDK4. However, other than FNCLCC grade, prognostic markers are limited. We aim to identify molecular prognostic markers for DDLS to help risk stratify patients. To this end, we studied 49 cases of DDLS in our institutional archives and performed cytogenomic microarray analysis on 47 cases. Gene copy numbers for 12 loci were evaluated and correlated with outcome data retrieved from our institutional electronic medical records. Using cut point analysis and comparison of Kaplan-Meier survival curves by log rank tests, high amplification levels of MDM2 (>38 copies) and CDK4 (>30 copies) correlated with decreased disease free survival (DFS) (P = .0168 and 0.0169 respectively) and disease specific survival (DSS) (P = .0082 and 0.0140 respectively). Additionally, MDM2 and CDK4 showed evidence of a synergistic effect so that each additional copy of one enhances the effect on prognosis of each additional copy of the other for decreased DFS (P = .0227, 0.1% hazard). High amplification of JUN (>16 copies) also correlated with decreased DFS (P = .0217), but not DSS. The presence of copy number alteration at 3q29 correlated with decreased DSS (P = .0192). The presence of >10 mitoses per 10 high power fields and FNCLCC grade 3 also correlated with decreased DFS (P = .0310 and 0.0254 respectively). MDM2 and CDK4 gene amplification levels, along with JUN amplification and copy alterations at 3q29, can be utilized for predicting outcome in patients with DDLS. Published by Elsevier Inc.

Characterization of Novel Genes Within 8P11-12 Amplicon in Breast Cancer

DTIC Science & Technology

2007-06-01

C-myc amplification in breast cancer: a meta - analysis of its occurrence and prognostic relevance. Br J Cancer, 83: 1688-1695, 2000. 2. Hui, R...Nass SJ, Dickson RB, Trock BJ. C-myc amplification in breast cancer: a meta - analysis of its occurrence and prognostic relevance. Br J Cancer 2000;83...a detailed genomic and expression analysis of the 8p11-p12 amplicon in breast cancer cell lines and identified several novel candidate genes

Analysis of artifacts suggests DGGE should not be used for quantitative diversity analysis.

PubMed

Neilson, Julia W; Jordan, Fiona L; Maier, Raina M

2013-03-01

PCR-denaturing gradient gel electrophoresis (PCR-DGGE) is widely used in microbial ecology for the analysis of comparative community structure. However, artifacts generated during PCR-DGGE of mixed template communities impede the application of this technique to quantitative analysis of community diversity. The objective of the current study was to employ an artificial bacterial community to document and analyze artifacts associated with multiband signatures and preferential template amplification and to highlight their impacts on the use of this technique for quantitative diversity analysis. Six bacterial species (three Betaproteobacteria, two Alphaproteobacteria, and one Firmicutes) were amplified individually and in combinations with primers targeting the V7/V8 region of the 16S rRNA gene. Two of the six isolates produced multiband profiles demonstrating that band number does not correlate directly with α-diversity. Analysis of the multiple bands from one of these isolates confirmed that both bands had identical sequences which lead to the hypothesis that the multiband pattern resulted from two distinct structural conformations of the same amplicon. In addition, consistent preferential amplification was demonstrated following pairwise amplifications of the six isolates. DGGE and real time PCR analysis identified primer mismatch and PCR inhibition due to 16S rDNA secondary structure as the most probable causes of preferential amplification patterns. Reproducible DGGE community profiles generated in this study confirm that PCR-DGGE provides an excellent high-throughput tool for comparative community structure analysis, but that method-specific artifacts preclude its use for accurate comparative diversity analysis. Copyright © 2013 Elsevier B.V. All rights reserved.

Role of Frequency Chirp and Energy Flow Directionality in the Strong Coupling Regime of Brillouin-Based Plasma Amplification.

PubMed

Chiaramello, M; Amiranoff, F; Riconda, C; Weber, S

2016-12-02

A detailed analysis is presented of the various stages of strong coupling Brillouin plasma amplification, emphasizing the importance of the chirp which can be of threefold origin: the intrinsic one driven by the amplification process, the one originating from the chirped-pulse-generated laser pulses, and the one associated with the plasma profile. Control of the overall chirp can optimize or quench the energy transfer. The time-dependent phase relation explains the energy flow direction during amplification and is characteristic for this strong coupling process. The study is also of potential importance to understand and maybe control cross-beam-energy transfer in inertial confinement fusion.

Thermal Analysis of a Disposable, Instrument-Free DNA Amplification Lab-on-a-Chip Platform.

PubMed

Pardy, Tamás; Rang, Toomas; Tulp, Indrek

2018-06-04

Novel second-generation rapid diagnostics based on nucleic acid amplification tests (NAAT) offer performance metrics on par with clinical laboratories in detecting infectious diseases at the point of care. The diagnostic assay is typically performed within a Lab-on-a-Chip (LoC) component with integrated temperature regulation. However, constraints on device dimensions, cost and power supply inherent with the device format apply to temperature regulation as well. Thermal analysis on simplified thermal models for the device can help overcome these barriers by speeding up thermal optimization. In this work, we perform experimental thermal analysis on the simplified thermal model for our instrument-free, single-use LoC NAAT platform. The system is evaluated further by finite element modelling. Steady-state as well as transient thermal analysis are performed to evaluate the performance of a self-regulating polymer resin heating element in the proposed device geometry. Reaction volumes in the target temperature range of the amplification reaction are estimated in the simulated model to assess compliance with assay requirements. Using the proposed methodology, we demonstrated our NAAT device concept capable of performing loop-mediated isothermal amplification in the 20⁻25 °C ambient temperature range with 32 min total assay time.

MOLECULAR AND CYTOGENETIC ANALYSIS OF LUNG TUMOR CELL LINES

EPA Science Inventory

We have measured the levels of amplification of oncogenes and tumor marker genes or other genes of interest in nine human lung tumor cell lines in comparison to normal human bronchial epithelial cells or normal blood lymphocytes to test the hypothesis that aberrant amplification ...

'Mitominis': multiplex PCR analysis of reduced size amplicons for compound sequence analysis of the entire mtDNA control region in highly degraded samples.

PubMed

Eichmann, Cordula; Parson, Walther

2008-09-01

The traditional protocol for forensic mitochondrial DNA (mtDNA) analyses involves the amplification and sequencing of the two hypervariable segments HVS-I and HVS-II of the mtDNA control region. The primers usually span fragment sizes of 300-400 bp each region, which may result in weak or failed amplification in highly degraded samples. Here we introduce an improved and more stable approach using shortened amplicons in the fragment range between 144 and 237 bp. Ten such amplicons were required to produce overlapping fragments that cover the entire human mtDNA control region. These were co-amplified in two multiplex polymerase chain reactions and sequenced with the individual amplification primers. The primers were carefully selected to minimize binding on homoplasic and haplogroup-specific sites that would otherwise result in loss of amplification due to mis-priming. The multiplexes have successfully been applied to ancient and forensic samples such as bones and teeth that showed a high degree of degradation.

Sensitivity analysis of reactive ecological dynamics.

PubMed

Verdy, Ariane; Caswell, Hal

2008-08-01

Ecological systems with asymptotically stable equilibria may exhibit significant transient dynamics following perturbations. In some cases, these transient dynamics include the possibility of excursions away from the equilibrium before the eventual return; systems that exhibit such amplification of perturbations are called reactive. Reactivity is a common property of ecological systems, and the amplification can be large and long-lasting. The transient response of a reactive ecosystem depends on the parameters of the underlying model. To investigate this dependence, we develop sensitivity analyses for indices of transient dynamics (reactivity, the amplification envelope, and the optimal perturbation) in both continuous- and discrete-time models written in matrix form. The sensitivity calculations require expressions, some of them new, for the derivatives of equilibria, eigenvalues, singular values, and singular vectors, obtained using matrix calculus. Sensitivity analysis provides a quantitative framework for investigating the mechanisms leading to transient growth. We apply the methodology to a predator-prey model and a size-structured food web model. The results suggest predator-driven and prey-driven mechanisms for transient amplification resulting from multispecies interactions.

Asymmetric exponential amplification reaction on a toehold/biotin featured template: an ultrasensitive and specific strategy for isothermal microRNAs analysis

PubMed Central

Chen, Jun; Zhou, Xueqing; Ma, Yingjun; Lin, Xiulian; Dai, Zong; Zou, Xiaoyong

2016-01-01

The sensitive and specific analysis of microRNAs (miRNAs) without using a thermal cycler instrument is significant and would greatly facilitate biological research and disease diagnostics. Although exponential amplification reaction (EXPAR) is the most attractive strategy for the isothermal analysis of miRNAs, its intrinsic limitations of detection efficiency and inevitable non-specific amplification critically restrict its use in analytical sensitivity and specificity. Here, we present a novel asymmetric EXPAR based on a new biotin/toehold featured template. A biotin tag was used to reduce the melting temperature of the primer/template duplex at the 5′ terminus of the template, and a toehold exchange structure acted as a filter to suppress the non-specific trigger of EXPAR. The asymmetric EXPAR exhibited great improvements in amplification efficiency and specificity as well as a dramatic extension of dynamic range. The limit of detection for the let-7a analysis was decreased to 6.02 copies (0.01 zmol), and the dynamic range was extended to 10 orders of magnitude. The strategy enabled the sensitive and accurate analysis of let-7a miRNA in human cancer tissues with clearly better precision than both standard EXPAR and RT-qPCR. Asymmetric EXPAR is expected to have an important impact on the development of simple and rapid molecular diagnostic applications for short oligonucleotides. PMID:27257058

c-MYC amplification and c-myc protein expression in pancreatic acinar cell carcinomas. New insights into the molecular signature of these rare cancers.

PubMed

La Rosa, Stefano; Bernasconi, Barbara; Vanoli, Alessandro; Sciarra, Amedeo; Notohara, Kenji; Albarello, Luca; Casnedi, Selenia; Billo, Paola; Zhang, Lizhi; Tibiletti, Maria Grazia; Sessa, Fausto

2018-05-02

The molecular alterations of pancreatic acinar cell carcinomas (ACCs) and mixed acinar-neuroendocrine carcinomas (MANECs) are not completely understood, and the possible role of c-MYC amplification in tumor development, progression, and prognosis is not known. We have investigated c-MYC gene amplification in a series of 35 ACCs and 4 MANECs to evaluate its frequency and a possible prognostic role. Gene amplification was investigated using interphasic fluorescence in situ hybridization analysis simultaneously hybridizing c-MYC and the centromere of chromosome 8 probes. Protein expression was immunohistochemically investigated using a specific monoclonal anti-c-myc antibody. Twenty cases had clones with different polysomies of chromosome 8 in absence of c-MYC amplification, and 5 cases had one amplified clone and other clones with chromosome 8 polysomy, while the remaining 14 cases were diploid for chromosome 8 and lacked c-MYC amplification. All MANECs showed c-MYC amplification and/or polysomy which were observed in 54% pure ACCs. Six cases (15.3%) showed nuclear immunoreactivity for c-myc, but only 4/39 cases showed simultaneous c-MYC amplification/polysomy and nuclear protein expression. c-myc immunoreactivity as well as c-MYC amplification and/or chromosome 8 polysomy was not statistically associated with prognosis. Our study demonstrates that a subset of ACCs shows c-MYC alterations including gene amplification and chromosome 8 polysomy. Although they are not associated with a different prognostic signature, the fact that these alterations are present in all MANECs suggests a role in the acinar-neuroendocrine differentiation possibly involved in the pathogenesis of MANECs.

Genomic Analysis Reveals a Common Breakpoint in Amplifications of the Plasmodium vivax Multidrug Resistance 1 Locus in Thailand.

PubMed

Auburn, Sarah; Serre, David; Pearson, Richard D; Amato, Roberto; Sriprawat, Kanlaya; To, Sheren; Handayuni, Irene; Suwanarusk, Rossarin; Russell, Bruce; Drury, Eleanor; Stalker, Jim; Miotto, Olivo; Kwiatkowski, Dominic P; Nosten, Francois; Price, Ric N

2016-10-15

In regions of coendemicity for Plasmodium falciparum and Plasmodium vivax where mefloquine is used to treat P. falciparum infection, drug pressure mediated by increased copy numbers of the multidrug resistance 1 gene (pvmdr1) may select for mefloquine-resistant P. vivax Surveillance is not undertaken routinely owing in part to methodological challenges in detection of gene amplification. Using genomic data on 88 P. vivax samples from western Thailand, we identified pvmdr1 amplification in 17 isolates, all exhibiting tandem copies of a 37.6-kilobase pair region with identical breakpoints. A novel breakpoint-specific polymerase chain reaction assay was designed to detect the amplification. The assay demonstrated high sensitivity, identifying amplifications in 13 additional, polyclonal infections. Application to 132 further samples identified the common breakpoint in all years tested (2003-2015), with a decline in prevalence after 2012 corresponding to local discontinuation of mefloquine regimens. Assessment of the structure of pvmdr1 amplification in other geographic regions will yield information about the population-specificity of the breakpoints and underlying amplification mechanisms. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.

A multi-method approach to the molecular diagnosis of overt and borderline 11p15.5 defects underlying Silver-Russell and Beckwith-Wiedemann syndromes.

PubMed

Russo, Silvia; Calzari, Luciano; Mussa, Alessandro; Mainini, Ester; Cassina, Matteo; Di Candia, Stefania; Clementi, Maurizio; Guzzetti, Sara; Tabano, Silvia; Miozzo, Monica; Sirchia, Silvia; Finelli, Palma; Prontera, Paolo; Maitz, Silvia; Sorge, Giovanni; Calcagno, Annalisa; Maghnie, Mohamad; Divizia, Maria Teresa; Melis, Daniela; Manfredini, Emanuela; Ferrero, Giovanni Battista; Pecile, Vanna; Larizza, Lidia

2016-01-01

Multiple (epi)genetic defects affecting the expression of the imprinted genes within the 11p15.5 chromosomal region underlie Silver-Russell (SRS) and Beckwith-Wiedemann (BWS) syndromes. The molecular diagnosis of these opposite growth disorders requires a multi-approach flowchart to disclose known primary and secondary (epi)genetic alterations; however, up to 20 and 30 % of clinically diagnosed BWS and SRS cases remain without molecular diagnosis. The complex structure of the 11p15 region with variable CpG methylation and low-rate mosaicism may account for missed diagnoses. Here, we demonstrate the relevance of complementary techniques for the assessment of different CpGs and the importance of testing multiple tissues to increase the SRS and BWS detection rate. Molecular testing of 147 and 450 clinically diagnosed SRS and BWS cases provided diagnosis in 34 SRS and 185 BWS patients, with 9 SRS and 21 BWS cases remaining undiagnosed and herein referred to as "borderline." A flowchart including complementary techniques and, when applicable, the analysis of buccal swabs, allowed confirmation of the molecular diagnosis in all borderline cases. Comparison of methylation levels by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) in borderline and control cases defined an interval of H19/IGF2:IG-DMR loss of methylation that was distinct between "easy to diagnose" and "borderline" cases, which were characterized by values ≤mean -3 standard deviations (SDs) compared to controls. Values ≥mean +1 SD at H19/IGF2: IG-DMR were assigned to borderline hypermethylated BWS cases and those ≤mean -2 SD at KCNQ1OT1: TSS-DMR to hypomethylated BWS cases; these were supported by quantitative pyrosequencing or Southern blot analysis. Six BWS cases suspected to carry mosaic paternal uniparental disomy of chromosome 11 were confirmed by SNP array, which detected mosaicism till 10 %. Regarding the clinical presentation, borderline SRS were representative of the syndromic phenotype, with exception of one patient, whereas BWS cases showed low frequency of the most common features except hemihyperplasia. A conclusive molecular diagnosis was reached in borderline methylation cases, increasing the detection rate by 6 % for SRS and 5 % for BWS cases. The introduction of complementary techniques and additional tissue analyses into routine diagnostic work-up should facilitate the identification of cases undiagnosed because of mosaicism, a distinctive feature of epigenetic disorders.

Whole-genome multiple displacement amplification from single cells.

PubMed

Spits, Claudia; Le Caignec, Cédric; De Rycke, Martine; Van Haute, Lindsey; Van Steirteghem, André; Liebaers, Inge; Sermon, Karen

2006-01-01

Multiple displacement amplification (MDA) is a recently described method of whole-genome amplification (WGA) that has proven efficient in the amplification of small amounts of DNA, including DNA from single cells. Compared with PCR-based WGA methods, MDA generates DNA with a higher molecular weight and shows better genome coverage. This protocol was developed for preimplantation genetic diagnosis, and details a method for performing single-cell MDA using the phi29 DNA polymerase. It can also be useful for the amplification of other minute quantities of DNA, such as from forensic material or microdissected tissue. The protocol includes the collection and lysis of single cells, and all materials and steps involved in the MDA reaction. The whole procedure takes 3 h and generates 1-2 microg of DNA from a single cell, which is suitable for multiple downstream applications, such as sequencing, short tandem repeat analysis or array comparative genomic hybridization.

Functional integration of PCR amplification and capillary eletrophoresis in a microfabricated DNA analysis device

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

Woolley, A.T.; deMello, A.J.; Mathies, R.A.

Microfabricated silicon PCR reactors and glass capillary electrophoresis (CE) chips have been successfully coupled to form an integrated DNA analysis system. This construct combines the rapid thermal cycling capabilities of microfabricated PCR devices (10{degree}C/s heating, 2.5{degree}C/s cooling) with the high-speed (<120 s) DNA separations provided by microfabricated CE chips. The PCR chamber and the CE chip were directly linked through a photolithographically fabricated channel filled with hydroxyethylcellulose sieving matrix. Electrophoretic injection directly from the PCR chamber through the cross injection channel was used as an `electrophoretic valve` to couple the PCR and CE devices on-chip. To demonstrate the functionality ofmore » this system, a 15 min PCR amplification of a {Beta}-globin target cloned in m13 was immediately followed by high-speed CE chip separation in under 120 s, providing a rapid PCR-CE analysis in under 20 min. A rapid assay for genomic Salmonella DNA was performed in under 45 min, demonstrating that challenging amplifications of diagnostically interesting targets can also be performed. Real-time monitoring of PCR target amplification in these integrated PCR-CE devices is also feasible. 33 refs., 6 figs.« less

Helicase dependent OnChip-amplification and its use in multiplex pathogen detection.

PubMed

Andresen, Dennie; von Nickisch-Rosenegk, Markus; Bier, Frank F

2009-05-01

The need for fast, specific and sensitive multiparametric detection methods is an ever growing demand in molecular diagnostics. Here we report on a newly developed method, the helicase dependent OnChip amplification (OnChip-HDA). This approach integrates the analysis and detection in one single reaction thus leading to time and cost savings in multiparametric analysis. HDA is an isothermal amplification method that is not depending on thermocycling as known from PCR due to the helicases' ability to unwind DNA double-strands. We have combined the HDA with microarray based detection, making it suitable for multiplex detection. As an example we used the OnChip HDA in single and multiplex amplifications for the detection of the two pathogens N. gonorrhoeae and S. aureus directly on surface bound primers. We have successfully shown the OnChip-HDA and applied it for single- and duplex-detection of the pathogens N. gonorrhoeae and S. aureus. We have developed a new method, the OnChip-HDA for the multiplex detection of pathogens. Its simplicity in reaction setup and potential for miniaturization and multiparametric analysis is advantageous for the integration in miniaturized Lab on Chip systems, e.g. needed in point of care diagnostics.

High levels of the AR-V7 Splice Variant and Co-Amplification of the Golgi Protein Coding YIPF6 in AR Amplified Prostate Cancer Bone Metastases.

PubMed

Djusberg, Erik; Jernberg, Emma; Thysell, Elin; Golovleva, Irina; Lundberg, Pia; Crnalic, Sead; Widmark, Anders; Bergh, Anders; Brattsand, Maria; Wikström, Pernilla

2017-05-01

The relation between androgen receptor (AR) gene amplification and other mechanisms behind castration-resistant prostate cancer (CRPC), such as expression of constitutively active AR variants and steroid-converting enzymes has been poorly examined. Specific aim was to examine AR amplification in PC bone metastases and to explore molecular and functional consequences of this, with the long-term goal of identifying novel molecular targets for treatment. Gene amplification was assessed by fluorescence in situ hybridization in cryo-sections of clinical PC bone metastases (n = 40) and by PCR-based copy number variation analysis. Whole genome mRNA expression was analyzed using H12 Illumina Beadchip arrays and specific transcript levels were quantified by qRT-PCR. Protein localization was analyzed using immunohistochemistry and confocal microscopy. The YIPF6 mRNA expression was transiently knocked down and stably overexpressed in the 22Rv1 cell line as representative for CRPC, and effects on cell proliferation, colony formation, migration, and invasion were determined in vitro. Extracellular vesicles (EVs) were isolated from cell cultures using size-exclusion chromatography and enumerated by nanoparticle tracking analysis. Protein content was identified by LC-MS/MS analysis. Blood coagulation was measured as activated partial thromboplastin time (APTT). Functional enrichment analysis was performed using the MetaCore software. AR amplification was detected in 16 (53%) of the bone metastases examined from CRPC patients (n = 30), and in none from the untreated patients (n = 10). Metastases with AR amplification showed high AR and AR-V7 mRNA levels, increased nuclear AR immunostaining, and co-amplification of genes such as YIPF6 in the AR proximity at Xq12. The YIPF6 protein was localized to the Golgi apparatus. YIPF6 overexpression in 22Rv1 cells resulted in reduced cell proliferation and colony formation, and in enhanced EV secretion. EVs from YIPF6 overproducing 22Rv1 cells were enriched for proteins involved in blood coagulation and, accordingly, decreased the APTT in a dose-dependent fashion. AR amplified CRPC bone metastases show high AR-V7 expression that probably gives resistance to AR-targeting drugs. Co-amplification of the Golgi protein coding YIPF6 gene with the AR may enhance the secretion of pro-coagulative EVs from cancer cells and thereby stimulate tumor progression and increase the coagulopathy risk in CRPC patients. Prostate 77: 625-638, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Microfluidic Devices for Forensic DNA Analysis: A Review.

PubMed

Bruijns, Brigitte; van Asten, Arian; Tiggelaar, Roald; Gardeniers, Han

2016-08-05

Microfluidic devices may offer various advantages for forensic DNA analysis, such as reduced risk of contamination, shorter analysis time and direct application at the crime scene. Microfluidic chip technology has already proven to be functional and effective within medical applications, such as for point-of-care use. In the forensic field, one may expect microfluidic technology to become particularly relevant for the analysis of biological traces containing human DNA. This would require a number of consecutive steps, including sample work up, DNA amplification and detection, as well as secure storage of the sample. This article provides an extensive overview of microfluidic devices for cell lysis, DNA extraction and purification, DNA amplification and detection and analysis techniques for DNA. Topics to be discussed are polymerase chain reaction (PCR) on-chip, digital PCR (dPCR), isothermal amplification on-chip, chip materials, integrated devices and commercially available techniques. A critical overview of the opportunities and challenges of the use of chips is discussed, and developments made in forensic DNA analysis over the past 10-20 years with microfluidic systems are described. Areas in which further research is needed are indicated in a future outlook.

RPA using a multiplexed cartridge for low cost point of care diagnostics in the field.

PubMed

Ereku, Luck Tosan; Mackay, Ruth E; Craw, Pascal; Naveenathayalan, Angel; Stead, Thomas; Branavan, Manorharanehru; Balachandran, Wamadeva

2018-04-15

A point of care device utilising Lab-on-a-Chip technologies that is applicable for biological pathogens was designed, fabricated and tested showing sample in to answer out capabilities. The purpose of the design was to develop a cartridge with the capability to perform nucleic acid extraction and purification from a sample using a chitosan membrane at an acidic pH. Waste was stored within the cartridge with the use of sodium polyacrylate to solidify or gelate the sample in a single chamber. Nucleic acid elution was conducted using the RPA amplification reagents (alkaline pH). Passive valves were used to regulate the fluid flow and a multiplexer was designed to distribute the fluid into six microchambers for amplification reactions. Cartridges were produced using soft lithography of silicone from 3D printed moulds, bonded to glass substrates. The isothermal technique, RPA is employed for amplification. This paper shows the results from two separate experiments: the first using the RPA control nucleic acid, the second showing successful amplification from Chlamydia Trachomatis. Endpoint analysis conducted for the RPA analysis was gel electrophoresis that showed 143 base pair DNA was amplified successfully for positive samples whilst negative samples did not show amplification. End point analysis for Chlamydia Trachomatis samples was fluorescence detection that showed successful detection of 1 copy/μL and 10 copies/μL spiked in a MES buffer. Crown Copyright © 2018. Published by Elsevier Inc. All rights reserved.

Development and Comparison of a Rapid Isothermal Nucleic Acid Amplification Test for Typing of Herpes Simplex Virus Types 1 and 2 on a Portable Fluorescence Detector

PubMed Central

Tong, Yanhong; McCarthy, Kaitlin; Kong, Huimin; Lemieux, Bertrand

2013-01-01

We have developed a rapid and simple molecular test, the IsoGlow HSV Typing assay, for the detection and typing of herpes simplex virus (type 1 and 2) from genital or oral lesions. Clinical samples suspended in viral transport mediums are simply diluted and then added to a helicase-dependent amplification master mix. The amplification and detection were performed on a portable fluorescence detector called the FireFly instrument. Detection of amplification products is based on end-point analysis using cycling probe technology. An internal control nucleic acid was included in the amplification master mix to monitor the presence of amplification inhibitors in the samples. Because the device has only two fluorescence detection channels, two strategies were developed and compared to detect the internal control template: internal control detected by melting curve analysis using a dual-labeled probe, versus internal control detection using end-point fluorescence release by a CPT probe at a lower temperature. Both have a total turnaround time of about 1 hour. Clinical performance relative to herpes viral culture was evaluated using 176 clinical specimens. Both formats of the IsoGlow HSV typing assay had sensitivities comparable to that of the Food and Drug Administration–cleared IsoAmp HSV (BioHelix Corp., Beverly MA) test and specificity for the two types of HSV comparable to that of ELVIS HSV (Diagnostic Hybrids, Athens, OH). PMID:22951487

Optimization of whole-transcriptome amplification from low cell density deep-sea microbial samples for metatranscriptomic analysis.

PubMed

Wu, Jieying; Gao, Weimin; Zhang, Weiwen; Meldrum, Deirdre R

2011-01-01

Limitation in sample quality and quantity is one of the big obstacles for applying metatranscriptomic technologies to explore gene expression and functionality of microbial communities in natural environments. In this study, several amplification methods were evaluated for whole-transcriptome amplification of deep-sea microbial samples, which are of low cell density and high impurity. The best amplification method was identified and incorporated into a complete protocol to isolate and amplify deep-sea microbial samples. In the protocol, total RNA was first isolated by a modified method combining Trizol (Invitrogen, CA) and RNeasy (QIAGEN, CA) method, amplified with a WT-Ovation™ Pico RNA Amplification System (NuGEN, CA), and then converted to double-strand DNA from single-strand cDNA with a WT-Ovation™ Exon Module (NuGEN, CA). The products from the whole-transcriptome amplification of deep-sea microbial samples were assessed first through random clone library sequencing. The BLAST search results showed that marine-based sequences are dominant in the libraries, consistent with the ecological source of the samples. The products were then used for next-generation Roche GS FLX Titanium sequencing to obtain metatranscriptome data. Preliminary analysis of the metatranscriptomic data showed good sequencing quality. Although the protocol was designed and demonstrated to be effective for deep-sea microbial samples, it should be applicable to similar samples from other extreme environments in exploring community structure and functionality of microbial communities. Copyright © 2010 Elsevier B.V. All rights reserved.

Product differentiation by analysis of DNA melting curves during the polymerase chain reaction.

PubMed

Ririe, K M; Rasmussen, R P; Wittwer, C T

1997-02-15

A microvolume fluorometer integrated with a thermal cycler was used to acquire DNA melting curves during polymerase chain reaction by fluorescence monitoring of the double-stranded DNA specific dye SYBR Green I. Plotting fluorescence as a function of temperature as the thermal cycler heats through the dissociation temperature of the product gives a DNA melting curve. The shape and position of this DNA melting curve are functions of the GC/AT ratio, length, and sequence and can be used to differentiate amplification products separated by less than 2 degrees C in melting temperature. Desired products can be distinguished from undesirable products, in many cases eliminating the need for gel electrophoresis. Analysis of melting curves can extend the dynamic range of initial template quantification when amplification is monitored with double-stranded DNA specific dyes. Complete amplification and analysis of products can be performed in less than 15 min.

Copy number analysis of NIPBL in a cohort of 510 patients reveals rare copy number variants and a mosaic deletion.

PubMed

Cheng, Yu-Wei; Tan, Christopher A; Minor, Agata; Arndt, Kelly; Wysinger, Latrice; Grange, Dorothy K; Kozel, Beth A; Robin, Nathaniel H; Waggoner, Darrel; Fitzpatrick, Carrie; Das, Soma; Del Gaudio, Daniela

2014-03-01

Cornelia de Lange syndrome (CdLS) is a genetically heterogeneous disorder characterized by growth retardation, intellectual disability, upper limb abnormalities, hirsutism, and characteristic facial features. In this study we explored the occurrence of intragenic NIPBL copy number variations (CNVs) in a cohort of 510 NIPBL sequence-negative patients with suspected CdLS. Copy number analysis was performed by custom exon-targeted oligonucleotide array-comparative genomic hybridization and/or MLPA. Whole-genome SNP array was used to further characterize rearrangements extending beyond the NIPBL gene. We identified NIPBL CNVs in 13 patients (2.5%) including one intragenic duplication and a deletion in mosaic state. Breakpoint sequences in two patients provided further evidence of a microhomology-mediated replicative mechanism as a potential predominant contributor to CNVs in NIPBL. Patients for whom clinical information was available share classical CdLS features including craniofacial and limb defects. Our experience in studying the frequency of NIBPL CNVs in the largest series of patients to date widens the mutational spectrum of NIPBL and emphasizes the clinical utility of performing NIPBL deletion/duplication analysis in patients with CdLS.

Quality control for quantitative PCR based on amplification compatibility test.

PubMed

Tichopad, Ales; Bar, Tzachi; Pecen, Ladislav; Kitchen, Robert R; Kubista, Mikael; Pfaffl, Michael W

2010-04-01

Quantitative qPCR is a routinely used method for the accurate quantification of nucleic acids. Yet it may generate erroneous results if the amplification process is obscured by inhibition or generation of aberrant side-products such as primer dimers. Several methods have been established to control for pre-processing performance that rely on the introduction of a co-amplified reference sequence, however there is currently no method to allow for reliable control of the amplification process without directly modifying the sample mix. Herein we present a statistical approach based on multivariate analysis of the amplification response data generated in real-time. The amplification trajectory in its most resolved and dynamic phase is fitted with a suitable model. Two parameters of this model, related to amplification efficiency, are then used for calculation of the Z-score statistics. Each studied sample is compared to a predefined reference set of reactions, typically calibration reactions. A probabilistic decision for each individual Z-score is then used to identify the majority of inhibited reactions in our experiments. We compare this approach to univariate methods using only the sample specific amplification efficiency as reporter of the compatibility. We demonstrate improved identification performance using the multivariate approach compared to the univariate approach. Finally we stress that the performance of the amplification compatibility test as a quality control procedure depends on the quality of the reference set. Copyright 2010 Elsevier Inc. All rights reserved.

Coexpression network analysis identifies transcriptional modules associated with genomic alterations in neuroblastoma.

PubMed

Yang, Liulin; Li, Yun; Wei, Zhi; Chang, Xiao

2018-06-01

Neuroblastoma is a highly complex and heterogeneous cancer in children. Acquired genomic alterations including MYCN amplification, 1p deletion and 11q deletion are important risk factors and biomarkers in neuroblastoma. Here, we performed a co-expression-based gene network analysis to study the intrinsic association between specific genomic changes and transcriptome organization. We identified multiple gene coexpression modules which are recurrent in two independent datasets and associated with functional pathways including nervous system development, cell cycle, immune system process and extracellular matrix/space. Our results also indicated that modules involved in nervous system development and cell cycle are highly associated with MYCN amplification and 1p deletion, while modules responding to immune system process are associated with MYCN amplification only. In summary, this integrated analysis provides novel insights into molecular heterogeneity and pathogenesis of neuroblastoma. This article is part of a Special Issue entitled: Accelerating Precision Medicine through Genetic and Genomic Big Data Analysis edited by Yudong Cai & Tao Huang. Copyright © 2017. Published by Elsevier B.V.

Signal Amplification Technologies for the Detection of Nucleic Acids: from Cell-Free Analysis to Live-Cell Imaging.

PubMed

Fozooni, Tahereh; Ravan, Hadi; Sasan, Hosseinali

2017-12-01

Due to their unique properties, such as programmability, ligand-binding capability, and flexibility, nucleic acids can serve as analytes and/or recognition elements for biosensing. To improve the sensitivity of nucleic acid-based biosensing and hence the detection of a few copies of target molecule, different modern amplification methodologies, namely target-and-signal-based amplification strategies, have already been developed. These recent signal amplification technologies, which are capable of amplifying the signal intensity without changing the targets' copy number, have resulted in fast, reliable, and sensitive methods for nucleic acid detection. Working in cell-free settings, researchers have been able to optimize a variety of complex and quantitative methods suitable for deploying in live-cell conditions. In this study, a comprehensive review of the signal amplification technologies for the detection of nucleic acids is provided. We classify the signal amplification methodologies into enzymatic and non-enzymatic strategies with a primary focus on the methods that enable us to shift away from in vitro detecting to in vivo imaging. Finally, the future challenges and limitations of detection for cellular conditions are discussed.

Limits on amplification by Aharonov-Albert-Vaidman weak measurement

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

Koike, Tatsuhiko; Tanaka, Saki

2011-12-15

We analyze the amplification by the Aharonov-Albert-Vaidman weak quantum measurement on a Sagnac interferometer [Dixon et al., Phys. Rev. Lett. 102, 173601 (2009)] up to all orders of the coupling strength between the measured system and the measuring device. The amplifier transforms a small tilt of a mirror into a large transverse displacement of the laser beam. The conventional analysis has shown that the measured value is proportional to the weak value, so that the amplification can be made arbitrarily large in the cost of decreasing output laser intensity. It is shown that the measured displacement and the amplification factormore » are in fact not proportional to the weak value and rather vanish in the limit of infinitesimal output intensity. We derive the optimal overlap of the pre- and postselected states with which the amplification become maximum. We also show that the nonlinear effects begin to arise in the performed experiments so that any improvements in the experiment, typically with an amplification greater than 100, should require the nonlinear theory in translating the observed value to the original displacement.« less

Polymerase Chain Reaction (PCR)-based methods for detection and identification of mycotoxigenic Penicillium species using conserved genes

USDA-ARS?s Scientific Manuscript database

Polymerase chain reaction amplification of conserved genes and sequence analysis provides a very powerful tool for the identification of toxigenic as well as non-toxigenic Penicillium species. Sequences are obtained by amplification of the gene fragment, sequencing via capillary electrophoresis of d...

Ancient DNA in human bone remains from Pompeii archaeological site.

PubMed

Cipollaro, M; Di Bernardo, G; Galano, G; Galderisi, U; Guarino, F; Angelini, F; Cascino, A

1998-06-29

aDNA extraction and amplification procedures have been optimized for Pompeian human bone remains whose diagenesis has been determined by histological analysis. Single copy genes amplification (X and Y amelogenin loci and Y specific alphoid repeat sequences) have been performed and compared with anthropometric data on sexing.

Clinical features and gene mutational spectrum of CDKL5-related diseases in a cohort of Chinese patients

PubMed Central

2014-01-01

Background Mutations in the cyclin-dependent kinase-like 5 (CDKL5) (NM_003159.2) gene have been associated with early-onset epileptic encephalopathies or Hanefeld variants of RTT(Rett syndrome). In order to clarify the CDKL5 genotype-phenotype correlations in Chinese patients, CDKL5 mutational screening in cases with early-onset epileptic encephalopathies and RTT without MECP2 mutation were performed. Methods The detailed clinical information including clinical manifestation, electroencephalogram (EEG), magnetic resonance imaging (MRI), blood, urine amino acid and organic acid screening of 102 Chinese patients with early-onset epileptic encephalopathies and RTT were collected. CDKL5 gene mutations were analyzed by PCR, direct sequencing and multiplex ligation-dependent probe amplification (MLPA). The patterns of X-chromosome inactivation (XCI) were studied in the female patients with CDKL5 gene mutation. Results De novo CDKL5 gene mutations were found in ten patients including one missense mutation (c.533G > A, p.R178Q) which had been reported, two splicing mutations (ISV6 + 1A > G, ISV13 + 1A > G), three micro-deletions (c.1111delC, c.2360delA, c.234delA), two insertions (c.1791 ins G, c.891_892 ins TT in a pair of twins) and one nonsense mutation (c.1375C > T, p.Q459X). Out of ten patients, 7 of 9 females with Hanefeld variants of RTT and the remaining 2 females with early onset epileptic encephalopathy, were detected while only one male with infantile spasms was detected. The common features of all female patients with CDKL5 gene mutations included refractory seizures starting before 4 months of age, severe psychomotor retardation, Rett-like features such as hand stereotypies, deceleration of head growth after birth and poor prognosis. In contrast, the only one male patient with CDKL5 mutation showed no obvious Rett-like features as females in our cohort. The X-chromosome inactivation patterns of all the female patients were random. Conclusions Mutations in CDKL5 gene are responsible for 7 with Hanefeld variants of RTT and 2 with early-onset epileptic encephalopathy in 71 girls as well as for 1 infantile spasms in 31 males. There are some differences in the phenotypes among genders with CDKL5 gene mutations and CDKL5 gene mutation analysis should be considered in both genders. PMID:24564546

Clinical features and gene mutational spectrum of CDKL5-related diseases in a cohort of Chinese patients.

PubMed

Zhao, Ying; Zhang, Xiaoying; Bao, Xinhua; Zhang, Qingping; Zhang, Jingjing; Cao, Guangna; Zhang, Jie; Li, Jiarui; Wei, Liping; Pan, Hong; Wu, Xiru

2014-02-25

Mutations in the cyclin-dependent kinase-like 5 (CDKL5) (NM_003159.2) gene have been associated with early-onset epileptic encephalopathies or Hanefeld variants of RTT(Rett syndrome). In order to clarify the CDKL5 genotype-phenotype correlations in Chinese patients, CDKL5 mutational screening in cases with early-onset epileptic encephalopathies and RTT without MECP2 mutation were performed. The detailed clinical information including clinical manifestation, electroencephalogram (EEG), magnetic resonance imaging (MRI), blood, urine amino acid and organic acid screening of 102 Chinese patients with early-onset epileptic encephalopathies and RTT were collected. CDKL5 gene mutations were analyzed by PCR, direct sequencing and multiplex ligation-dependent probe amplification (MLPA). The patterns of X-chromosome inactivation (XCI) were studied in the female patients with CDKL5 gene mutation. De novo CDKL5 gene mutations were found in ten patients including one missense mutation (c.533G > A, p.R178Q) which had been reported, two splicing mutations (ISV6 + 1A > G, ISV13 + 1A > G), three micro-deletions (c.1111delC, c.2360delA, c.234delA), two insertions (c.1791 ins G, c.891_892 ins TT in a pair of twins) and one nonsense mutation (c.1375C > T, p.Q459X). Out of ten patients, 7 of 9 females with Hanefeld variants of RTT and the remaining 2 females with early onset epileptic encephalopathy, were detected while only one male with infantile spasms was detected. The common features of all female patients with CDKL5 gene mutations included refractory seizures starting before 4 months of age, severe psychomotor retardation, Rett-like features such as hand stereotypies, deceleration of head growth after birth and poor prognosis. In contrast, the only one male patient with CDKL5 mutation showed no obvious Rett-like features as females in our cohort. The X-chromosome inactivation patterns of all the female patients were random. Mutations in CDKL5 gene are responsible for 7 with Hanefeld variants of RTT and 2 with early-onset epileptic encephalopathy in 71 girls as well as for 1 infantile spasms in 31 males. There are some differences in the phenotypes among genders with CDKL5 gene mutations and CDKL5 gene mutation analysis should be considered in both genders.

Optimization of multiplexed PCR on an integrated microfluidic forensic platform for rapid DNA analysis.

PubMed

Estes, Matthew D; Yang, Jianing; Duane, Brett; Smith, Stan; Brooks, Carla; Nordquist, Alan; Zenhausern, Frederic

2012-12-07

This study reports the design, prototyping, and assay development of multiplexed polymerase chain reaction (PCR) on a plastic microfluidic device. Amplification of 17 DNA loci is carried out directly on-chip as part of a system for continuous workflow processing from sample preparation (SP) to capillary electrophoresis (CE). For enhanced performance of on-chip PCR amplification, improved control systems have been developed making use of customized Peltier assemblies, valve actuators, software, and amplification chemistry protocols. Multiple enhancements to the microfluidic chip design have been enacted to improve the reliability of sample delivery through the various on-chip modules. This work has been enabled by the encapsulation of PCR reagents into a solid phase material through an optimized Solid Phase Encapsulating Assay Mix (SPEAM) bead-based hydrogel fabrication process. SPEAM bead technology is reliably coupled with precise microfluidic metering and dispensing for efficient amplification and subsequent DNA short tandem repeat (STR) fragment analysis. This provides a means of on-chip reagent storage suitable for microfluidic automation, with the long shelf-life necessary for point-of-care (POC) or field deployable applications. This paper reports the first high quality 17-plex forensic STR amplification from a reference sample in a microfluidic chip with preloaded solid phase reagents, that is designed for integration with up and downstream processing.

REVIEW ARTICLE: A comparison of site response techniques using earthquake data and ambient seismic noise analysis in the large urban areas of Santiago de Chile

NASA Astrophysics Data System (ADS)

Pilz, Marco; Parolai, Stefano; Leyton, Felipe; Campos, Jaime; Zschau, Jochen

2009-08-01

Situated in an active tectonic region, Santiago de Chile, the country's capital with more than six million inhabitants, faces tremendous earthquake risk. Macroseismic data for the 1985 Valparaiso event show large variations in the distribution of damage to buildings within short distances, indicating strong effects of local sediments on ground motion. Therefore, a temporary seismic network was installed in the urban area for recording earthquake activity and a study was carried out aiming to estimate site amplification derived from horizontal-to-vertical (H/V) spectral ratios from earthquake data (EHV) and ambient noise (NHV), as well as using the standard spectral ratio (SSR) technique with a nearby reference station located on igneous rock. The results lead to the following conclusions: (1) The analysis of earthquake data shows significant dependence on the local geological structure with respect to amplitude and duration. (2) An amplification of ground motion at frequencies higher than the fundamental one can be found. This amplification would not be found when looking at NHV ratios alone. (3) The analysis of NHV spectral ratios shows that they can only provide a lower bound in amplitude for site amplification. (4) P-wave site responses always show lower amplitudes than those derived by S waves, and sometimes even fail to provide some frequencies of amplification. (5) No variability in terms of time and amplitude is observed in the analysis of the H/V ratio of noise. (6) Due to the geological conditions in some parts of the investigated area, the fundamental resonance frequency of a site is difficult to estimate following standard criteria proposed by the SESAME consortium, suggesting that these are too restrictive under certain circumstances.

Evaluation of HER-2/neu status in breast cancer specimens using immunohistochemistry (IHC) & fluorescence in-situ hybridization (FISH) assay

PubMed Central

Goud, Kalal Iravathy; Dayakar, Seetha; Vijayalaxmi, Kolanupaka; Babu, Saidam Jangu; Vijay, Anand Reddy P.

2012-01-01

Background & objectives: Fluorescence in situ hybridization (FISH) is increasingly being recognized as the most accurate and predictive test for HER2/neu gene amplification and response to therapy in breast cancer. In the present study we investigated HER-2/neu gene amplification by FISH in breast carcinoma tissue specimens and compared the results with that of immunohistochemical (IHC) analysis. Methods: A total of 90 breast carcinoma tissue samples were used for immunohistochemical (IHC) and FISH analysis. IHC was performed by using mouse monoclonal antibody to the intracellular domain of HER-2/neu protein. Each slide was scored in a blinded fashion by two pathologists according to the manufacturer's recommended criteria. FISH analysis was performed on paraffin embedded breast tumour tissue sections. The polysomy for centromere 17 (Spec green signal) was read as green signals less than 4 as moderate polysomy, and more than 4 as highly polysomy. Results: Thirty of the 90 patients had negative results by IHC and FISH. Of the 28 patients with the score of 2+ by IHC, 20 were FISH positive for HER-2/neu gene amplification, three were FISH negative and five patients showed equivocal (1.8-2.2) results by FISH. These five cases were retested for IHC and FISH on different paraffin embedded tissue blocks, and all five were found positive for HER-2/neu gene amplification. Twenty five patients with the score of 3+ by IHC were FISH positive for HER-2/neu gene amplification (>2.2). Seven cases with the score of 3+ by IHC were FISH negative for HER-2/neu gene amplification (>2.2), and showed polysomy of chromosome number 17 high polysomy > 4. Interpretation & conclusions: Our results indicated that HER-2/neu status by FISH should be performed in all cases of breast tumour with a 2+ score by IHC. Cases demonstrating a 3+ score by IHC may be subjected to FISH to rule out polysomy of chromosome 17 which could be falsely interpreted as HER-2/neu overexpression by IHC analysis. There is also a need for establishing a clinically validated cut-off value for HER-2/neu FISH amplification against IHC which may be further compared and calibrated. PMID:22561616

[Practice value of whole genome amplification technology to be used in forensic science and analysis of its result].

PubMed

Deng, Jian-qiang; Hou, Yi-ping

2005-08-01

Genetic analysis from forensic microsamples is a urgent, difficult task in forensic science, because it is frequently limited by the amount of specimen available in forensic practice, much effort has been carried out to resolve this difficulty. Whole genome amplification (WGA) technology, which was developing quickly in these years, has been thought to be a powerful, reliable and efficient strategy in analysis of minute amount DNA on many fields. In this review, we discuss its application in forensic science.

Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis

PubMed Central

Zhao, Hongjuan; Hastie, Trevor; Whitfield, Michael L; Børresen-Dale, Anne-Lise; Jeffrey, Stefanie S

2002-01-01

Background T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays. Results Using an optimized protocol, the average correlation coefficient of gene expression of 11,123 cDNA clones between amplified and unamplified samples is 0.82 (0.85 when a virtual array was created using repeatedly amplified samples to minimize experimental variation). Less than 4% of genes show changes in expression level by 2-fold or greater after amplification compared to unamplified samples. Most changes due to amplification are not systematic both within one tumor sample and between different tumors. Amplification appears to dampen the variation of gene expression for some genes when compared to unamplified poly(A)+ RNA. The reproducibility between repeatedly amplified samples is 0.97 when performed on the same day, but drops to 0.90 when performed weeks apart. The fidelity and reproducibility of amplification is not affected by decreasing the amount of input total RNA in the 0.3–3 micrograms range. Adding template-switching primer, DNA ligase, or column purification of double-stranded cDNA does not improve the fidelity of amplification. The correlation coefficient between amplified and unamplified samples is higher when total RNA is used as template for both experimental and reference RNA amplification. Conclusion T7 based linear amplification reproducibly generates amplified RNA that closely approximates original sample for gene expression profiling using cDNA microarrays. PMID:12445333

Next-generation sequencing reveals the mutational landscape of clinically diagnosed Usher syndrome: copy number variations, phenocopies, a predominant target for translational read-through, and PEX26 mutated in Heimler syndrome.

PubMed

Neuhaus, Christine; Eisenberger, Tobias; Decker, Christian; Nagl, Sandra; Blank, Cornelia; Pfister, Markus; Kennerknecht, Ingo; Müller-Hofstede, Cornelie; Charbel Issa, Peter; Heller, Raoul; Beck, Bodo; Rüther, Klaus; Mitter, Diana; Rohrschneider, Klaus; Steinhauer, Ute; Korbmacher, Heike M; Huhle, Dagmar; Elsayed, Solaf M; Taha, Hesham M; Baig, Shahid M; Stöhr, Heidi; Preising, Markus; Markus, Susanne; Moeller, Fabian; Lorenz, Birgit; Nagel-Wolfrum, Kerstin; Khan, Arif O; Bolz, Hanno J

2017-09-01

Combined retinal degeneration and sensorineural hearing impairment is mostly due to autosomal recessive Usher syndrome (USH1: congenital deafness, early retinitis pigmentosa (RP); USH2: progressive hearing impairment, RP). Sanger sequencing and NGS of 112 genes (Usher syndrome, nonsyndromic deafness, overlapping conditions), MLPA, and array-CGH were conducted in 138 patients clinically diagnosed with Usher syndrome. A molecular diagnosis was achieved in 97% of both USH1 and USH2 patients, with biallelic mutations in 97% (USH1) and 90% (USH2), respectively. Quantitative readout reliably detected CNVs (confirmed by MLPA or array-CGH), qualifying targeted NGS as one tool for detecting point mutations and CNVs. CNVs accounted for 10% of identified USH2A alleles, often in trans to seemingly monoallelic point mutations. We demonstrate PTC124-induced read-through of the common p.Trp3955* nonsense mutation (13% of detected USH2A alleles), a potential therapy target. Usher gene mutations were found in most patients with atypical Usher syndrome, but the diagnosis was adjusted in case of double homozygosity for mutations in OTOA and NR2E3 , genes implicated in isolated deafness and RP. Two patients with additional enamel dysplasia had biallelic PEX26 mutations, for the first time linking this gene to Heimler syndrome. Targeted NGS not restricted to Usher genes proved beneficial in uncovering conditions mimicking Usher syndrome.

MET expression and amplification in patients with localized gastric cancer

PubMed Central

Janjigian, Yelena Y.; Tang, Laura H.; Coit, Daniel G.; Kelsen, David P.; Francone, Todd D.; Weiser, Martin R.; Jhanwar, Suresh C.; Shah, Manish A.

2013-01-01

Background MET, the receptor for hepatocyte growth factor has been proposed as a therapeutic target in gastric cancer. This study assessed the incidence of MET expression and gene amplification in tumors of Western patients with gastric cancer. Methods Tumor specimens from patients enrolled on a preoperative chemotherapy study (NCI 5700) were examined for presence of MET gene amplification by fluorescence in situ hybridization (FISH), MET mRNA expression by quantitative polymerase chain reaction, MET overexpression by immunohistochemistry (IHC), and for evidence of MET pathway activation by p-MET IHC. Results Although high-level of MET protein and mRNA were commonly encountered (in 63% and 50% of resected tumor specimens, respectively), none of these tumors had MET gene amplification by FISH, and only 6.6% had evidence of MET tyrosine kinase activity by p-MET IHC. Conclusions In this cohort of patients with localized gastric cancer, the presence of high MET protein and RNA expression does not correlate with MET gene amplification or pathway activation as evidenced by the absence of amplification by FISH and negative p-MET IHC analysis. Impact This paper demonstrates a lack of MET amplification and pathway activation in a cohort of 38 patients with localized gastric cancer, suggesting that MET-driven gastric cancers are relatively rare in Western patients. PMID:21393565

Real-time electrochemical monitoring of isothermal helicase-dependent amplification of nucleic acids.

PubMed

Kivlehan, Francine; Mavré, François; Talini, Luc; Limoges, Benoît; Marchal, Damien

2011-09-21

We described an electrochemical method to monitor in real-time the isothermal helicase-dependent amplification of nucleic acids. The principle of detection is simple and well-adapted to the development of portable, easy-to-use and inexpensive nucleic acids detection technologies. It consists of monitoring a decrease in the electrochemical current response of a reporter DNA intercalating redox probe during the isothermal DNA amplification. The method offers the possibility to quantitatively analyze target nucleic acids in less than one hour at a single constant temperature, and to perform at the end of the isothermal amplification a DNA melt curve analysis for differentiating between specific and non-specific amplifications. To illustrate the potentialities of this approach for the development of a simple, robust and low-cost instrument with high throughput capability, the method was validated with an electrochemical system capable of monitoring up to 48 real-time isothermal HDA reactions simultaneously in a disposable microplate consisting of 48-electrochemical microwells. Results obtained with this approach are comparable to that obtained with a well-established but more sophisticated and expensive fluorescence-based method. This makes for a promising alternative detection method not only for real-time isothermal helicase-dependent amplification of nucleic acid, but also for other isothermal DNA amplification strategies.

Familial co-segregation of Coffin-Lowry syndrome inherited from the mother and autosomal dominant Waardenburg type IV syndrome due to deletion of EDNRB inherited from the father.

PubMed

Loupe, Jacob; Sampath, Srirangan; Lacassie, Yves

2014-10-01

We report an African-American family that was identified after the proposita was referred for diagnostic evaluation at 4½ months with a history of Hirschsprung and dysmorphic features typical of Waardenburg syndrome (WS). Family evaluation revealed that the father had heterochromidia irides and hypertelorism supporting the clinical diagnosis of WS; however, examination of the mother revealed characteristic facial and digital features of Coffin-Lowry syndrome (CLS). Molecular testing of the mother identified a novel 2 bp deletion (c.865_866delCA) in codon 289 of RPS6KA3 leading to a frame-shift and premature termination of translation 5 codons downstream (NM_004586.2:p.Gln289ValfsX5). This deletion also was identified in the proposita and her three sisters with a clinical suspicion of CLS, all of whom as carriers for this X-linked disorder had very subtle manifestations. The molecular confirmation of WS type 4 (Shah-Waardenburg; WS4) was not as straightforward. To evaluate WS types 1-4, multiple sequential molecular tests were requested, including Sanger sequencing of all exons, and deletion/duplication analysis using MLPA for PAX3, MITF, SOX10, EDN3 and EDNRB. Although sequencing did not identify any disease causing variants, MLPA identified a heterozygous deletion of the entire EDNRB in the father. This deletion was also found in the proposita and the oldest child. Since the heterozygous deletion was the only change identified in EDNRB, this family represents one of the few cases of an autosomal dominant inheritance of WS4 involving the endothelin pathway. Altogether, clinical evaluation of the family revealed one child to be positive for WS4 and two positive for CLS, while two children were positive for both diseases simultaneously (including the proposita) while another pair test negative for either disease. This kinship is an example of the coincidence of two conditions co-segregating in one family, with variable phenotypes requiring molecular testing to confirm the clinical diagnoses. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Detection of DNA Sequences Refractory to PCR Amplification Using a Biophysical SERRS Assay (Surface Enhanced Resonant Raman Spectroscopy)

PubMed Central

Feuillie, Cécile; Merheb, Maxime M.; Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

2014-01-01

The analysis of ancient or processed DNA samples is often a great challenge, because traditional Polymerase Chain Reaction – based amplification is impeded by DNA damage. Blocking lesions such as abasic sites are known to block the bypass of DNA polymerases, thus stopping primer elongation. In the present work, we applied the SERRS-hybridization assay, a fully non-enzymatic method, to the detection of DNA refractory to PCR amplification. This method combines specific hybridization with detection by Surface Enhanced Resonant Raman Scattering (SERRS). It allows the detection of a series of double-stranded DNA molecules containing a varying number of abasic sites on both strands, when PCR failed to detect the most degraded sequences. Our SERRS approach can quickly detect DNA molecules without any need for DNA repair. This assay could be applied as a pre-requisite analysis prior to enzymatic reparation or amplification. A whole new set of samples, both forensic and archaeological, could then deliver information that was not yet available due to a high degree of DNA damage. PMID:25502338

Detection of DNA sequences refractory to PCR amplification using a biophysical SERRS assay (Surface Enhanced Resonant Raman Spectroscopy).

PubMed

Feuillie, Cécile; Merheb, Maxime M; Gillet, Benjamin; Montagnac, Gilles; Daniel, Isabelle; Hänni, Catherine

2014-01-01

The analysis of ancient or processed DNA samples is often a great challenge, because traditional Polymerase Chain Reaction - based amplification is impeded by DNA damage. Blocking lesions such as abasic sites are known to block the bypass of DNA polymerases, thus stopping primer elongation. In the present work, we applied the SERRS-hybridization assay, a fully non-enzymatic method, to the detection of DNA refractory to PCR amplification. This method combines specific hybridization with detection by Surface Enhanced Resonant Raman Scattering (SERRS). It allows the detection of a series of double-stranded DNA molecules containing a varying number of abasic sites on both strands, when PCR failed to detect the most degraded sequences. Our SERRS approach can quickly detect DNA molecules without any need for DNA repair. This assay could be applied as a pre-requisite analysis prior to enzymatic reparation or amplification. A whole new set of samples, both forensic and archaeological, could then deliver information that was not yet available due to a high degree of DNA damage.

MET amplification, expression, and exon 14 mutations in colorectal adenocarcinoma.

PubMed

Zhang, Meng; Li, Guichao; Sun, Xiangjie; Ni, Shujuan; Tan, Cong; Xu, Midie; Huang, Dan; Ren, Fei; Li, Dawei; Wei, Ping; Du, Xiang

2018-04-08

MET amplification, expression, and splice mutations at exon 14 result in dysregulation of the MET signaling pathway. The aim of this study was to identify the relationship between MET amplification, protein or mRNA expression, and mutations in colorectal cancer (CRC). MET immunohistochemistry (IHC) was used for MET protein expression analysis and fluorescence in situ hybridization (FISH) was used for MET amplification detection. Both analyses were performed in tissue microarrays (TMA) containing 294 of colorectal adenocarcinoma tissue samples and 131 samples of adjacent normal epithelial tissue. MET mRNA expression was examined by real-time quantitative polymerase chain reaction (qRT-PCR) in 72 fresh colorectal adenocarcinoma tissue samples and adjacent normal colon tissue. PCR sequencing was performed to screen for MET exon 14 splice mutations in 59 fresh CRC tissue samples. Our results showed that MET protein expression was higher in colorectal tumor tissue than in adjacent normal intestinal epithelium. Positive MET protein expression was associated with significantly poorer overall survival (OS) and disease-free survival (DFS). Multivariate analysis revealed that positive MET protein expression was an independent risk factor for DFS, but not for OS. MET mRNA expression was upregulated in tumor tissues compared with the adjacent normal tissues. The incidence of MET amplification was 4.4%. None of the patients was positive for MET mutation. Collectively, MET was overexpressed in colorectal adenocarcinoma, and its positive protein expression predicted a poorer outcome in CRC patients. Furthermore, according to our results, MET amplification and 14 exon mutation are extremely rare events in colorectal adenocarcinoma. Copyright © 2018. Published by Elsevier Inc.

Development and comparison of a rapid isothermal nucleic acid amplification test for typing of herpes simplex virus types 1 and 2 on a portable fluorescence detector.

PubMed

Tong, Yanhong; McCarthy, Kaitlin; Kong, Huimin; Lemieux, Bertrand

2012-11-01

We have developed a rapid and simple molecular test, the IsoGlow HSV Typing assay, for the detection and typing of herpes simplex virus (type 1 and 2) from genital or oral lesions. Clinical samples suspended in viral transport mediums are simply diluted and then added to a helicase-dependent amplification master mix. The amplification and detection were performed on a portable fluorescence detector called the FireFly instrument. Detection of amplification products is based on end-point analysis using cycling probe technology. An internal control nucleic acid was included in the amplification master mix to monitor the presence of amplification inhibitors in the samples. Because the device has only two fluorescence detection channels, two strategies were developed and compared to detect the internal control template: internal control detected by melting curve analysis using a dual-labeled probe, versus internal control detection using end-point fluorescence release by a CPT probe at a lower temperature. Both have a total turnaround time of about 1 hour. Clinical performance relative to herpes viral culture was evaluated using 176 clinical specimens. Both formats of the IsoGlow HSV typing assay had sensitivities comparable to that of the Food and Drug Administration-cleared IsoAmp HSV (BioHelix Corp., Beverly MA) test and specificity for the two types of HSV comparable to that of ELVIS HSV (Diagnostic Hybrids, Athens, OH). Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

[Individual Identification of Cartilage by Direct Amplification in Mass Disasters].

PubMed

Wang, C H; Xu, C; Li, X Q; Wu, Y; Du, Z

2017-06-01

To explore the effectiveness of direct amplification for the STR analysis of cartilage, and to accelerate the effectiveness of disaster victim identification. Eighty-eight cartilage samples were directly amplified by PowerPle® 21 kit, and the results of genotyping were compared with that obtained by the magnetic beads method. In 88 cartilage samples, the STR genotypes were successfully detected from 84 samples by direct amplification and magnetic beads method, and both the results of genotyping by two method were consistent. Direct amplification with PowerPlex® 21 kit can be used for STR genotyping of cartilages. This method is operated easily and promptly, which has a potential application in the individual identification of mass disasters. Copyright© by the Editorial Department of Journal of Forensic Medicine

Amplification of bovine papillomavirus DNA by N-methyl-N'-nitro-N-nitrosoguanidine, ultraviolet irradiation, or infection with herpes simplex virus

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

Schmitt, J.; Schlehofer, J.R.; Mergener, K.

1989-09-01

Treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or irradiation with ultraviolet light (uv254 nm) induces amplification of integrated as well as episomal sequences of bovine papillomavirus (BPV) type 1 DNA in BPV-1-transformed mouse C127 cells (i.e., ID13 cells). This is shown by filter in situ hybridization and Southern blot analysis of cellular DNA. Similarly, infection of ID13 cells with herpes simplex virus (HSV) type 1 which has been shown to be mutagenic for host cell DNA leads to amplification of BPV DNA sequences. In contrast to this induction of DNA amplification by initiators, treatment of ID13 cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)more » does not result in increased synthesis of BPV DNA nor does TPA treatment modulate the initiator-induced DNA amplification. Similar to other cell systems infection with adeno-associated virus (AAV) type 2 inhibits BPV-1 DNA amplification irrespective of the inducing agent. In contrast to initiator-induced DNA amplification, treatment with carcinogen (MNNG) or tumor promoters or combination of MNNG and promoter of C127 cells prior to transformation by BPV-1 does not lead to an increase in the number of transformed foci. The induction of amplification of papillomavirus DNA by initiating agents possibly represents one of the mechanisms by which the observed synergism between papillomavirus infection and initiators in tumorigenesis might occur.« less

Word Recognition and Learning: Effects of Hearing Loss and Amplification Feature

PubMed Central

Stewart, Elizabeth C.; Willman, Amanda P.; Odgear, Ian S.

2017-01-01

Two amplification features were examined using auditory tasks that varied in stimulus familiarity. It was expected that the benefits of certain amplification features would increase as the familiarity with the stimuli decreased. A total of 20 children and 15 adults with normal hearing as well as 21 children and 17 adults with mild to severe hearing loss participated. Three models of ear-level devices were selected based on the quality of the high-frequency amplification or the digital noise reduction (DNR) they provided. The devices were fitted to each participant and used during testing only. Participants completed three tasks: (a) word recognition, (b) repetition and lexical decision of real and nonsense words, and (c) novel word learning. Performance improved significantly with amplification for both the children and the adults with hearing loss. Performance improved further with wideband amplification for the children more than for the adults. In steady-state noise and multitalker babble, performance decreased for both groups with little to no benefit from amplification or from the use of DNR. When compared with the listeners with normal hearing, significantly poorer performance was observed for both the children and adults with hearing loss on all tasks with few exceptions. Finally, analysis of across-task performance confirmed the hypothesis that benefit increased as the familiarity of the stimuli decreased for wideband amplification but not for DNR. However, users who prefer DNR for listening comfort are not likely to jeopardize their ability to detect and learn new information when using this feature. PMID:29169314

Real-time DNA Amplification and Detection System Based on a CMOS Image Sensor.

PubMed

Wang, Tiantian; Devadhasan, Jasmine Pramila; Lee, Do Young; Kim, Sanghyo

2016-01-01

In the present study, we developed a polypropylene well-integrated complementary metal oxide semiconductor (CMOS) platform to perform the loop mediated isothermal amplification (LAMP) technique for real-time DNA amplification and detection simultaneously. An amplification-coupled detection system directly measures the photon number changes based on the generation of magnesium pyrophosphate and color changes. The photon number decreases during the amplification process. The CMOS image sensor observes the photons and converts into digital units with the aid of an analog-to-digital converter (ADC). In addition, UV-spectral studies, optical color intensity detection, pH analysis, and electrophoresis detection were carried out to prove the efficiency of the CMOS sensor based the LAMP system. Moreover, Clostridium perfringens was utilized as proof-of-concept detection for the new system. We anticipate that this CMOS image sensor-based LAMP method will enable the creation of cost-effective, label-free, optical, real-time and portable molecular diagnostic devices.

Site characterization and site response in Port-au-Prince, Haiti

USGS Publications Warehouse

Hough, Susan E.; Yong, Alan K.; Altidor, Jean Robert; Anglade, Dieuseul; Given, Douglas D.; Mildor, Saint-Louis

2011-01-01

Waveform analysis of aftershocks of the Mw7.0 Haiti earthquake of 12 January 2010 reveals amplification of ground motions at sites within the Cul de Sac valley in which Port-au-Prince is situated. Relative to ground motions recorded at a hard-rock reference site, peak acceleration values are amplified by a factor of approximately 1.8 at sites on low-lying Mio-Pliocene deposits in central Port-au-Prince and by a factor of approximately 2.5–3 on a steep foothill ridge in the southern Port-au-Prince metropolitan region. The observed amplitude, predominant periods, variability, and polarization of amplification are consistent with predicted topographic amplification by a steep, narrow ridge. A swath of unusually high damage in this region corresponds with the extent of the ridge where high weak-motion amplifications are observed. We use ASTER (Advanced Spaceborne Thermal Emission and Reflection Radiometer) imagery to map local geomorphology, including characterization of both near-surface and of small-scale topographic structures that correspond to zones of inferred amplification.

Frequency-domain nonlinear optics in two-dimensionally patterned quasi-phase-matching media.

PubMed

Phillips, C R; Mayer, B W; Gallmann, L; Keller, U

2016-07-11

Advances in the amplification and manipulation of ultrashort laser pulses have led to revolutions in several areas. Examples include chirped pulse amplification for generating high peak-power lasers, power-scalable amplification techniques, pulse shaping via modulation of spatially-dispersed laser pulses, and efficient frequency-mixing in quasi-phase-matched nonlinear crystals to access new spectral regions. In this work, we introduce and demonstrate a new platform for nonlinear optics which has the potential to combine these separate functionalities (pulse amplification, frequency transfer, and pulse shaping) into a single monolithic device that is bandwidth- and power-scalable. The approach is based on two-dimensional (2D) patterning of quasi-phase-matching (QPM) gratings combined with optical parametric interactions involving spatially dispersed laser pulses. Our proof of principle experiment demonstrates this technique via mid-infrared optical parametric chirped pulse amplification of few-cycle pulses. Additionally, we present a detailed theoretical and numerical analysis of such 2D-QPM devices and how they can be designed.

Microfluidic Devices for Forensic DNA Analysis: A Review

PubMed Central

Bruijns, Brigitte; van Asten, Arian; Tiggelaar, Roald; Gardeniers, Han

2016-01-01

Microfluidic devices may offer various advantages for forensic DNA analysis, such as reduced risk of contamination, shorter analysis time and direct application at the crime scene. Microfluidic chip technology has already proven to be functional and effective within medical applications, such as for point-of-care use. In the forensic field, one may expect microfluidic technology to become particularly relevant for the analysis of biological traces containing human DNA. This would require a number of consecutive steps, including sample work up, DNA amplification and detection, as well as secure storage of the sample. This article provides an extensive overview of microfluidic devices for cell lysis, DNA extraction and purification, DNA amplification and detection and analysis techniques for DNA. Topics to be discussed are polymerase chain reaction (PCR) on-chip, digital PCR (dPCR), isothermal amplification on-chip, chip materials, integrated devices and commercially available techniques. A critical overview of the opportunities and challenges of the use of chips is discussed, and developments made in forensic DNA analysis over the past 10–20 years with microfluidic systems are described. Areas in which further research is needed are indicated in a future outlook. PMID:27527231

VS30, site amplifications and some comparisons: The Adapazari (Turkey) case

NASA Astrophysics Data System (ADS)

Ozcep, Tazegul; Ozcep, Ferhat; Ozel, Oguz

The aim of this study was to investigate the role of VS30 in site amplifications in the Adapazari region, Turkey. To fulfil this aim, amplifications from VS30 measurements were compared with earthquake data for different soil types in the seismic design codes. The Adapazari area was selected as the study area, and shear-wave velocity distribution was obtained by the multichannel analysis of surface waves (MASWs) method at 100 sites for the top 50 m of soil. Aftershock data following the Mw 7.4 Izmit earthquake of 17 August 1999 gave magnitudes between 4.0 and 5.6 at six stations installed in and around the Adapazari Basin, at Babalı, Şeker, Genç, Hastane, Toyota and Imar. This data was used to estimate site amplifications by the reference-station method. In addition, the fundamental periods of the station sites were estimated by the single station method. Site classifications based on VS30 in the seismic design codes were compared with the fundamental periods and amplification values. It was found that site amplifications (from earthquake data) and relevant spectra (from VS30) are not in good agreement for soils in Adapazari (Turkey).

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs.

PubMed

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Zel, Jana; Gruden, Kristina

2008-10-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1-25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification.

NAIMA: target amplification strategy allowing quantitative on-chip detection of GMOs

PubMed Central

Morisset, Dany; Dobnik, David; Hamels, Sandrine; Žel, Jana; Gruden, Kristina

2008-01-01

We have developed a novel multiplex quantitative DNA-based target amplification method suitable for sensitive, specific and quantitative detection on microarray. This new method named NASBA Implemented Microarray Analysis (NAIMA) was applied to GMO detection in food and feed, but its application can be extended to all fields of biology requiring simultaneous detection of low copy number DNA targets. In a first step, the use of tailed primers allows the multiplex synthesis of template DNAs in a primer extension reaction. A second step of the procedure consists of transcription-based amplification using universal primers. The cRNA product is further on directly ligated to fluorescent dyes labelled 3DNA dendrimers allowing signal amplification and hybridized without further purification on an oligonucleotide probe-based microarray for multiplex detection. Two triplex systems have been applied to test maize samples containing several transgenic lines, and NAIMA has shown to be sensitive down to two target copies and to provide quantitative data on the transgenic contents in a range of 0.1–25%. Performances of NAIMA are comparable to singleplex quantitative real-time PCR. In addition, NAIMA amplification is faster since 20 min are sufficient to achieve full amplification. PMID:18710880

Generation of sequence signatures from DNA amplification fingerprints with mini-hairpin and microsatellite primers.

PubMed

Caetano-Anollés, G; Gresshoff, P M

1996-06-01

DNA amplification fingerprinting (DAF) with mini-hairpins harboring arbitrary "core" sequences at their 3' termini were used to fingerprint a variety of templates, including PCR products and whole genomes, to establish genetic relationships between plant tax at the interspecific and intraspecific level, and to identify closely related fungal isolates and plant accessions. No correlation was observed between the sequence of the arbitrary core, the stability of the mini-hairpin structure and DAF efficiency. Mini-hairpin primers with short arbitrary cores and primers complementary to simple sequence repeats present in microsatellites were also used to generate arbitrary signatures from amplification profiles (ASAP). The ASAP strategy is a dual-step amplification procedure that uses at least one primer in each fingerprinting stage. ASAP was able to reproducibly amplify DAF products (representing about 10-15 kb of sequence) following careful optimization of amplification parameters such as primer and template concentration. Avoidance of primer sequences partially complementary to DAF product termini was necessary in order to produce distinct fingerprints. This allowed the combinatorial use of oligomers in nucleic acid screening, with numerous ASAP fingerprinting reactions based on a limited number of primer sequences. Mini-hairpin primers and ASAP analysis significantly increased detection of polymorphic DNA, separating closely related bermudagrass (Cynodon) cultivars and detecting putatively linked markers in bulked segregant analysis of the soybean (Glycine max) supernodulation (nitrate-tolerant symbiosis) locus.

Computational method and system for modeling, analyzing, and optimizing DNA amplification and synthesis

DOEpatents

Vandersall, Jennifer A.; Gardner, Shea N.; Clague, David S.

2010-05-04

A computational method and computer-based system of modeling DNA synthesis for the design and interpretation of PCR amplification, parallel DNA synthesis, and microarray chip analysis. The method and system include modules that address the bioinformatics, kinetics, and thermodynamics of DNA amplification and synthesis. Specifically, the steps of DNA selection, as well as the kinetics and thermodynamics of DNA hybridization and extensions, are addressed, which enable the optimization of the processing and the prediction of the products as a function of DNA sequence, mixing protocol, time, temperature and concentration of species.

Prognostic relevance of Fédération Nationale des Centres de Lutte Contre le Cancer grade and MDM2 amplification levels in dedifferentiated liposarcoma: a study of 50 cases.

PubMed

Jour, George; Gullet, Ashley; Liu, Mingdong; Hoch, Benjamin L

2015-01-01

Dedifferentiated liposarcoma represents a form of liposarcoma composed of a non-lipogenic sarcoma associated with well-differentiated liposarcoma. The prognostic significance of histological grading of the dedifferentiated component remains to be elucidated due to vague grading criteria employed in previous studies. Molecular markers of tumor behavior, including amplification levels of murine double minute-2 (MDM2) and cyclin-dependent kinase-4 (CDK4) genes, have been explored in a limited number of cases. Here we investigate whether 'Fédération Nationale des Centres de Lutte Contre le Cancer' (FNCLCC) grade and MDM2 gene amplification levels have prognostic value in dedifferentiated liposarcoma in terms of local recurrence and disease-specific survival. Fifty cases were retrieved, reviewed and FNCLCC grade was scored for the dedifferentiated component. Testing for MDM2 gene amplification was performed by fluorescence in situ hybridization. Amplification was categorized as high level (≥20 copies) and as low level (<20 copies). Follow-up data was obtained through chart review. Log-rank test and Cox proportional hazard models were used to determine the effect of grade and level of MDM2 amplification on outcomes. Our series includes 50 patients (male n=28, female n=22) with an average age of 63 years (range, 28-88) and a median follow-up of 28 months (range, 2-120). Tumors were graded as grade 1 (6%), grade 2 (58%), and grade 3 (36%). When adjusted for age, sex, site, tumor size, and margin status, grade 3 patients had a higher recurrence rate than grades 1 and 2 (HR=2.07, 95% CI: 1.24, 7.62; P=0.015). Patients with high-level MDM2 amplification had higher recurrence rate on univariate analysis (P=0.028), but not on multivariate analysis (HR=1.69, 95% CI: 0.73, 3.94; P=0.221). FNCLCC grade 3 dedifferentiation confers a worse prognosis in dedifferentiated liposarcoma in terms of local recurrence. MDM2 amplification level remains a useful diagnostic tool in dedifferentiated liposarcoma, but has no prognostic value in terms of local recurrence.

Recurrent amplification of RTEL1 and ABCA13 and its synergistic effect associated with clinicopathological data of gastric adenocarcinoma.

PubMed

Araújo, T M; Seabra, A D; Lima, E M; Assumpção, P P; Montenegro, R C; Demachki, S; Burbano, R M; Khayat, A S

2016-01-01

Despite progression in treatment of gastric cancer, prognosis of patients remains poor, in part due to the low rate of diagnosis during its early stages. This paradigm implies the necessity to identify molecular biomarkers for early gastric cancer diagnosis, as well as for disease monitoring, thus contributing to the development of new therapeutic approaches. In a previous study, performed by array-Comparative Genomic Hybridization, we described for the first time in literature recurrent amplification of RTEL1 and ABCA13 genes in gastric cancer. Thus, the aim of the present study was to validate recurrent amplification of RTEL1 and ABCA13 genes and associate CNV status with clinicopathological data. Results showed RTEL1 and ABCA13 amplification in 38 % of samples. Statistical analysis demonstrated that RTEL amplification is more common in older patients and more associated with intestinal type and ABCA13 amplification increases the risk of lymph node metastasis and is more common in men. Co-amplification of these genes showed a significant association with advanced staging. aCGH is a very useful tool for investigating novel genes associated with carcinogenesis and RTEL1 amplification may be important for the development of gastric cancer in older patients, besides being a probable event contributing for chromosomal instability in intestinal gastric carcinogenesis. ABCA13 amplification may have age-specific function and could be considered a useful marker for predicting lymph node metastasis in resected gastric cancer patients in early stage. Lastly, RTEL1 and ABCA13 synergistic effect may be considered as a putative marker for advanced staging in gastric cancer patients.

On-chip electrical detection of parallel loop-mediated isothermal amplification with DG-BioFETs for the detection of foodborne bacterial pathogens

USDA-ARS?s Scientific Manuscript database

The use of field effect transistors (FETs) as the transduction element for the detection of DNA amplification reactions will enable portable and inexpensive nucleic acid analysis. Transistors used as biological sensors,or BioFETs, minimize the cost and size of detection platforms by leveraging fabri...

Designing a Micromixer for Rolling Circle Amplification in Cancer Biomarker Detection

NASA Astrophysics Data System (ADS)

Altural, Hayriye

2015-03-01

Rolling circle amplification (RCA) is an alternative method to the Polymerase Chain Reaction based amplification for point-of-care (POC) diagnosis. In future personalized cancer diagnostic for POC applications, smaller, faster and cheaper methods are needed instead of costly and time-consuming laboratory tests. Microfluidic chips can perform the detection of cancer biomarkers within less analysis time, and provide for improvement in the sensitivity and specificity required for biochemical analysis as well. Rapid mixing is essential in the chips used in cancer diagnostic. The goal of this study is to design a micromixer for rapid RCA-based analysis and develop the assay time in cancer biomarker detection. By combining assays with micromixers, multi-step bioreactions in microfluidic chips may be achieved with minimal external control. Here, simulation results related to the micromixer are obtained by COMSOL software. The Scientific and Technological Research Council of Turkey (TUBITAK) is acknowledged for granting of H. Altural postdoctoral study in the framework of TUBITAK-BIDEB 2219-International Postdoctoral Research Scholarship Program.

Direct identification of non-polio enteroviruses in residual paralysis cases by analysis of VP1 sequences.

PubMed

Rahimi, Pooneh; Tabatabaie, H; Gouya, Mohammad M; Mahmudi, M; Musavi, T; Rad, K Samimi; Azad, T Mokhtari; Nategh, R

2009-06-01

The 66 serotypes of human enteroviruses (EVs) are classified into four species A-D, based on phylogenetic relationships in multiple genome regions. Partial VP(1) amplification and sequence analysis are reliable methods for identifying non-polio enterovirus serotypes, especially in negative cell culture specimens from patients with residual paralysis. In Iran during the years 2000-2002, there were 29 residual paralysis cases with negative cell (RD, HEp(2) and L(20)B) culture results. The genomic RNA was extracted from stool specimens from cases of residual paralysis and detected by amplification of the 5'-nontranslated region using RT-PCR with Pan-EV primers. Partial VP(1) amplification by semi-nested RT-PCR (snRT-PCR) and sequence analysis were done. Specimens from the 29 culture-negative cases contained echoviruses of six different serotypes. The global eradication of wild polioviruses is near and study of non-polio enteroviruses, which can cause poliomyelitis, is increasingly important to understand their pathogenesis. The VP(1) sequences, derived from the snRT-PCR products, allowed rapid molecular analysis of these non-polio strains.

Assessment of palindromes as platforms for DNA amplification in breast cancer

PubMed Central

Guenthoer, Jamie; Diede, Scott J.; Tanaka, Hisashi; Chai, Xiaoyu; Hsu, Li; Tapscott, Stephen J.; Porter, Peggy L.

2012-01-01

DNA amplification, particularly of chromosomes 8 and 11, occurs frequently in breast cancer and is a key factor in tumorigenesis, often associated with poor prognosis. The mechanisms involved in the amplification of these regions are not fully understood. Studies from model systems have demonstrated that palindrome formation can be an early step in DNA amplification, most notably seen in the breakage–fusion–bridge (BFB) cycle. Therefore, palindromes might be associated with gene amplicons in breast cancer. To address this possibility, we coupled high-resolution palindrome profiling by the Genome-wide Analysis of Palindrome Formation (GAPF) assay with genome-wide copy-number analyses on a set of breast cancer cell lines and primary tumors to spatially associate palindromes and copy-number gains. We identified GAPF-positive regions distributed nonrandomly throughout cell line and tumor genomes, often in clusters, and associated with copy-number gains. Commonly amplified regions in breast cancer, chromosomes 8q and 11q, had GAPF-positive regions flanking and throughout the copy-number gains. We also identified amplification-associated GAPF-positive regions at similar locations in subsets of breast cancers with similar characteristics (e.g., ERBB2 amplification). These shared positive regions offer the potential to evaluate the utility of palindromes as prognostic markers, particularly in premalignant breast lesions. Our results implicate palindrome formation in the amplification of regions with key roles in breast tumorigenesis, particularly in subsets of breast cancers. PMID:21752925

A Model for Amplification of Hair-Bundle Motion by Cyclical Binding of Ca2+ to Mechanoelectrical-Transduction Channels

NASA Astrophysics Data System (ADS)

Choe, Yong; Magnasco, Marcelo O.; Hudspeth, A. J.

1998-12-01

Amplification of auditory stimuli by hair cells augments the sensitivity of the vertebrate inner ear. Cell-body contractions of outer hair cells are thought to mediate amplification in the mammalian cochlea. In vertebrates that lack these cells, and perhaps in mammals as well, active movements of hair bundles may underlie amplification. We have evaluated a mathematical model in which amplification stems from the activity of mechanoelectrical-transduction channels. The intracellular binding of Ca2+ to channels is posited to promote their closure, which increases the tension in gating springs and exerts a negative force on the hair bundle. By enhancing bundle motion, this force partially compensates for viscous damping by cochlear fluids. Linear stability analysis of a six-state kinetic model reveals Hopf bifurcations for parameter values in the physiological range. These bifurcations signal conditions under which the system's behavior changes from a damped oscillatory response to spontaneous limit-cycle oscillation. By varying the number of stereocilia in a bundle and the rate constant for Ca2+ binding, we calculate bifurcation frequencies spanning the observed range of auditory sensitivity for a representative receptor organ, the chicken's cochlea. Simulations using prebifurcation parameter values demonstrate frequency-selective amplification with a striking compressive nonlinearity. Because transduction channels occur universally in hair cells, this active-channel model describes a mechanism of auditory amplification potentially applicable across species and hair-cell types.

Waddell non-organic signs: new evidence suggests somatic amplification among outpatient chronic pain patients.

PubMed

Wygant, Dustin B; Arbisi, Paul A; Bianchini, Kevin J; Umlauf, Robert L

2017-04-01

Waddell et al. identified a set of eight non-organic signs in 1980. There has been controversy about their meaning, particularly with respect to their use as validity indicators. The current study examined the Waddell signs in relation to measures of somatic amplification or over-reporting in a sample of outpatient chronic pain patients. We examined the degree to which these signs were associated with measures of over-reporting. This study examined scores on the Waddell signs in relation to over-reporting indicators in an outpatient chronic pain sample. We examined 230 chronic pain patients treated at a multidisciplinary pain clinic. The majority of these patients presented with primary back or spinal injuries. The outcome measures used in the study were Waddell signs, Modified Somatic Perception Questionnaire, Pain Disability Index, and the Minnesota Multiphasic Personality Inventory-2 Restructured Form. We examined Waddell signs using multivariate analysis of variance (MANOVA) and analysis of variance (ANOVA), receiver operating characteristic analysis, classification accuracy, and relative risk ratios. Multivariate analysis of variance and ANOVA showed a significant association between Waddell signs and somatic amplification. Classification analyses showed increased odds of somatic amplification at a Waddell score of 2 or 3. Our results found significant evidence of an association between Waddell signs and somatic over-reporting. Elevated scores on the Waddell signs (particularly scores higher than 2 and 3) were associated with increased odds of exhibiting somatic over-reporting. Copyright © 2016 Elsevier Inc. All rights reserved.

An Intrinsically Digital Amplification Scheme for Hearing Aids

NASA Astrophysics Data System (ADS)

Blamey, Peter J.; Macfarlane, David S.; Steele, Brenton R.

2005-12-01

Results for linear and wide-dynamic range compression were compared with a new 64-channel digital amplification strategy in three separate studies. The new strategy addresses the requirements of the hearing aid user with efficient computations on an open-platform digital signal processor (DSP). The new amplification strategy is not modeled on prior analog strategies like compression and linear amplification, but uses statistical analysis of the signal to optimize the output dynamic range in each frequency band independently. Using the open-platform DSP processor also provided the opportunity for blind trial comparisons of the different processing schemes in BTE and ITE devices of a high commercial standard. The speech perception scores and questionnaire results show that it is possible to provide improved audibility for sound in many narrow frequency bands while simultaneously improving comfort, speech intelligibility in noise, and sound quality.

Real-time correction of tsunami site effect by frequency-dependent tsunami-amplification factor

NASA Astrophysics Data System (ADS)

Tsushima, H.

2017-12-01

For tsunami early warning, I developed frequency-dependent tsunami-amplification factor and used it to design a recursive digital filter that can be applicable for real-time correction of tsunami site response. In this study, I assumed that a tsunami waveform at an observing point could be modeled by convolution of source, path and site effects in time domain. Under this assumption, spectral ratio between offshore and the nearby coast can be regarded as site response (i.e. frequency-dependent amplification factor). If the amplification factor can be prepared before tsunamigenic earthquakes, its temporal convolution to offshore tsunami waveform provides tsunami prediction at coast in real time. In this study, tsunami waveforms calculated by tsunami numerical simulations were used to develop frequency-dependent tsunami-amplification factor. Firstly, I performed numerical tsunami simulations based on nonlinear shallow-water theory from many tsuanmigenic earthquake scenarios by varying the seismic magnitudes and locations. The resultant tsunami waveforms at offshore and the nearby coastal observing points were then used in spectral-ratio analysis. An average of the resulted spectral ratios from the tsunamigenic-earthquake scenarios is regarded as frequency-dependent amplification factor. Finally, the estimated amplification factor is used in design of a recursive digital filter that can be applicable in time domain. The above procedure is applied to Miyako bay at the Pacific coast of northeastern Japan. The averaged tsunami-height spectral ratio (i.e. amplification factor) between the location at the center of the bay and the outside show a peak at wave-period of 20 min. A recursive digital filter based on the estimated amplification factor shows good performance in real-time correction of tsunami-height amplification due to the site effect. This study is supported by Japan Society for the Promotion of Science (JSPS) KAKENHI grant 15K16309.

The effects of host diversity on vector-borne disease: the conditions under which diversity will amplify or dilute the disease risk.

PubMed

Miller, Ezer; Huppert, Amit

2013-01-01

Multihost vector-borne infectious diseases form a significant fraction of the global infectious disease burden. In this study we explore the relationship between host diversity, vector behavior, and disease risk. To this end, we have developed a new dynamic model which includes two distinct host species and one vector species with variable preferences. With the aid of the model we were able to compute the basic reproductive rate, R 0, a well-established measure of disease risk that serves as a threshold parameter for disease outbreak. The model analysis reveals that the system has two different qualitative behaviors: (i) the well-known dilution effect, where the maximal R0 is obtained in a community which consists a single host (ii) a new amplification effect, denoted by us as diversity amplification, where the maximal R0 is attained in a community which consists both hosts. The model analysis extends on previous results by underlining the mechanism of both, diversity amplification and the dilution, and specifies the exact conditions for their occurrence. We have found that diversity amplification occurs where the vector prefers the host with the highest transmission ability, and dilution is obtained when the vector does not show any preference, or it prefers to bite the host with the lower transmission ability. The mechanisms of dilution and diversity amplification are able to account for the different and contradictory patterns often observed in nature (i.e., in some cases disease risk is increased while in other is decreased when the diversity is increased). Implication of the diversity amplification mechanism also challenges current premises about the interaction between biodiversity, climate change, and disease risk and calls for retrospective thinking in planning intervention policies aimed at protecting the preferred host species.

Limited genomic heterogeneity of circulating melanoma cells in advanced stage patients

NASA Astrophysics Data System (ADS)

Ruiz, Carmen; Li, Julia; Luttgen, Madelyn S.; Kolatkar, Anand; Kendall, Jude T.; Flores, Edna; Topp, Zheng; Samlowski, Wolfram E.; McClay, Edward; Bethel, Kelly; Ferrone, Soldano; Hicks, James; Kuhn, Peter

2015-02-01

Purpose. Circulating melanoma cells (CMCs) constitute a potentially important representation of time-resolved tumor biology in patients. To date, genomic characterization of CMCs has been limited due to the lack of a robust methodology capable of identifying them in a format suitable for downstream characterization. Here, we have developed a methodology to detect intact CMCs that enables phenotypic, morphometric and genomic analysis at the single cell level. Experimental design. Blood samples from 40 metastatic melanoma patients and 10 normal blood donors were prospectively collected. A panel of 7 chondroitin sulfate proteoglycan 4 (CSPG4)-specific monoclonal antibodies (mAbs) was used to immunocytochemically label CMCs. Detection was performed by automated digital fluorescence microscopy and multi-parametric computational analysis. Individual CMCs were captured by micromanipulation for whole genome amplification and copy number variation (CNV) analysis. Results. Based on CSPG4 expression and nuclear size, 1-250 CMCs were detected in 22 (55%) of 40 metastatic melanoma patients (0.5-371.5 CMCs ml-1). Morphometric analysis revealed that CMCs have a broad spectrum of morphologies and sizes but exhibit a relatively homogeneous nuclear size that was on average 1.5-fold larger than that of surrounding PBMCs. CNV analysis of single CMCs identified deletions of CDKN2A and PTEN, and amplification(s) of TERT, BRAF, KRAS and MDM2. Furthermore, novel chromosomal amplifications in chr12, 17 and 19 were also found. Conclusions. Our findings show that CSPG4 expressing CMCs can be found in the majority of advanced melanoma patients. High content analysis of this cell population may contribute to the design of effective personalized therapies in patients with melanoma.

Clinical significance of hTERC gene amplification detection by FISH in the screening of cervical lesions.

PubMed

Zhang, Yuan; Wang, Xiaobei; Ma, Ling; Wang, Zehua; Hu, Lihua

2009-06-01

This study evaluated the clinical significance of hTERC gene amplification detection by fluorescence in situ hybridization (FISH) in the screening of cervical lesions. Cervical specimens of 50 high risk patients were detected by thin liquid-based cytology. The patients whose cytological results were classified as ASCUS or above were subjected to the subsequent colposcopic biopsies. Slides prepared from these 50 cervical specimens were analyzed for hTERC gene amplification using interphase FISH with the two-color hTERC probe. The results of the cytological analysis and those of subsequent biopsies, when available, were compared with the FISH-detected hTERC abnormalities. It was found that the positive rates of hTERC gene amplification in NILM, ASCUS, LSIL, HSIL, and SCC groups were 0.00, 28.57%, 57.14%, 100%, and 100%, respectively. The positive rates of hTERC gene amplification in HSIL and SCC groups were significantly higher than those in NILM, ASCUS and LSIL groups (all P<0.05). The mean percentages of cells with hTERC gene amplification in NILM, ASCUS, LSIL, HSIL, and SCC groups were 0.00, 10.50%, 36.00%, 79.00%, and 96.50%, respectively. Patients with HSIL or SCC cytological diagnoses had significantly higher mean percentages of cells with hTERC gene amplification than did patients with NILM, ASCUS or LSIL cytological diagnoses (all P<0.05). It was concluded that two-color interphase FISH could detect hTERC gene amplification to accurately distinguish HSIL and ISIL of cervical cells. It may be an adjunct to cytology screening, especially high-risk patients.

A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification.

PubMed

Li, Xia; Song, Juan; Xue, Qing-Wang; You, Fu-Heng; Lu, Xia; Kong, Yan-Cong; Ma, Shu-Yi; Jiang, Wei; Li, Chen-Zhong

2016-10-21

Bisphenol A (BPA) detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA)/Exonuclease III (Exo III)-combined cascade amplification strategy. First, the duplex DNA probe (RP) with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of "G-quadruplex" in lantern-like structures. Finally, the continuously enriched "G-quadruplex lanterns" were lightened by zinc(II)-protoporphyrin IX (ZnPPIX) generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10 -17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX) provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring.

A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification

PubMed Central

Li, Xia; Song, Juan; Xue, Qing-Wang; You, Fu-Heng; Lu, Xia; Kong, Yan-Cong; Ma, Shu-Yi; Jiang, Wei; Li, Chen-Zhong

2016-01-01

Bisphenol A (BPA) detection in drinking water and food packaging materials has attracted much attention since the discovery that BPA can interfere with normal physiological processes and cause adverse health effects. Here, we constructed a label-free aptamer fluorescent assay for selective and sensitive detection of BPA based on the rolling circle amplification (RCA)/Exonuclease III (Exo III)-combined cascade amplification strategy. First, the duplex DNA probe (RP) with anti-BPA aptamer and trigger sequence was designed for BPA recognition and signal amplification. Next, under the action of BPA, the trigger probe was liberated from RP to initiate RCA reaction as primary amplification. Subsequently, the RCA products were used to trigger Exo III assisted secondary amplification with the help of hairpin probes, producing plenty of “G-quadruplex” in lantern-like structures. Finally, the continuously enriched “G-quadruplex lanterns” were lightened by zinc(II)-protoporphyrin IX (ZnPPIX) generating enhanced fluorescence signals. By integrating the primary RCA and secondary Exo III mediated cascade amplification strategy, this method displayed an excellent sensitivity with the detection limits of 5.4 × 10−17 M. In addition, the anti-BPA aptamer exhibits high recognition ability with BPA, guaranteeing the specificity of detection. The reporter signal probe (G-quadruplex with ZnPPIX) provides a label-free fluorescence signals readout without complicated labeling procedures, making the method simple in design and cost-effective in operation. Moreover, environmental samples analysis was also performed, suggesting that our strategy was reliable and had a great potential application in environmental monitoring. PMID:28335318

Methods for applying accurate digital PCR analysis on low copy DNA samples.

PubMed

Whale, Alexandra S; Cowen, Simon; Foy, Carole A; Huggett, Jim F

2013-01-01

Digital PCR (dPCR) is a highly accurate molecular approach, capable of precise measurements, offering a number of unique opportunities. However, in its current format dPCR can be limited by the amount of sample that can be analysed and consequently additional considerations such as performing multiplex reactions or pre-amplification can be considered. This study investigated the impact of duplexing and pre-amplification on dPCR analysis by using three different assays targeting a model template (a portion of the Arabidopsis thaliana alcohol dehydrogenase gene). We also investigated the impact of different template types (linearised plasmid clone and more complex genomic DNA) on measurement precision using dPCR. We were able to demonstrate that duplex dPCR can provide a more precise measurement than uniplex dPCR, while applying pre-amplification or varying template type can significantly decrease the precision of dPCR. Furthermore, we also demonstrate that the pre-amplification step can introduce measurement bias that is not consistent between experiments for a sample or assay and so could not be compensated for during the analysis of this data set. We also describe a model for estimating the prevalence of molecular dropout and identify this as a source of dPCR imprecision. Our data have demonstrated that the precision afforded by dPCR at low sample concentration can exceed that of the same template post pre-amplification thereby negating the need for this additional step. Our findings also highlight the technical differences between different templates types containing the same sequence that must be considered if plasmid DNA is to be used to assess or control for more complex templates like genomic DNA.

Methods for Applying Accurate Digital PCR Analysis on Low Copy DNA Samples

PubMed Central

Whale, Alexandra S.; Cowen, Simon; Foy, Carole A.; Huggett, Jim F.

2013-01-01

Digital PCR (dPCR) is a highly accurate molecular approach, capable of precise measurements, offering a number of unique opportunities. However, in its current format dPCR can be limited by the amount of sample that can be analysed and consequently additional considerations such as performing multiplex reactions or pre-amplification can be considered. This study investigated the impact of duplexing and pre-amplification on dPCR analysis by using three different assays targeting a model template (a portion of the Arabidopsis thaliana alcohol dehydrogenase gene). We also investigated the impact of different template types (linearised plasmid clone and more complex genomic DNA) on measurement precision using dPCR. We were able to demonstrate that duplex dPCR can provide a more precise measurement than uniplex dPCR, while applying pre-amplification or varying template type can significantly decrease the precision of dPCR. Furthermore, we also demonstrate that the pre-amplification step can introduce measurement bias that is not consistent between experiments for a sample or assay and so could not be compensated for during the analysis of this data set. We also describe a model for estimating the prevalence of molecular dropout and identify this as a source of dPCR imprecision. Our data have demonstrated that the precision afforded by dPCR at low sample concentration can exceed that of the same template post pre-amplification thereby negating the need for this additional step. Our findings also highlight the technical differences between different templates types containing the same sequence that must be considered if plasmid DNA is to be used to assess or control for more complex templates like genomic DNA. PMID:23472156

Improved multiple displacement amplification (iMDA) and ultraclean reagents.

PubMed

Motley, S Timothy; Picuri, John M; Crowder, Chris D; Minich, Jeremiah J; Hofstadler, Steven A; Eshoo, Mark W

2014-06-06

Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA). A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance and representation of the genome. The iMDA protocol in combination with DNA-free laboratory consumables, significantly improved the ability to sequence specimens with low levels of DNA. iMDA has broad utility in metagenomics, diagnostics, ancient DNA analysis, pre-implantation embryo screening, single-cell genomics, whole genome sequencing of unculturable organisms, and forensic applications for both human and microbial targets.

Biologically relevant effects of mRNA amplification on gene expression profiles.

PubMed

van Haaften, Rachel I M; Schroen, Blanche; Janssen, Ben J A; van Erk, Arie; Debets, Jacques J M; Smeets, Hubert J M; Smits, Jos F M; van den Wijngaard, Arthur; Pinto, Yigal M; Evelo, Chris T A

2006-04-11

Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other.Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P <0.001). Those reporters were also predicted to form significantly more (0.76+/-0.07 versus 0.38+/-0.1) and longer (9.4+/-0.3 versus 8.4+/-0.4) hairpins as compared to representative control reporters present before and after amplification. This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular biopsies is representative for the whole left ventricle of the rat heart. However, specific gene transcripts present in parental tissues were undetectable in the minute left ventricular biopsies. Transcripts that were lost due to the amplification process were not randomly distributed, but had higher GC-content and hairpins in the sequence and were mainly found in the lower intensity range which includes many transcription factors from specific signalling pathways.

Biologically relevant effects of mRNA amplification on gene expression profiles

PubMed Central

van Haaften, Rachel IM; Schroen, Blanche; Janssen, Ben JA; van Erk, Arie; Debets, Jacques JM; Smeets, Hubert JM; Smits, Jos FM; van den Wijngaard, Arthur; Pinto, Yigal M; Evelo, Chris TA

2006-01-01

Background Gene expression microarray technology permits the analysis of global gene expression profiles. The amount of sample needed limits the use of small excision biopsies and/or needle biopsies from human or animal tissues. Linear amplification techniques have been developed to increase the amount of sample derived cDNA. These amplified samples can be hybridised on microarrays. However, little information is available whether microarrays based on amplified and unamplified material yield comparable results. In the present study we compared microarray data obtained from amplified mRNA derived from biopsies of rat cardiac left ventricle and non-amplified mRNA derived from the same organ. Biopsies were linearly amplified to acquire enough material for a microarray experiment. Both amplified and unamplified samples were hybridized to the Rat Expression Set 230 Array of Affymetrix. Results Analysis of the microarray data showed that unamplified material of two different left ventricles had 99.6% identical gene expression. Gene expression patterns of two biopsies obtained from the same parental organ were 96.3% identical. Similarly, gene expression pattern of two biopsies from dissimilar organs were 92.8% identical to each other. Twenty-one percent of reporters called present in parental left ventricular tissue disappeared after amplification in the biopsies. Those reporters were predominantly seen in the low intensity range. Sequence analysis showed that reporters that disappeared after amplification had a GC-content of 53.7+/-4.0%, while reporters called present in biopsy- and whole LV-samples had an average GC content of 47.8+/-5.5% (P <0.001). Those reporters were also predicted to form significantly more (0.76+/-0.07 versus 0.38+/-0.1) and longer (9.4+/-0.3 versus 8.4+/-0.4) hairpins as compared to representative control reporters present before and after amplification. Conclusion This study establishes that the gene expression profile obtained after amplification of mRNA of left ventricular biopsies is representative for the whole left ventricle of the rat heart. However, specific gene transcripts present in parental tissues were undetectable in the minute left ventricular biopsies. Transcripts that were lost due to the amplification process were not randomly distributed, but had higher GC-content and hairpins in the sequence and were mainly found in the lower intensity range which includes many transcription factors from specific signalling pathways. PMID:16608515

CCNE1 amplification is associated with aggressive potential in endometrioid endometrial carcinomas.

PubMed

Nakayama, Kentaro; Rahman, Mohammed Tanjimur; Rahman, Munmun; Nakamura, Kohei; Ishikawa, Masako; Katagiri, Hiroshi; Sato, Emi; Ishibashi, Tomoka; Iida, Kouji; Ishikawa, Noriyuki; Kyo, Satoru

2016-02-01

The clinicopathological significance of amplification was investigated of the gene encoding cyclin E (CCNE1) and we assessed whether CCNE1 was a potential target in endometrioid endometrial carcinomas. CCNE1 amplification and CCNE1 or F-box and WD repeat domain-containing 7 (FBXW7) expression in endometrial endometrioid carcinoma was assessed by immunohistochemistry and fluorescence in situ hybridization. CCNE1 knockdown by small interfering RNA (siRNA) was used to assess the CCNE1 function. The results showed that CCNE1 amplification was present in 9 (8.3%) of 108 endometrial carcinomas. CCNE1 amplification was correlated with high histological grade (Grade 3; p=0.0087) and lymphovascular space invasion (p=0.0258). No significant association was observed between CCNE1 amplification and FIGO stage (p=0.851), lymph node metastasis (p=0.078), body mass index (p=0.265), deep myometrial invasion (p=0.256), menopausal status (p=0.289) or patient age (p=0.0817). CCNE1 amplification was significantly correlated with shorter progression-free and overall survival (p=0.0081 and 0.0073, respectively). CCNE1 protein expression or loss of FBXW7 expression in endometrial endometrioid carcinoma tended to be correlated with shorter progression-free and overall survival; however, this difference was not statistically significant. Multivariate analysis showed that CCNE1 amplification was an independent prognostic factor for overall survival but not for progression-free survival (P=0.0454 and 0.2175, respectively). Profound growth inhibition was observed in siRNA-transfected cancer cells with endogenous CCNE1 overexpression compared with that in cancer cells having low CCNE1 expression. CCNE1 amplification was independent of p53, HER2, MLH1 and ARID1A expression but dependent on PTEN expression in endometrial carcinomas. These findings indicated that CCNE1 amplification was critical for the survival of endometrial endometrioid carcinomas. Furthermore, the effects of CCNE1 knockdown were dependent on the CCNE1 expression status, suggesting that CCNE1-targeted therapy may be beneficial for patients with endometrial endometrioid carcinoma having CCNE1 amplification.

CCNE1 amplification is associated with aggressive potential in endometrioid endometrial carcinomas

PubMed Central

NAKAYAMA, KENTARO; RAHMAN, MOHAMMED TANJIMUR; RAHMAN, MUNMUN; NAKAMURA, KOHEI; ISHIKAWA, MASAKO; KATAGIRI, HIROSHI; SATO, EMI; ISHIBASHI, TOMOKA; IIDA, KOUJI; ISHIKAWA, NORIYUKI; KYO, SATORU

2016-01-01

The clinicopathological significance of amplification was investigated of the gene encoding cyclin E (CCNE1) and we assessed whether CCNE1 was a potential target in endometrioid endometrial carcinomas. CCNE1 amplification and CCNE1 or F-box and WD repeat domain-containing 7 (FBXW7) expression in endometrial endometrioid carcinoma was assessed by immunohistochemistry and fluorescence in situ hybridization. CCNE1 knockdown by small interfering RNA (siRNA) was used to assess the CCNE1 function. The results showed that CCNE1 amplification was present in 9 (8.3%) of 108 endometrial carcinomas. CCNE1 amplification was correlated with high histological grade (Grade 3; P=0.0087) and lymphovascular space invasion (P=0.0258). No significant association was observed between CCNE1 amplification and FIGO stage (P=0.851), lymph node metastasis (P=0.078), body mass index (P=0.265), deep myometrial invasion (P=0.256), menopausal status (P=0.289) or patient age (P=0.0817). CCNE1 amplification was significantly correlated with shorter progression-free and overall survival (P=0.0081 and 0.0073, respectively). CCNE1 protein expression or loss of FBXW7 expression in endometrial endometrioid carcinoma tended to be correlated with shorter progression-free and overall survival; however, this difference was not statistically significant. Multivariate analysis showed that CCNE1 amplification was an independent prognostic factor for overall survival but not for progression-free survival (P=0.0454 and 0.2175, respectively). Profound growth inhibition was observed in siRNA-transfected cancer cells with endogenous CCNE1 overexpression compared with that in cancer cells having low CCNE1 expression. CCNE1 amplification was independent of p53, HER2, MLH1 and ARID1A expression but dependent on PTEN expression in endometrial carcinomas. These findings indicated that CCNE1 amplification was critical for the survival of endometrial endometrioid carcinomas. Furthermore, the effects of CCNE1 knockdown were dependent on the CCNE1 expression status, suggesting that CCNE1-targeted therapy may be beneficial for patients with endometrial endometrioid carcinoma having CCNE1 amplification. PMID:26647729

Whole genome amplification of single pollen grains from a sugarcane cultivar and analysis of the genetic relatedness based on SCoT markers

USDA-ARS?s Scientific Manuscript database

Single pollen grains were isolated from an intact anther of a sugarcane cultivar and collected using a pair of special forceps. The single pollen grains were lysed in an alkali/detergent solution respectively. The resulting solution was used as the template for Whole Genome Amplification. Genomic DN...

Effect of Arctic Amplification on Design Snow Loads in Alaska

DTIC Science & Technology

2016-09-01

snow water equivalent UFC Unified Facilities Criteria UTC Coordinated Universal Time Keywords: Alaska, Arctic amplification, climate change...extreme value analysis, snow loads, snow water equivalent , SWE Acknowledgements: This work was conducted with support from the Strategic... equivalent (SWE) of the snowpack. We acquired SWE data from a number of sources that provide automatic or manual observations, reanalysis data, or

Identification and removal of low-complexity sites in allele-specific analysis of ChIP-seq data.

PubMed

Waszak, Sebastian M; Kilpinen, Helena; Gschwind, Andreas R; Orioli, Andrea; Raghav, Sunil K; Witwicki, Robert M; Migliavacca, Eugenia; Yurovsky, Alisa; Lappalainen, Tuuli; Hernandez, Nouria; Reymond, Alexandre; Dermitzakis, Emmanouil T; Deplancke, Bart

2014-01-15

High-throughput sequencing technologies enable the genome-wide analysis of the impact of genetic variation on molecular phenotypes at unprecedented resolution. However, although powerful, these technologies can also introduce unexpected artifacts. We investigated the impact of library amplification bias on the identification of allele-specific (AS) molecular events from high-throughput sequencing data derived from chromatin immunoprecipitation assays (ChIP-seq). Putative AS DNA binding activity for RNA polymerase II was determined using ChIP-seq data derived from lymphoblastoid cell lines of two parent-daughter trios. We found that, at high-sequencing depth, many significant AS binding sites suffered from an amplification bias, as evidenced by a larger number of clonal reads representing one of the two alleles. To alleviate this bias, we devised an amplification bias detection strategy, which filters out sites with low read complexity and sites featuring a significant excess of clonal reads. This method will be useful for AS analyses involving ChIP-seq and other functional sequencing assays. The R package abs filter for library clonality simulations and detection of amplification-biased sites is available from http://updepla1srv1.epfl.ch/waszaks/absfilter

The Development and Use of Internal Amplification Controls (IACs) with DNA Profiling Kits for Forensic DNA Analysis.

PubMed

Zahra, Nathalie; Goodwin, William

2016-01-01

Biological samples recovered for forensic investigations are often degraded and/or have low amounts of DNA; in addition, in some instances the samples may be contaminated with chemicals that can act as PCR inhibitors. As a consequence this can make interpretation of the results challenging with the possibility of having partial profiles and false negative results. Because of the impact of DNA analysis on forensic investigations, it is important to monitor the process of DNA profiling, in particular the amplification reaction. In this chapter we describe a method for the in-house generation and use of internal amplification controls (IACs) with DNA profiling kits to monitor the success of the PCR proces. In the example we show the use of the SGM Plus® kit. These controls can also be used to aid the interpretation of the DNA profile.

Enzymatic signal amplification for sensitive detection of intracellular antigens by flow cytometry.

PubMed

Karkmann, U; Radbruch, A; Hölzel, V; Scheffold, A

1999-11-19

Flow cytometry is the method of choice for the analysis of single cells with respect to the expression of specific antigens. Antigens can be detected with specific antibodies either on the cell surface or within the cells, after fixation and permeabilization of the cell membrane. Using conventional fluorochrome-labeled antibodies several thousand antigens are required for clear-cut separation of positive and negative cells. More sensitive reagents, e.g., magnetofluorescent liposomes conjugated to specific antibodies permit the detection of less than 200 molecules per cell but cannot be used for the detection of intracellular antigens. Here, we describe an enzymatic amplification technique (intracellular tyramine-based signal amplification, ITSA) for the sensitive cytometric analysis of intracellular cytokines by immunofluorescence. This approach results in a 10 to 15-fold improvement of the signal-to-noise ratio compared to conventional fluorochrome labeled antibodies and permits the detection of as few as 300-400 intracellular antigens per cell.

Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing.

PubMed

Castle, John; Garrett-Engele, Phil; Armour, Christopher D; Duenwald, Sven J; Loerch, Patrick M; Meyer, Michael R; Schadt, Eric E; Stoughton, Roland; Parrish, Mark L; Shoemaker, Daniel D; Johnson, Jason M

2003-01-01

Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.

Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing

PubMed Central

Castle, John; Garrett-Engele, Phil; Armour, Christopher D; Duenwald, Sven J; Loerch, Patrick M; Meyer, Michael R; Schadt, Eric E; Stoughton, Roland; Parrish, Mark L; Shoemaker, Daniel D; Johnson, Jason M

2003-01-01

Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing. PMID:14519201

Improvement of Method for Estimation of Site Amplification Factor Based on Average Shear-wave Velocity of Ground

NASA Astrophysics Data System (ADS)

Yamaguchi, Makoto; Midorikawa, Saburoh

The empirical equation for estimating the site amplification factor of ground motion by the average shear-wave velocity of ground (AVS) is examined. In the existing equations, the coefficient on dependence of the amplification factor on the AVS was treated as constant. The analysis showed that the coefficient varies with change of the AVS for short periods. A new estimation equation was proposed considering the dependence on the AVS. The new equation can represent soil characteristics that the softer soil has the longer predominant period, and can make better estimations for short periods than the existing method.

Multiplex Allele-Specific Amplification from Whole Blood for Detecting Multiple Polymorphisms Simultaneously

PubMed Central

Zhu, Jianjie; Chen, Lanxin; Mao, Yong; Zhou, Huan

2013-01-01

Allele-specific amplification on the basis of polymerase chain reaction (PCR) has been widely used for single-nucleotide polymorphism (SNP) genotyping. However, the extraction of PCR-compatible genomic DNA from whole blood is usually required. This process is complicated and tedious, and is prone to cause cross-contamination between samples. To facilitate direct PCR amplification from whole blood without the extraction of genomic DNA, we optimized the pH value of PCR solution and the concentrations of magnesium ions and facilitator glycerol. Then, we developed multiplex allele-specific amplifications from whole blood and applied them to a case–control study. In this study, we successfully established triplex, five-plex, and eight-plex allele-specific amplifications from whole blood for determining the distribution of genotypes and alleles of 14 polymorphisms in 97 gastric cancer patients and 141 healthy controls. Statistical analysis results showed significant association of SNPs rs9344, rs1799931, and rs1800629 with the risk of gastric cancer. This method is accurate, time-saving, cost-effective, and easy-to-do, especially suitable for clinical prediction of disease susceptibility. PMID:23072573

Amplification of the entire kanamycin biosynthetic gene cluster during empirical strain improvement of Streptomyces kanamyceticus.

PubMed

Yanai, Koji; Murakami, Takeshi; Bibb, Mervyn

2006-06-20

Streptomyces kanamyceticus 12-6 is a derivative of the wild-type strain developed for industrial kanamycin (Km) production. Southern analysis and DNA sequencing revealed amplification of a large genomic segment including the entire Km biosynthetic gene cluster in the chromosome of strain 12-6. At 145 kb, the amplifiable unit of DNA (AUD) is the largest AUD reported in Streptomyces. Striking repetitive DNA sequences belonging to the clustered regularly interspaced short palindromic repeats family were found in the AUD and may play a role in its amplification. Strain 12-6 contains a mixture of different chromosomes with varying numbers of AUDs, sometimes exceeding 36 copies and producing an amplified region >5.7 Mb. The level of Km production depended on the copy number of the Km biosynthetic gene cluster, suggesting that DNA amplification occurred during strain improvement as a consequence of selection for increased Km resistance. Amplification of DNA segments including entire antibiotic biosynthetic gene clusters might be a common mechanism leading to increased antibiotic production in industrial strains.

Indel analysis by droplet digital PCR: a sensitive method for DNA mixture detection and chimerism analysis.

PubMed

Santurtún, Ana; Riancho, José A; Arozamena, Jana; López-Duarte, Mónica; Zarrabeitia, María T

2017-01-01

Several methods have been developed to determinate genetic profiles from a mixed samples and chimerism analysis in transplanted patients. The aim of this study was to explore the effectiveness of using the droplet digital PCR (ddPCR) for mixed chimerism detection (a mixture of genetic profiles resulting after allogeneic hematopoietic stem cell transplantation (HSCT)). We analyzed 25 DNA samples from patients who had undergone HSCT and compared the performance of ddPCR and two established methods for chimerism detection, based upon the Indel and STRs analysis, respectively. Additionally, eight artificial mixture DNA samples were created to evaluate the sensibility of ddPCR. Our results show that the chimerism percentages estimated by the analysis of a single Indel using ddPCR were very similar to those calculated by the amplification of 15 STRs (r 2  = 0.970) and with the results obtained by the amplification of 38 Indels (r 2  = 0.975). Moreover, the amplification of a single Indel by ddPCR was sensitive enough to detect a minor DNA contributor comprising down to 0.5 % of the sample. We conclude that ddPCR can be a powerful tool for the determination of a genetic profile of forensic mixtures and clinical chimerism analysis when traditional techniques are not sensitive enough.

Identification of the first de novo PAR1 deletion downstream of SHOX in an individual diagnosed with Léri-Weill dyschondrosteosis (LWD).

PubMed

Barroso, Eva; Benito-Sanz, Sara; Belinchón, Alberta; Yuste-Checa, Patricia; Gracia, Ricardo; Aragones, Angel; Campos-Barros, Angel; Heath, Karen E

2010-01-01

Léri-Weill dyschondrosteosis (LWD, MIM 127300), is a dominantly inherited skeletal dysplasia with disproportionate short stature, mesomelic limb shortening, and the characteristic Madelung deformity. Two regions of the pseudoautosomal region 1 (PAR1) have been shown to be involved in LWD, SHOX (short-stature homeobox-containing gene) and the downstream enhancer region. We report our genetic findings of a young girl clinically diagnosed with LWD. We analyzed the proband and her family using MLPA and microsatellite analysis. We identified a deletion, 726-866 kb in size, of the downstream SHOX enhancer region in the proband. Neither parent carried the deletion. Microsatellite analysis showed that the deleted allele was of paternal origin. The mutation is more likely to have arisen from a de novo event but paternal gonadal mosaicism cannot be excluded. In conclusion, we report the clinical and molecular details of the first case of a de novo deletion of the downstream PAR1 region in an LWD individual. De novo deletions of SHOX and the downstream enhancer region must be therefore considered in cases of isolated LWD. Copyright 2010 Elsevier Masson SAS. All rights reserved.

Genetic alteration profiling of patients with resected squamous cell lung carcinomas

PubMed Central

Zhang, Ningning; Lin, Dongmei; Wu, Di; Zhu, Xinxin; Song, Wenya; Shi, Yuankai

2016-01-01

In this study, we analyzed the genetic profiles of squamous cell lung carcinoma (SqCLC) to identify potential therapeutic targets. Approximately 2,800 COSMIC mutations from 50 genes were determined by next-generation sequencing. Amplification/deletion of SOX2, CDKN2A, PTEN, FGFR1, EGFR, CCND1, HER2 and PDGFRA were detected by FISH and expression of VEGFR2, PD-L1 and PTEN were examined by IHC. One hundred and fifty-seven samples of SqCLC were collected. Somatic mutations was identified in 73.9% of cases, with TP53 (56.1%), CDKN2A (8.9%), PIK3CA (8.9%), KRAS (4.5%) and EGFR (3.2%). Gene copy number alterations were identified in 75.8% of cases, including SOX2 amplification (31.2%), CDKN2A deletion (21.7%), PTEN deletion (16.6%), FGFR1 amplification (15.9%), EGFR amplification (14.0%), CCND1 amplification (14.0%), HER2 amplification (9.6%) and PDGFRA amplification (7.6%). Positive expression of VEGFR2 and PD-L1 and loss of PTEN expression were observed in 80.5%, 47.2%, and 42.7% of cases, respectively. Multivariate analysis showed that positive expression of PD-L1 was an independent favorable prognostic factor for DFS (HR = 0.610; P = 0.044). In conclusion, nearly all (93.6%) SqCLC cases harbored at least one potential druggable target. The findings of this study could facilitate the identification of therapeutic target candidates for precision medicine of SqCLC. PMID:27145277

Development of a rapid assay for the diagnosis of Myxobolus cerebralis in fish and oligochaetes using loop-mediated isothermal amplification.

PubMed

El-Matbouli, M; Soliman, H

2005-09-01

A loop-mediated isothermal amplification assay was developed for the rapid detection of Myxobolus cerebralis in both fish and oligochaete hosts. The assay was optimized to amplify parasitic DNA by incubation with Bst DNA polymerase and a set of six specially constructed primers at 65 degrees C for 60 min. The amplification products were detected visually using SYBR Green I dye which gave identical results to gel electrophoresis analysis. Parasite DNA was detected from infected oligochaetes, and from the anal fin, caudal fin, dorsal fin and operculum of clinically infected fish. This 'Myxo-LAMP' assay has a detection limit similar to that of a polymerase chain reaction assay (10(-6)), but is more rapid and only requires a water bath for amplification and is therefore practical for simple and rapid diagnosis of infected tissue.

Sequencing of the large dsDNA genome of Oryctes rhinoceros nudivirus using multiple displacement amplification of nanogram amounts of virus DNA.

PubMed

Wang, Yongjie; Kleespies, Regina G; Ramle, Moslim B; Jehle, Johannes A

2008-09-01

The genomic sequence analysis of many large dsDNA viruses is hampered by the lack of enough sample materials. Here, we report a whole genome amplification of the Oryctes rhinoceros nudivirus (OrNV) isolate Ma07 starting from as few as about 10 ng of purified viral DNA by application of phi29 DNA polymerase- and exonuclease-resistant random hexamer-based multiple displacement amplification (MDA) method. About 60 microg of high molecular weight DNA with fragment sizes of up to 25 kbp was amplified. A genomic DNA clone library was generated using the product DNA. After 8-fold sequencing coverage, the 127,615 bp of OrNV whole genome was sequenced successfully. The results demonstrate that the MDA-based whole genome amplification enables rapid access to genomic information from exiguous virus samples.

Whole Genome Amplification of Labeled Viable Single Cells Suited for Array-Comparative Genomic Hybridization.

PubMed

Kroneis, Thomas; El-Heliebi, Amin

2015-01-01

Understanding details of a complex biological system makes it necessary to dismantle it down to its components. Immunostaining techniques allow identification of several distinct cell types thereby giving an inside view of intercellular heterogeneity. Often staining reveals that the most remarkable cells are the rarest. To further characterize the target cells on a molecular level, single cell techniques are necessary. Here, we describe the immunostaining, micromanipulation, and whole genome amplification of single cells for the purpose of genomic characterization. First, we exemplify the preparation of cell suspensions from cultured cells as well as the isolation of peripheral mononucleated cells from blood. The target cell population is then subjected to immunostaining. After cytocentrifugation target cells are isolated by micromanipulation and forwarded to whole genome amplification. For whole genome amplification, we use GenomePlex(®) technology allowing downstream genomic analysis such as array-comparative genomic hybridization.

Simple Approaches to Minimally-Instrumented, Microfluidic-Based Point-of-Care Nucleic Acid Amplification Tests

PubMed Central

Mauk, Michael G.; Song, Jinzhao; Liu, Changchun; Bau, Haim H.

2018-01-01

Designs and applications of microfluidics-based devices for molecular diagnostics (Nucleic Acid Amplification Tests, NAATs) in infectious disease testing are reviewed, with emphasis on minimally instrumented, point-of-care (POC) tests for resource-limited settings. Microfluidic cartridges (‘chips’) that combine solid-phase nucleic acid extraction; isothermal enzymatic nucleic acid amplification; pre-stored, paraffin-encapsulated lyophilized reagents; and real-time or endpoint optical detection are described. These chips can be used with a companion module for separating plasma from blood through a combined sedimentation-filtration effect. Three reporter types: Fluorescence, colorimetric dyes, and bioluminescence; and a new paradigm for end-point detection based on a diffusion-reaction column are compared. Multiplexing (parallel amplification and detection of multiple targets) is demonstrated. Low-cost detection and added functionality (data analysis, control, communication) can be realized using a cellphone platform with the chip. Some related and similar-purposed approaches by others are surveyed. PMID:29495424

Targeting Conserved Genes in Penicillium Species.

PubMed

Peterson, Stephen W

2017-01-01

Polymerase chain reaction amplification of conserved genes and sequence analysis provides a very powerful tool for the identification of toxigenic as well as non-toxigenic Penicillium species. Sequences are obtained by amplification of the gene fragment, sequencing via capillary electrophoresis of dideoxynucleotide-labeled fragments or NGS. The sequences are compared to a database of validated isolates. Identification of species indicates the potential of the fungus to make particular mycotoxins.

Prognostic roles for fibroblast growth factor receptor family members in malignant peripheral nerve sheath tumor.

PubMed

Zhou, Wenya; Du, Xiaoling; Song, Fengju; Zheng, Hong; Chen, Kexin; Zhang, Wei; Yang, Jilong

2016-04-19

Malignant peripheral nerve sheath tumors (MPNST) are rare, highly malignant, and poorly understood sarcomas. The often poor outcome of MPNST highlights the necessity of identifying prognostic predictors for this aggressive sarcoma. Here, we investigate the role of fibroblast growth factor receptor (FGFR) family members in human MPNSTs. aCGH and bioinformatics analysis identified frequent amplification of the FGFR1 gene. FISH analysis revealed that 26.9% MPNST samples had amplification of FGFR1, with both focal and polysomy patterns observed. IHC identified that FGFR1 protein expression was positively correlated with FGFR1 gene amplification. High expression of FGFR1 protein was associated with better overall survival (OS) and was an independent prognostic predictor for OS of MPNST patients. Additionally, combined expression of FGFR1 and FGFR2 protein characterized a subtype of MPNST with better OS. FGFR4 protein was expressed 82.3% of MPNST samples, and was associated with poor disease-free survival. We performed microarray-based comparative genomic hybridization (aCGH) profiling of two cohorts of primary MPNST tissue samples including 25 patients treated at The University of Texas MD Anderson Cancer Center and 26 patients from Tianjin Medical University Cancer Institute and Hospital. Fluorescence in situ hybridization (FISH) was used to validate the gene amplification detected by aCGH analysis. Another cohort of 63 formalin-fixed paraffin-embedded MPNST samples (including 52 samples for FISH assay) was obtained to explore FGFR1, 2, 3, and 4 protein expression by immunohistochemical (IHC) analysis. Our integrated genomic and molecular studies provide evidence that FGFRs play different prognostic roles in MPNST.

Detection of the 35S promoter in transgenic maize via various isothermal amplification techniques: a practical approach.

PubMed

Zahradnik, Celine; Kolm, Claudia; Martzy, Roland; Mach, Robert L; Krska, Rudolf; Farnleitner, Andreas H; Brunner, Kurt

2014-11-01

In 2003 the European Commission introduced a 0.9% threshold for food and feed products containing genetically modified organism (GMO)-derived components. For commodities containing GMO contents higher than this threshold, labelling is mandatory. To provide a DNA-based rapid and simple detection method suitable for high-throughput screening of GMOs, several isothermal amplification approaches for the 35S promoter were tested: strand displacement amplification, nicking-enzyme amplification reaction, rolling circle amplification, loop-mediated isothermal amplification (LAMP) and helicase-dependent amplification (HDA). The assays developed were tested for specificity in order to distinguish between samples containing genetically modified (GM) maize and non-GM maize. For those assays capable of this discrimination, tests were performed to determine the lower limit of detection. A false-negative rate was determined to rule out whether GMO-positive samples were incorrectly classified as GMO-negative. A robustness test was performed to show reliable detection independent from the instrument used for amplification. The analysis of three GM maize lines showed that only LAMP and HDA were able to differentiate between the GMOs MON810, NK603, and Bt11 and non-GM maize. Furthermore, with the HDA assay it was possible to realize a detection limit as low as 0.5%. A false-negative rate of only 5% for 1% GM maize for all three maize lines shows that HDA has the potential to be used as an alternative strategy for the detection of transgenic maize. All results obtained with the LAMP and HDA assays were compared with the results obtained with a previously reported real-time PCR assay for the 35S promoter in transgenic maize. This study presents two new screening assays for detection of the 35S promoter in transgenic maize by applying the isothermal amplification approaches HDA and LAMP.

Clinical relevance of TRKA expression on neuroblastoma: comparison with N-MYC amplification and CD44 expression.

PubMed Central

Combaret, V.; Gross, N.; Lasset, C.; Balmas, K.; Bouvier, R.; Frappaz, D.; Beretta-Brognara, C.; Philip, T.; Favrot, M. C.; Coll, J. L.

1997-01-01

TRKA expression was evaluated on 122 untreated neuroblastomas by immunohistochemistry using an antibody with predetermined specificity. This procedure is simple and reliable for protein detection at cellular level in a routine clinical setting. Fourteen tumours were classified as benign ganglioneuroma with a restricted expression of TRKA on ganglion cells; these patients were excluded from the following analysis. A total of 108 tumours were classified as neuroblastoma or ganglioneuroblastoma; 74 expressed TRKA protein, which strongly correlated with low stage, absence of N-MYC amplification, age (<1 year), CD44 expression and favourable clinical outcome. In a univariate analysis including tumour stage, age, histology, N-MYC amplification, CD44 and TRKA expression, all parameters had significant prognostic value. The absence of TRKA expression on CD44-positive or N-MYC non-amplified tumours permits the characterization of a subgroup of patients with intermediate prognosis. However, in a multivariate analysis taking into consideration the prognostic factors mentioned above, CD44 and tumour stage were the only independent prognostic factors for the prediction of patients' event-free survival. PMID:9099963

Pseudoautosomal abnormalities in terminal AZFb+c deletions are associated with isochromosomes Yp and may lead to abnormal growth and neuropsychiatric function.

PubMed

Castro, A; Rodríguez, F; Flórez, M; López, P; Curotto, B; Martínez, D; Maturana, A; Lardone, M C; Palma, C; Mericq, V; Ebensperger, M; Cassorla, F

2017-02-01

Are copy number variations (CNVs) in the pseudoautosomal regions (PARs) frequent in subjects with Y-chromosome microdeletions and can they lead to abnormal stature and/or neuropsychiatric disorders? Only subjects diagnosed with azoospermia factor (AZF)b+c deletions spanning to the end of the Y chromosome (i.e. terminal deletions) harbor Y isochromosomes and/or cells 45,X that lead to pseudoautosomal gene CNVs, which were associated with abnormal stature and/or neuropsychiatric disorders. The microdeletions in the long arm of the Y chromosome (Yq) that include the loss of one to three AZF regions, referred to as Yq microdeletions, constitute the most important known etiological factor for primary spermatogenic failure. Recently, controversy has arisen about whether Yq microdeletions are associated with gain or loss of PAR genes, which are implicated in skeletal development and neuropsychiatric function. We studied a cohort of 42 Chilean patients with complete AZF deletions (4 AZFa, 4 AZFb, 23 AZFc, 11 AZFb+c) from a university medical center, diagnosed over a period of 15 years. The subjects underwent complete medical examinations with special attention to their stature and neuropsychiatric function. All subjects were characterized for Yq breakpoints by PCR, and for CNVs in PARs by multiplex ligation-dependent probe amplification (MLPA), followed by qPCR analysis for genes in PAR1 (SHOX and ZBED1), PAR2 (IL9R) and two single copy genes (SRY and DDX3Y, respectively located in Yp11.3 and AZFa). In addition, karyotypes revision and fluorescence in situ hybridization (FISH) for SRY and centromeric probes for X (DXZ1) and Y (DYZ3) chromosomes were performed in males affected with CNVs. We did not detect CNVs in any of the 35 AZF-deleted men with interstitial deletions (AZFa, AZFb, AZFc or AZFb+c). However, six of the seven patients with terminal AZFb+c deletions showed CNVs: two patients showed a loss and four patients showed a gain of PAR1 genes, with the expected loss of VAMP-7 in PAR2. In these patients, the Yq breakpoints localized to the palindromes P8, P5 or P4. In the four cases with gain of PAR1, qPCR analysis showed duplicated signals for SRY and DDX3Y and one copy of IL9R, indicating isodicentric Yp chromosomes [idic(Y)] with breakpoint in Yq11.22. The two patients who had loss of PAR1, as shown by MLPA, had an additional reduction for SRY and DDX3Y, as shown by qPCR, associated with a high proportion of 45,X cells, as determined by FISH and karyotype. In agreement with the karyotype analysis, we detected DYZ3++ and DYZ3+ cells by FISH in the six patients, confirming idic(Y) and revealing additional monocentric Y chromosome [i(Y)]. Five patients had a history of major depressive disorders or bipolar disorder, and three had language impairment, whereas two patients showed severe short stature (Z score: -2.75 and -2.62), while a man with bipolar disorder was very tall (Z score: +2.56). N/A. The number of males studied with Y-chromosome microdeletions and normozoospermic controls with normal karyotypes may not be enough to rule out an association between AZF deletions and PAR abnormalities. The prevalence of Y isochromosomes and/or 45,X cells detected in peripheral blood does not necessarily reflect the variations of PAR genes in target tissues. This study shows that CNVs in PARs were present exclusively in patients with terminal AZFb+c deletions associated with the presence of Y isochromosomes and 45,X cells, and may lead to neuropsychiatric and growth disorders. In contrast, we show that men with interstitial Yq microdeletions with normal karyotypes do not have an increased risk of PAR abnormalities and of phenotypical consequences. Moreover, our results highlight the importance of performing molecular studies, which are not considered in the usual screening for patients with Yq microdeletions. This work was supported by the National Fund for Scientific and Technological Development of Chile (FONDECYT), grant no. 1120176 (A.C.). The authors declare that no conflicting interests exist. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

An evaluation of tyramide signal amplification and archived fixed and frozen tissue in microarray gene expression analysis

PubMed Central

Karsten, Stanislav L.; Van Deerlin, Vivianna M. D.; Sabatti, Chiara; Gill, Lisa H.; Geschwind, Daniel H.

2002-01-01

Archival formalin-fixed, paraffin-embedded and ethanol-fixed tissues represent a potentially invaluable resource for gene expression analysis, as they are the most widely available material for studies of human disease. Little data are available evaluating whether RNA obtained from fixed (archival) tissues could produce reliable and reproducible microarray expression data. Here we compare the use of RNA isolated from human archival tissues fixed in ethanol and formalin to frozen tissue in cDNA microarray experiments. Since an additional factor that can limit the utility of archival tissue is the often small quantities available, we also evaluate the use of the tyramide signal amplification method (TSA), which allows the use of small amounts of RNA. Detailed analysis indicates that TSA provides a consistent and reproducible signal amplification method for cDNA microarray analysis, across both arrays and the genes tested. Analysis of this method also highlights the importance of performing non-linear channel normalization and dye switching. Furthermore, archived, fixed specimens can perform well, but not surprisingly, produce more variable results than frozen tissues. Consistent results are more easily obtainable using ethanol-fixed tissues, whereas formalin-fixed tissue does not typically provide a useful substrate for cDNA synthesis and labeling. PMID:11788730

Gene amplification of the transcription factor DP1 and CTNND1 in human lung cancer.

PubMed

Castillo, Sandra D; Angulo, Barbara; Suarez-Gauthier, Ana; Melchor, Lorenzo; Medina, Pedro P; Sanchez-Verde, Lydia; Torres-Lanzas, Juan; Pita, Guillermo; Benitez, Javier; Sanchez-Cespedes, Montse

2010-09-01

The search for novel oncogenes is important because they could be the target of future specific anticancer therapies. In the present paper we report the identification of novel amplified genes in lung cancer by means of global gene expression analysis. To screen for amplicons, we aligned the gene expression data according to the position of transcripts in the human genome and searched for clusters of over-expressed genes. We found several clusters with gene over-expression, suggesting an underlying genomic amplification. FISH and microarray analysis for DNA copy number in two clusters, at chromosomes 11q12 and 13q34, confirmed the presence of amplifications spanning about 0.4 and 1 Mb for 11q12 and 13q34, respectively. Amplification at these regions each occurred at a frequency of 3%. Moreover, quantitative RT-PCR of each individual transcript within the amplicons allowed us to verify the increased in gene expression of several genes. The p120ctn and DP1 proteins, encoded by two candidate oncogenes, CTNND1 and TFDP1, at 11q12 and 13q amplicons, respectively, showed very strong immunostaining in lung tumours with gene amplification. We then focused on the 13q34 amplicon and in the TFDP1 candidate oncogene. To further determine the oncogenic properties of DP1, we searched for lung cancer cell lines carrying TFDP1 amplification. Depletion of TFDP1 expression by small interference RNA in a lung cancer cell line (HCC33) with TFDP1 amplification and protein over-expression reduced cell viability by 50%. In conclusion, we report the identification of two novel amplicons, at 13q34 and 11q12, each occurring at a frequency of 3% of non-small cell lung cancers. TFDP1, which encodes the E2F-associated transcription factor DP1 is a candidate oncogene at 13q34. The data discussed in this publication have been deposited in NCBIs Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series Accession No. GSE21168.

Optimization of interaction conditions for efficient short laser pulse amplification by stimulated Brillouin scattering in the strongly coupled regime

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

Chiaramello, M.; Riconda, C.; Amiranoff, F.

Plasma amplification of low energy, a short (∼100–500 fs) laser pulse by an energetic long (∼10 ps) pulse via strong coupling Stimulated Brillouin Backscattering is investigated with an extensive analysis of one-dimensional particle-in-cell simulations. Parameters relevant to nowadays experimental conditions are investigated. The obtained seed pulse spectra are analyzed as a function of the interaction conditions such as plasma profile, pulses delay, and seed or pulse duration. The factors affecting the amount of energy transferred are determined, and the competition between Brillouin-based amplification and parasitic Raman backscattering is analyzed, leading to the optimization of the interaction conditions.

Distinguishing Raman from strongly coupled Brillouin amplification for short pulses

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

Jia, Qing; Barth, Ido; Edwards, Matthew R.

2016-05-15

Plasma-based amplification by strongly coupled Brillouin scattering has recently been suggested for the compression of a short seed laser to ultrahigh intensities in sub-quarter-critical-density plasmas. However, by employing detailed spectral analysis of particle-in-cell simulations in the same parameter regime, we demonstrate that, in fact, Raman backscattering amplification is responsible for the growth and compression of the high-intensity, leading spike, where most of the energy compression occurs, while the ion mode only affects the low-intensity tail of the amplified pulse. The critical role of the initial seed shape is identified. A number of subtleties in the numerical simulations are also pointedmore » out.« less

Mathematical simulation of the amplification of 1790-nm laser radiation in a nuclear-excited He - Ar plasma containing nanoclusters of uranium compounds

NASA Astrophysics Data System (ADS)

Kosarev, V. A.; Kuznetsova, E. E.

2014-02-01

The possibility of applying dusty active media in nuclearpumped lasers has been considered. The amplification of 1790-nm radiation in a nuclear-excited dusty He - Ar plasma is studied by mathematical simulation. The influence of nanoclusters on the component composition of the medium and the kinetics of the processes occurring in it is analysed using a specially developed kinetic model, including 72 components and more than 400 reactions. An analysis of the results indicates that amplification can in principle be implemented in an active laser He - Ar medium containing 10-nm nanoclusters of metallic uranium and uranium dioxide.

Genetic amplification of PPME1 in gastric and lung cancer and its potential as a novel therapeutic target

PubMed Central

Li, Jing; Han, Sufang; Qian, Ziliang; Su, Xinying; Fan, Shuqiong; Fu, Jiangang; Liu, Yuanjie; Yin, Xiaolu; Gao, Zeren; Zhang, Jingchuan; Yu, De-Hua; Ji, Qunsheng

2014-01-01

Protein phosphatase methylesterase 1 (PPME1) is a protein phosphatase 2A (PP2A)-specific methyl esterase that negatively regulates PP2A through demethylation at its carboxy terminal leucine 309 residue. Emerging evidence shows that the upregulation of PPME1 is associated with poor prognosis in glioblastoma patients. By performing an array comparative genomic hybridization analysis to detect copy number changes, we have been the first to identify PPME1 gene amplification in 3.8% (5/131) of Chinese gastric cancer (GC) samples and 3.1% (4/124) of Chinese lung cancer (LC) samples. This PPME1 gene amplification was confirmed by fluorescence in situ hybridization analysis and is correlated with elevated protein expression, as determined by immunohistochemistry analysis. To further investigate the role of PPME1 amplification in tumor growth, short-hairpin RNA-mediated gene silencing was employed. A knockdown of PPME1 expression resulted in a significant inhibition of cell proliferation and induction of cell apoptosis in PPME1-amplified human cancer cell lines SNU668 (GC) and Oka-C1 (LC), but not in nonamplified MKN1 (GC) and HCC95 (LC) cells. The PPME1 gene knockdown also led to a consistent decrease in PP2A demethylation at leucine 309, which was correlated with the downregulation of cellular Erk and AKT phosphorylation. Our data indicate that PPME1 could be an attractive therapeutic target for a subset of GCs and LCs. PMID:24253382

A Sound Pressure-level Meter Without Amplification

NASA Technical Reports Server (NTRS)

Stowell, E Z

1937-01-01

The N.A.C.A. has developed a simple pressure-level meter for the measurement of sound-pressure levels above 70 db. The instrument employs a carbon microphone but has no amplification. The source of power is five flashlight batteries. Measurements may be made up to the threshold of feeling with an accuracy of plus or minus 2 db; band analysis of complex spectra may be made if desired.

The Role of Excitons on Light Amplification in Lead Halide Perovskites.

PubMed

Lü, Quan; Wei, Haohan; Sun, Wenzhao; Wang, Kaiyang; Gu, Zhiyuan; Li, Jiankai; Liu, Shuai; Xiao, Shumin; Song, Qinghai

2016-12-01

The role of excitons on the amplifications of lead halide perovskites has been explored. Unlike the photoluminescence, the intensity of amplified spontaneous emission is partially suppressed at low temperature. The detailed analysis and experiments show that the inhibition is attributed to the existence of exciton and a quantitative model has been built to explain the experimental observations. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Discovery of natural gain amplification in the 10-micrometer carbon dioxide laser bands on Mars - A natural laser

NASA Technical Reports Server (NTRS)

Mumma, M. J.; Buhl, D.; Chin, G.; Deming, D.; Espenak, F.; Kostiuk, T.; Zipoy, D.

1981-01-01

Fully resolved intensity profiles of various lines in the carbon dioxide band at 10.4 micrometers have been measured on Mars with an infrared heterodyne spectrometer. Analysis of the line shapes shows that the Mars atmosphere exhibits positive gain in these lines. The detection of natural optical gain amplification enables identification of these lines as a definite natural laser.

Optimization of ultrahigh-speed multiplex PCR for forensic analysis.

PubMed

Gibson-Daw, Georgiana; Crenshaw, Karin; McCord, Bruce

2018-01-01

In this paper, we demonstrate the design and optimization of an ultrafast PCR amplification technique, used with a seven-locus multiplex that is compatible with conventional capillary electrophoresis systems as well as newer microfluidic chip devices. The procedure involves the use of a high-speed polymerase and a rapid cycling protocol to permit multiplex PCR amplification of forensic short tandem repeat loci in 6.5 min. We describe the selection and optimization of master mix reagents such as enzyme, buffer, MgCl 2 , and dNTPs, as well as primer ratios, total volume, and cycle conditions, in order to get the best profile in the shortest time possible. Sensitivity and reproducibility studies are also described. The amplification process utilizes a small high-speed thermocycler and compact laptop, making it portable and potentially useful for rapid, inexpensive on-site genotyping. The seven loci of the multiplex were taken from conventional STR genotyping kits and selected for their size and lack of overlap. Analysis was performed using conventional capillary electrophoresis and microfluidics with fluorescent detection. Overall, this technique provides a more rapid method for rapid sample screening of suspects and victims. Graphical abstract Rapid amplification of forensic DNA using high speed thermal cycling followed by capillary or microfluidic electrophoresis.

Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data.

PubMed

Olova, Nelly; Krueger, Felix; Andrews, Simon; Oxley, David; Berrens, Rebecca V; Branco, Miguel R; Reik, Wolf

2018-03-15

Whole-genome bisulfite sequencing (WGBS) is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research. Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing. We perform a comparative analysis of several commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se is the main trigger of pronounced sequencing biases, and PCR amplification builds on these underlying artefacts. The majority of standard library preparation methods yield a significantly biased sequence output and overestimate global methylation. Importantly, both absolute and relative methylation levels at specific genomic regions vary substantially between methods, with clear implications for DNA methylation studies. We show that amplification-free library preparation is the least biased approach for WGBS. In protocols with amplification, the choice of bisulfite conversion protocol or polymerase can significantly minimize artefacts. To aid with the quality assessment of existing WGBS datasets, we have integrated a bias diagnostic tool in the Bismark package and offer several approaches for consideration during the preparation and analysis of WGBS datasets.

Observational Evidence for Desert Amplification Using Multiple Satellite Datasets.

PubMed

Wei, Nan; Zhou, Liming; Dai, Yongjiu; Xia, Geng; Hua, Wenjian

2017-05-17

Desert amplification identified in recent studies has large uncertainties due to data paucity over remote deserts. Here we present observational evidence using multiple satellite-derived datasets that desert amplification is a real large-scale pattern of warming mode in near surface and low-tropospheric temperatures. Trend analyses of three long-term temperature products consistently confirm that near-surface warming is generally strongest over the driest climate regions and this spatial pattern of warming maximizes near the surface, gradually decays with height, and disappears in the upper troposphere. Short-term anomaly analyses show a strong spatial and temporal coupling of changes in temperatures, water vapor and downward longwave radiation (DLR), indicating that the large increase in DLR drives primarily near surface warming and is tightly associated with increasing water vapor over deserts. Atmospheric soundings of temperature and water vapor anomalies support the results of the long-term temperature trend analysis and suggest that desert amplification is due to comparable warming and moistening effects of the troposphere. Likely, desert amplification results from the strongest water vapor feedbacks near the surface over the driest deserts, where the air is very sensitive to changes in water vapor and thus efficient in enhancing the longwave greenhouse effect in a warming climate.

Increasing the sensitivity of reverse phase protein arrays by antibody-mediated signal amplification

PubMed Central

2010-01-01

Background Reverse phase protein arrays (RPPA) emerged as a useful experimental platform to analyze biological samples in a high-throughput format. Different signal detection methods have been described to generate a quantitative readout on RPPA including the use of fluorescently labeled antibodies. Increasing the sensitivity of RPPA approaches is important since many signaling proteins or posttranslational modifications are present at a low level. Results A new antibody-mediated signal amplification (AMSA) strategy relying on sequential incubation steps with fluorescently-labeled secondary antibodies reactive against each other is introduced here. The signal quantification is performed in the near-infrared range. The RPPA-based analysis of 14 endogenous proteins in seven different cell lines demonstrated a strong correlation (r = 0.89) between AMSA and standard NIR detection. Probing serial dilutions of human cancer cell lines with different primary antibodies demonstrated that the new amplification approach improved the limit of detection especially for low abundant target proteins. Conclusions Antibody-mediated signal amplification is a convenient and cost-effective approach for the robust and specific quantification of low abundant proteins on RPPAs. Contrasting other amplification approaches it allows target protein detection over a large linear range. PMID:20569466

Novel Degenerate PCR Method for Whole-Genome Amplification Applied to Peru Margin (ODP Leg 201) Subsurface Samples

PubMed Central

Martino, Amanda J.; Rhodes, Matthew E.; Biddle, Jennifer F.; Brandt, Leah D.; Tomsho, Lynn P.; House, Christopher H.

2011-01-01

A degenerate polymerase chain reaction (PCR)-based method of whole-genome amplification, designed to work fluidly with 454 sequencing technology, was developed and tested for use on deep marine subsurface DNA samples. While optimized here for use with Roche 454 technology, the general framework presented may be applicable to other next generation sequencing systems as well (e.g., Illumina, Ion Torrent). The method, which we have called random amplification metagenomic PCR (RAMP), involves the use of specific primers from Roche 454 amplicon sequencing, modified by the addition of a degenerate region at the 3′ end. It utilizes a PCR reaction, which resulted in no amplification from blanks, even after 50 cycles of PCR. After efforts to optimize experimental conditions, the method was tested with DNA extracted from cultured E. coli cells, and genome coverage was estimated after sequencing on three different occasions. Coverage did not vary greatly with the different experimental conditions tested, and was around 62% with a sequencing effort equivalent to a theoretical genome coverage of 14.10×. The GC content of the sequenced amplification product was within 2% of the predicted values for this strain of E. coli. The method was also applied to DNA extracted from marine subsurface samples from ODP Leg 201 site 1229 (Peru Margin), and results of a taxonomic analysis revealed microbial communities dominated by Proteobacteria, Chloroflexi, Firmicutes, Euryarchaeota, and Crenarchaeota, among others. These results were similar to those obtained previously for those samples; however, variations in the proportions of taxa identified illustrates well the generally accepted view that community analysis is sensitive to both the amplification technique used and the method of assigning sequences to taxonomic groups. Overall, we find that RAMP represents a valid methodology for amplifying metagenomes from low-biomass samples. PMID:22319519

Site effects in Port-au-Prince (Haiti) from the analysis of spectral ratio and numerical simulations.

USGS Publications Warehouse

St. Fleur, Sadrac; Bertrand, Etienne; Courboulex, Francoise; Mercier de Lépinay, Bernard; Deschamps, Anne; Hough, Susan E.; Cultrera, Giovanna; Boisson, Dominique; Prepetit, Claude

2016-01-01

To provide better insight into seismic ground motion in the Port‐au‐Prince metropolitan area, we investigate site effects at 12 seismological stations by analyzing 78 earthquakes with magnitude smaller than 5 that occurred between 2010 and 2013. Horizontal‐to‐vertical spectral ratio on earthquake recordings and a standard spectral ratio were applied to the seismic data. We also propose a simplified lithostratigraphic map and use available geotechnical and geophysical data to construct representative soil columns in the vicinity of each station that allow us to compute numerical transfer functions using 1D simulations. At most of the studied sites, spectral ratios are characterized by weak‐motion amplification at frequencies above 5 Hz, in good agreement with the numerical transfer functions. A mismatch between the observed amplifications and simulated response at lower frequencies shows that the considered soil columns could be missing a deeper velocity contrast. Furthermore, strong amplification between 2 and 10 Hz linked to local topographic features is found at one station located in the south of the city, and substantial amplification below 5 Hz is detected near the coastline, which we attribute to deep and soft sediments as well as the presence of surface waves. We conclude that for most investigated sites in Port‐au‐Prince, seismic amplifications due to site effects are highly variable but seem not to be important at high frequencies. At some specific locations, however, they could strongly enhance the low‐frequency content of the seismic ground shaking. Although our analysis does not consider nonlinear effects, we thus conclude that, apart from sites close to the coast, sediment‐induced amplification probably had only a minor impact on the level of strong ground motion, and was not the main reason for the high level of damage in Port‐au‐Prince.

Geometric Effects on the Amplification of First Mode Instability Waves

NASA Technical Reports Server (NTRS)

Kirk, Lindsay C.; Candler, Graham V.

2013-01-01

The effects of geometric changes on the amplification of first mode instability waves in an external supersonic boundary layer were investigated using numerical techniques. Boundary layer stability was analyzed at Mach 6 conditions similar to freestream conditions obtained in quiet ground test facilities so that results obtained in this study may be applied to future test article design to measure first mode instability waves. The DAKOTA optimization software package was used to optimize an axisymmetric geometry to maximize the amplification of the waves at first mode frequencies as computed by the 2D STABL hypersonic boundary layer stability analysis tool. First, geometric parameters such as nose radius, cone half angle, vehicle length, and surface curvature were examined separately to determine the individual effects on the first mode amplification. Finally, all geometric parameters were allowed to vary to produce a shape optimized to maximize the amplification of first mode instability waves while minimizing the amplification of second mode instability waves. Since first mode waves are known to be most unstable in the form of oblique wave, the geometries were optimized using a broad range of wave frequencies as well as a wide range of oblique wave angles to determine the geometry that most amplifies the first mode waves. Since first mode waves are seen most often in flows with low Mach numbers at the edge of the boundary layer, the edge Mach number for each geometry was recorded to determine any relationship between edge Mach number and the stability of first mode waves. Results indicate that an axisymmetric cone with a sharp nose and a slight flare at the aft end under the Mach 6 freestream conditions used here will lower the Mach number at the edge of the boundary layer to less than 4, and the corresponding stability analysis showed maximum first mode N factors of 3.

Paternity testing in case of brother-sister incest.

PubMed

Macan, Marijana; Uvodić, Petra; Botica, Vladimir

2003-06-01

We performed a paternity test in a case of incest between brother and sister. DNA from blood samples of the alleged parents and their two children was obtained with Chelex DNA extraction method and quantified with Applied Biosystems QuantiBlot quantitation kit. Polymerase chain reaction (PCR) amplification of DNA samples was performed with AmpFlSTR SGM Plus PCR amplification kit and GenePrint PowerPlex PCR amplification kit. The amplified products were separated and detected by using the Perkin Elmer's ABI PRISM trade mark 310 Genetic Analyser. DNA and data analysis of 17 loci and Amelogenin confirmed the suspicion of brother-sister incest. Since both children had inherited all of the obligate alleles from the alleged father, we could confirm with certainty of 99.999999% that the oldest brother in the family was the biological father of both children. Calculated data showed that even in a case of brother-sister incest, paternity could be proved by the analysis of Amelogenin and 17 DNA loci.

Developmental validation of a Cannabis sativa STR multiplex system for forensic analysis.

PubMed

Howard, Christopher; Gilmore, Simon; Robertson, James; Peakall, Rod

2008-09-01

A developmental validation study based on recommendations of the Scientific Working Group on DNA Analysis Methods (SWGDAM) was conducted on a multiplex system of 10 Cannabis sativa short tandem repeat loci. Amplification of the loci in four multiplex reactions was tested across DNA from dried root, stem, and leaf sources, and DNA from fresh, frozen, and dried leaf tissue with a template DNA range of 10.0-0.01 ng. The loci were amplified and scored consistently for all DNA sources when DNA template was in the range of 10.0-1.0 ng. Some allelic dropout and PCR failure occurred in reactions with lower template DNA amounts. Overall, amplification was best using 10.0 ng of template DNA from dried leaf tissue indicating that this is the optimal source material. Cross species amplification was observed in Humulus lupulus for three loci but there was no allelic overlap. This is the first study following SWGDAM validation guidelines to validate short tandem repeat markers for forensic use in plants.

Systematic Site Characterization at Seismic Stations combined with Empirical Spectral Modeling: critical data for local hazard analysis

NASA Astrophysics Data System (ADS)

Michel, Clotaire; Hobiger, Manuel; Edwards, Benjamin; Poggi, Valerio; Burjanek, Jan; Cauzzi, Carlo; Kästli, Philipp; Fäh, Donat

2016-04-01

The Swiss Seismological Service operates one of the densest national seismic networks in the world, still rapidly expanding (see http://www.seismo.ethz.ch/monitor/index_EN). Since 2009, every newly instrumented site is characterized following an established procedure to derive realistic 1D VS velocity profiles. In addition, empirical Fourier spectral modeling is performed on the whole network for each recorded event with sufficient signal-to-noise ratio. Besides the source characteristics of the earthquakes, statistical real time analyses of the residuals of the spectral modeling provide a seamlessly updated amplification function w.r. to Swiss rock conditions at every station. Our site characterization procedure is mainly based on the analysis of surface waves from passive experiments and includes cross-checks of the derived amplification functions with those obtained through spectral modeling. The systematic use of three component surface-wave analysis, allowing the derivation of both Rayleigh and Love waves dispersion curves, also contributes to the improved quality of the retrieved profiles. The results of site characterisation activities at recently installed strong-motion stations depict the large variety of possible effects of surface geology on ground motion in the Alpine context. Such effects range from de-amplification at hard-rock sites to amplification up to a factor of 15 in lacustrine sediments with respect to the Swiss reference rock velocity model. The derived velocity profiles are shown to reproduce observed amplification functions from empirical spectral modeling. Although many sites are found to exhibit 1D behavior, our procedure allows the detection and qualification of 2D and 3D effects. All data collected during the site characterization procedures in the last 20 years are gathered in a database, implementing a data model proposed for community use at the European scale through NERA and EPOS (www.epos-eu.org). A web stationbook derived from it can be accessed through the interface www.stations.seismo.ethz.ch.

The effects of density gradients on the convective amplification of upper hybrid waves in the magnetosphere

NASA Technical Reports Server (NTRS)

Engel, J.; Kennel, C.

1985-01-01

Intense (at least 10 mV/m) electrostatic plasma waves have been detected near the upper hybrid frequency between + or -50 deg MLAT during recent GEOS-1 crossings. Wave growth rate and convective amplification calculations were carried out in order to explain the occurrence of intense upper hybrid (IUH) events over such a wide range of latitudes. The effects of wave refractions were taken into account in the convective amplification calculations. Specific results are presented for the upper hybrid wave growth of an IUH event occurring at 10 deg MLAT. It is shown that a density gradient may be necessary to explain the observed amplification at 10 deg MLAT. At the equator, however, the long scale length of the magnetic field gradient enables large amplitudes to be attained without a density gradient. The results of a UH ray tracing analysis are discussed within the framework of current theories concerning magnetospheric continuum radiation.

Disruption of the APC gene by t(5;7) translocation in a Turcot family.

PubMed

Sahnane, Nora; Bernasconi, Barbara; Carnevali, Ileana; Furlan, Daniela; Viel, Alessandra; Sessa, Fausto; Tibiletti, Maria Grazia

2016-03-01

Turcot syndrome (TS) refers to the combination of colorectal polyps and primary tumours of the central nervous system. TS is a heterogeneous genetic condition due to APC and/or mismatch repair germline mutations. When APC is involved the vast majority of mutations are truncating, but in approximately 20%-30% of patients with familial polyposis no germline mutation can be found. A 30-year-old Caucasian woman with a positive pedigree for TS was referred to our Genetic Counselling Service. She was negative for APC and MUTYH but showed a reciprocal balanced translocation t(5;7)(q22;p15) at chromosome analysis. FISH analysis using specific BAC probes demonstrated that 5q22 breakpoint disrupted the APC gene. Transcript analysis by MLPA and digital PCR revealed that the cytogenetic rearrangement involving the 3' end of the APC gene caused a defective expression of a truncated transcript. This result allowed cytogenetic analysis to be offered to all the other family members and segregation analysis clearly demonstrated that all the carriers were affected, whereas non-carriers did not have the polyposis. A cytogenetic approach permitted the identification of the mutation-causing disease in this family, and the segregation analysis together with the transcript study supported the pathogenetic role of this mutation. Karyotype analysis was used as a predictive test in all members of this family. This family suggests that clinically positive TS and FAP cases, which test negative with standard molecular analysis, could be easily and cost-effectively resolved by a classical and molecular cytogenetic approach. Copyright © 2015 Elsevier Inc. All rights reserved.

Droplet Microfluidic Device Fabrication and Use for Isothermal Amplification and Detection of MicroRNA.

PubMed

Giuffrida, Maria Chiara; D'Agata, Roberta; Spoto, Giuseppe

2017-01-01

Droplet microfluidics combined with the isothermal circular strand displacement polymerization (ICSDP) represents a powerful new technique to detect both single-stranded DNA and microRNA sequences. The method here described helps in overcoming some drawbacks of the lately introduced droplet polymerase chain reaction (PCR) amplification when implemented in microfluidic devices. The method also allows the detection of nanoliter droplets of nucleic acids sequences solutions, with a particular attention to microRNA sequences that are detected at the picomolar level. The integration of the ICSDP amplification protocol in droplet microfluidic devices reduces the time of analysis and the amount of sample required. In addition, there is also the possibility to design parallel analyses to be integrated in portable devices.

The effects of variable sample biomass on comparative metagenomics.

PubMed

Chafee, Meghan; Maignien, Loïs; Simmons, Sheri L

2015-07-01

Longitudinal studies that integrate samples with variable biomass are essential to understand microbial community dynamics across space or time. Shotgun metagenomics is widely used to investigate these communities at the functional level, but little is known about the effects of combining low and high biomass samples on downstream analysis. We investigated the interacting effects of DNA input and library amplification by polymerase chain reaction on comparative metagenomic analysis using dilutions of a single complex template from an Arabidopsis thaliana-associated microbial community. We modified the Illumina Nextera kit to generate high-quality large-insert (680 bp) paired-end libraries using a range of 50 pg to 50 ng of input DNA. Using assembly-based metagenomic analysis, we demonstrate that DNA input level has a significant impact on community structure due to overrepresentation of low-GC genomic regions following library amplification. In our system, these differences were largely superseded by variations between biological replicates, but our results advocate verifying the influence of library amplification on a case-by-case basis. Overall, this study provides recommendations for quality filtering and de-replication prior to analysis, as well as a practical framework to address the issue of low biomass or biomass heterogeneity in longitudinal metagenomic surveys. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

MET Amplification Identifies a Small and Aggressive Subgroup of Esophagogastric Adenocarcinoma With Evidence of Responsiveness to Crizotinib

PubMed Central

Lennerz, Jochen K.; Kwak, Eunice L.; Ackerman, Allison; Michael, Michael; Fox, Stephen B.; Bergethon, Kristin; Lauwers, Gregory Y.; Christensen, James G.; Wilner, Keith D.; Haber, Daniel A.; Salgia, Ravi; Bang, Yung-Jue; Clark, Jeffrey W.; Solomon, Benjamin J.; Iafrate, A. John

2011-01-01

Purpose Amplification of the MET proto-oncogene in gastroesophageal cancer (GEC) may constitute a molecular marker for targeted therapy. We examined a GEC cohort with follow-up and reported the clinical response of four additional patients with MET-amplified tumors to the small molecule inhibitor crizotinib as part of an expanded phase I cohort study. Patients and Methods From 2007 to 2009, patients with GEC were genetically screened as a consecutive series of 489 tumors (stages 0, I, and II, 39%; III, 25%; IV, 36%; n = 222 esophageal, including n = 21 squamous carcinomas). MET, EGFR, and HER2 amplification status was assessed by using fluorescence in situ hybridization. Results Ten (2%) of 489 patients screened harbored MET amplification; 23 (4.7%) harbored EGFR amplification; 45 (8.9%) harbored HER2 amplification; and 411 (84%) were wild type for all three genes (ie, negative). MET-amplified tumors were typically high-grade adenocarcinomas that presented at advanced stages (5%; n = 4 of 80). EGFR-amplified tumors showed the highest fraction of squamous cell carcinoma (17%; n = 4 of 23). HER2, MET, and EGFR amplification were, with one exception (MET and EGFR positive), mutually exclusive events. Survival analysis in patients with stages III and IV disease showed substantially shorter median survival in MET/EGFR-amplified groups, with a rank order for all groups by median survival (from most to least aggressive): MET (7.1 months; P < .001) less than EGFR (11.2 months; P = .16) less than HER2 (16.9 months; P = .89) when compared with the negative group (16.2 months). Two of four patients with MET-amplified tumors treated with crizotinib experienced tumor shrinkage (−30% and −16%) and experienced progression after 3.7 and 3.5 months. Conclusion MET amplification defines a small and aggressive subset of GEC with indications of transient sensitivity to the targeted MET inhibitor crizotinib (PF-02341066). PMID:22042947

MET amplification identifies a small and aggressive subgroup of esophagogastric adenocarcinoma with evidence of responsiveness to crizotinib.

PubMed

Lennerz, Jochen K; Kwak, Eunice L; Ackerman, Allison; Michael, Michael; Fox, Stephen B; Bergethon, Kristin; Lauwers, Gregory Y; Christensen, James G; Wilner, Keith D; Haber, Daniel A; Salgia, Ravi; Bang, Yung-Jue; Clark, Jeffrey W; Solomon, Benjamin J; Iafrate, A John

2011-12-20

Amplification of the MET proto-oncogene in gastroesophageal cancer (GEC) may constitute a molecular marker for targeted therapy. We examined a GEC cohort with follow-up and reported the clinical response of four additional patients with MET-amplified tumors to the small molecule inhibitor crizotinib as part of an expanded phase I cohort study. From 2007 to 2009, patients with GEC were genetically screened as a consecutive series of 489 tumors (stages 0, I, and II, 39%; III, 25%; IV, 36%; n = 222 esophageal, including n = 21 squamous carcinomas). MET, EGFR, and HER2 amplification status was assessed by using fluorescence in situ hybridization. Ten (2%) of 489 patients screened harbored MET amplification; 23 (4.7%) harbored EGFR amplification; 45 (8.9%) harbored HER2 amplification; and 411 (84%) were wild type for all three genes (ie, negative). MET-amplified tumors were typically high-grade adenocarcinomas that presented at advanced stages (5%; n = 4 of 80). EGFR-amplified tumors showed the highest fraction of squamous cell carcinoma (17%; n = 4 of 23). HER2, MET, and EGFR amplification were, with one exception (MET and EGFR positive), mutually exclusive events. Survival analysis in patients with stages III and IV disease showed substantially shorter median survival in MET/EGFR-amplified groups, with a rank order for all groups by median survival (from most to least aggressive): MET (7.1 months; P < .001) less than EGFR (11.2 months; P = .16) less than HER2 (16.9 months; P = .89) when compared with the negative group (16.2 months). Two of four patients with MET-amplified tumors treated with crizotinib experienced tumor shrinkage (-30% and -16%) and experienced progression after 3.7 and 3.5 months. MET amplification defines a small and aggressive subset of GEC with indications of transient sensitivity to the targeted MET inhibitor crizotinib (PF-02341066).

Differential impact of ionic and coordinate covalent chromium (Cr)-DNA binding on DNA replication.

PubMed

Fornsaglio, Jamie L; O'Brien, Travis J; Patierno, Steven R

2005-11-01

The reactive species produced by the reduction of Cr(VI), particularly Cr(III), can form both ionic and coordinate covalent complexes with DNA. These Cr(III)-DNA interactions consist of Cr-DNA monoadducts, Cr-DNA ternary adducts, and Cr-DNA interstrand cross-links (Cr-ICLs), the latter of which are DNA polymerase arresting lesions (PALs). We sought to determine the impact of Cr-DNA interactions on the formation of replication blocking lesions in S. cerevisiae using a PCR-based method. We found that target sequence (TS) amplification using DNA isolated from Cr(VI)-treated yeast actually increased as a function of Cr(VI) concentration. Moreover, the enhanced TS amplification was reproduced in vitro using Cr(III)-treated DNA. In contrast, PCR amplification of TS from DNA isolated from yeast exposed to equitoxic doses of the inorganic DNA cross-linking agent cisplatin (CDDP), was decreased in a concentration-dependent manner. This paradox suggested that a specific Cr-DNA interaction, such as an ionic Cr-DNA complex, was responsible for the enhanced TS amplification, thereby masking the replication-blocking effect of certain ternary Cr-DNA adducts (i.e. interstrand cross-links). To test this possibility, we removed ionically associated Cr from the DNA using salt extraction prior to PCR analysis. This procedure obviated the increased amplification and revealed a dose-dependent decrease in TS amplification and an increase in Cr-PALs. These data from DNA analyzed ex vivo after treatment of intact cells indicate that ionic interactions of Cr with DNA result in increased DNA amplification whereas coordinate-covalent Cr-DNA complexes lead to formation of Cr-PALs. Thus, these results suggest that treatment of living cells with Cr(VI) leads to two modes of Cr-binding, which may have conflicting effects on DNA replication.

Photocleavable DNA barcode-antibody conjugates allow sensitive and multiplexed protein analysis in single cells.

PubMed

Agasti, Sarit S; Liong, Monty; Peterson, Vanessa M; Lee, Hakho; Weissleder, Ralph

2012-11-14

DNA barcoding is an attractive technology, as it allows sensitive and multiplexed target analysis. However, DNA barcoding of cellular proteins remains challenging, primarily because barcode amplification and readout techniques are often incompatible with the cellular microenvironment. Here we describe the development and validation of a photocleavable DNA barcode-antibody conjugate method for rapid, quantitative, and multiplexed detection of proteins in single live cells. Following target binding, this method allows DNA barcodes to be photoreleased in solution, enabling easy isolation, amplification, and readout. As a proof of principle, we demonstrate sensitive and multiplexed detection of protein biomarkers in a variety of cancer cells.

PMS2 gene mutational analysis: direct cDNA sequencing to circumvent pseudogene interference.

PubMed

Wimmer, Katharina; Wernstedt, Annekatrin

2014-01-01

The presence of highly homologous pseudocopies can compromise the mutation analysis of a gene of interest. In particular, when using PCR-based strategies, pseudogene co-amplification has to be effectively prevented. This is often achieved by using primers designed to be parental gene specific according to the reference sequence and by applying stringent PCR conditions. However, there are cases in which this approach is of limited utility. For example, it has been shown that the PMS2 gene exchanges sequences with one of its pseudogenes, named PMS2CL. This results in functional PMS2 alleles containing pseudogene-derived sequences at their 3'-end and in nonfunctional PMS2CL pseudogene alleles that contain gene-derived sequences. Hence, the paralogues cannot be distinguished according to the reference sequence. This shortcoming can be effectively circumvented by using direct cDNA sequencing. This approach is based on the selective amplification of PMS2 transcripts in two overlapping 1.6-kb RT-PCR products. In addition to avoiding pseudogene co-amplification and allele dropout, this method has also the advantage that it allows to effectively identify deletions, splice mutations, and de novo retrotransposon insertions that escape the detection of most DNA-based mutation analysis protocols.

Prognostic significance of ESR1 gene amplification, mRNA/protein expression and functional profiles in high-risk early breast cancer: a translational study of the Hellenic Cooperative Oncology Group (HeCOG).

PubMed

Pentheroudakis, George; Kotoula, Vassiliki; Eleftheraki, Anastasia G; Tsolaki, Eleftheria; Wirtz, Ralph M; Kalogeras, Konstantine T; Batistatou, Anna; Bobos, Mattheos; Dimopoulos, Meletios A; Timotheadou, Eleni; Gogas, Helen; Christodoulou, Christos; Papadopoulou, Kyriaki; Efstratiou, Ioannis; Scopa, Chrisoula D; Papaspyrou, Irene; Vlachodimitropoulos, Dimitrios; Linardou, Helena; Samantas, Epaminontas; Pectasides, Dimitrios; Pavlidis, Nicholas; Fountzilas, George

2013-01-01

Discrepant data have been published on the incidence and prognostic significance of ESR1 gene amplification in early breast cancer. Formalin-fixed paraffin-embedded tumor blocks were collected from women with early breast cancer participating in two HeCOG adjuvant trials. Messenger RNA was studied by quantitative PCR, ER protein expression was centrally assessed using immunohistochemistry (IHC) and ESR1 gene copy number by dual fluorescent in situ hybridization probes. In a total of 1010 women with resected node-positive early breast adenocarcinoma, the tumoral ESR1/CEP6 gene ratio was suggestive of deletion in 159 (15.7%), gene gain in 551 (54.6%) and amplification in 42 cases (4.2%), with only 30 tumors (3%) harboring five or more ESR1 copies. Gene copy number ratio showed a significant, though weak correlation to mRNA and protein expression (Spearman's Rho <0.23, p = 0.01). ESR1 clusters were observed in 9.5% (57 gain, 38 amplification) of cases. In contrast to mRNA and protein expression, which were favorable prognosticators, gene copy number changes did not obtain prognostic significance. When ESR1/CEP6 gene ratio was combined with function (as defined by ER protein and mRNA expression) in a molecular classifier, the Gene Functional profile, it was functional status that impacted on prognosis. In univariate analysis, patients with functional tumors (positive ER protein expression and gene ratio normal or gain/amplification) fared better than those with non-functional tumors with ESR1 gain (HR for relapse or death 0.49-0.64, p = 0.003). Significant interactions were observed between gene gain/amplification and paclitaxel therapy (trend for DFS benefit from paclitaxel only in patients with ESR1 gain/amplification, p = 0.066) and Gene Functional profile with HER2 amplification (Gene Functional profile prognostic only in HER2-normal cases, p = 0.029). ESR1 gene deletion and amplification do not constitute per se prognostic markers, instead they can be classified to distinct prognostic groups according to their protein-mediated functional status.

[Prognostic significance of MYCN amplification in children neuroblastic tumors].

PubMed

Niu, Huilin; Xu, Tao; Wang, Fenghua; Chen, Zhengrong; Gao, Qiu; Yi, Peng; Xia, Jianqing

2015-02-01

To summarize the clinicopathologic features of neuroblastic tumors (NT), and to explore the prognostic significance of MYCN amplification in NT. The clinicopathologic data of 267 NT were reviewed. MYCN gene amplification was detected by fluorescence in situ hybridization (FISH) in 119 cases and the relationship with pathological characteristics and prognostic significance were analyzed. The study included 267 cases of children NT from patients aged from 1 day to 13 years (median 27 months). The male to female ratio was 1.43. There were 38 cases (14.2%), 43 cases (16.1%), 71 cases (26.6%), and 115 cases (43.1%) of INSS stages I, II, III and IV respectively.Favorable histology group had 157 cases (59.9%); unfavorable histology group had 110 cases (40.1%).Of the 119 NT cases with MYCN FISH performed, 18 cases (15.1%) showed amplification and the signal ratio of MYCN to CEP2 was 4.08-43.29. One hundred and one cases of non-amplified MYCN included MYCN gain in 79 cases (66.3%) and MYCN negative in 22 cases (18.5%). MYCN expression showed significant difference (P = 0.000) between ages, gender, NT type and MKI, but not INPC and clinical stage (P > 0.05).Of the 18 cases with MYCN amplification, 3 were undifferentiated, and 15 poorly differentiated; 17 had high MKI and one moderate MKI. All 18 cases were in unfavorable histology group; the overall survival rate was 3/18, with an average survival time of (17.9 ± 2.4) months.Of the 101 MYCN non-amplification cases, the overall survival rate was 68.3% (69/101), with an average survival time of (29.8 ± 1.3) months. Survival analysis showed the cases with MYCN amplification had worse prognosis (P < 0.05). NT were commonly diagnosed in early ages and easily to metastasize. Most of cases with favorable histology. The cases of MYCN amplification showed unfavorable histology, and the majority cases with high MKI; The patients with MYCN gene amplification had poor prognosis.

A systematic analysis of directional site effects at stations of the Italian Seismic Network to test the role of local topography

NASA Astrophysics Data System (ADS)

Pischiutta, Marta; Cianfarra, Paola; Salvini, Francesco; Cara, Fabrizio; Vannoli, Paola

2018-03-01

Directional site effects observed at seismological stations on pronounced relief are analyzed. We investigate the ground motion properties calculating horizontal-to-vertical spectral ratios and horizontal polarization of both ambient vibrations and earthquake records using broadband seismograms of the Italian Seismic Network. We find that a subset of 47 stations with pronounced relief, results in a significant (>2) directional amplification of the horizontal component, with a well defined, site-specific direction of motion. However, the horizontal spectral response of sites is not uniform, varying from an isolated (resonant) frequency peak to a broadband amplification, interesting frequency bands as large as 1-10 Hz in many cases. Using the 47 selected stations, we have tried to establish a relation between directional amplification and topography geometry in a 2D-vision, when applicable, through a morphological analysis of the Digital Elevation Model using Geographic Information Systems. The procedure computes the parameters that characterize the geometry of topographic irregularities (size and slope), in combination with a principal component analysis that automatically yields the orientation of the elongated ridges. In seeking a relation between directional amplification and the surface morphology, we have found that it is impossible to fit the variety of observations with a resonant topography model as well as to identify common features in the ground motion behavior for stations with similar topography typologies. We conclude that, rather than the shape of the topography, local structural complexities and details of the near-surface structure must play a predominant role in controlling ground motion properties at sites with pronounced relief.

Muscle RAS oncogene homolog (MRAS) recurrent mutation in Borrmann type IV gastric cancer.

PubMed

Yasumoto, Makiko; Sakamoto, Etsuko; Ogasawara, Sachiko; Isobe, Taro; Kizaki, Junya; Sumi, Akiko; Kusano, Hironori; Akiba, Jun; Torimura, Takuji; Akagi, Yoshito; Itadani, Hiraku; Kobayashi, Tsutomu; Hasako, Shinichi; Kumazaki, Masafumi; Mizuarai, Shinji; Oie, Shinji; Yano, Hirohisa

2017-01-01

The prognosis of patients with Borrmann type IV gastric cancer (Type IV) is extremely poor. Thus, there is an urgent need to elucidate the molecular mechanisms underlying the oncogenesis of Type IV and to identify new therapeutic targets. Although previous studies using whole-exome and whole-genome sequencing have elucidated genomic alterations in gastric cancer, none has focused on comprehensive genetic analysis of Type IV. To discover cancer-relevant genes in Type IV, we performed whole-exome sequencing and genome-wide copy number analysis on 13 patients with Type IV. Exome sequencing identified 178 somatic mutations in protein-coding sequences or at splice sites. Among the mutations, we found a mutation in muscle RAS oncogene homolog (MRAS), which is predicted to cause molecular dysfunction. MRAS belongs to the Ras subgroup of small G proteins, which includes the prototypic RAS oncogenes. We analyzed an additional 46 Type IV samples to investigate the frequency of MRAS mutation. There were eight nonsynonymous mutations (mutation frequency, 17%), showing that MRAS is recurrently mutated in Type IV. Copy number analysis identified six focal amplifications and one homozygous deletion, including insulin-like growth factor 1 receptor (IGF1R) amplification. The samples with IGF1R amplification had remarkably higher IGF1R mRNA and protein expression levels compared with the other samples. This is the first report of MRAS recurrent mutation in human tumor samples. Our results suggest that MRAS mutation and IGF1R amplification could drive tumorigenesis of Type IV and could be new therapeutic targets. © 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

A systematic analysis of directional site effects at stations of the Italian seismic network to test the role of local topography

NASA Astrophysics Data System (ADS)

Pischiutta, Marta; Cianfarra, Paola; Salvini, Francesco; Cara, Fabrizio; Vannoli, Paola

2018-07-01

Directional site effects observed at seismological stations on pronounced relief are analysed. We investigate the ground motion properties calculating horizontal-to-vertical spectral ratios and horizontal polarization of both ambient vibrations and earthquake records using broad-band seismograms of the Italian seismic network. We find that a subset of 47 stations with pronounced relief results in a significant (>2) directional amplification of the horizontal component, with a well-defined, site-specific direction of motion. However, the horizontal spectral response of sites is not uniform, varying from an isolated (resonant) frequency peak to a broad-band amplification, interesting frequency bands as large as 1-10 Hz in many cases. Using 47 selected stations, we have tried to establish a relation between directional amplification and topography geometry in a 2-D vision, when applicable, through a morphological analysis of the digital elevation model using geographic information systems. The procedure computes the parameters that characterize the geometry of topographic irregularities (size and slope), in combination with a principal component analysis that automatically yields the orientation of the elongated ridges. In seeking a relation between directional amplification and the surface morphology, we have found that it is impossible to fit the variety of observations with a resonant topography model as well as to identify common features in the ground motion behaviour for stations with similar topography typologies. We conclude that, rather than the shape of the topography, local structural complexities and details of the near-surface structure must play a predominant role in controlling ground motion properties at sites with pronounced relief.

Somatically Acquired Isodicentric Y and Mosaic Loss of Chromosome Y in a Boy with Hypospadias.

PubMed

Miyado, Mami; Muroya, Koji; Katsumi, Momori; Saito, Kazuki; Kon, Masafumi; Fukami, Maki

2018-04-07

Isodicentric Y chromosome [idic(Y)] represents a relatively common subtype of Y chromosomal rearrangements in the germline; however, limited evidence supports the postzygotic occurrence of idic(Y). Here, we report a boy with hypospadias and somatically acquired idic(Y). The 3.5-year-old boy has been identified in our previous study for patients with hypospadias. In the present study, cytogenetic analysis including FISH revealed a 45,X[5]/46,X,idic(Y)[7]/46,XY[8] karyotype. MLPA showed a mosaic deletion involving PPP1R12BP1 and RBMY2DP. The idic(Y) was likely to have been formed through aberrant recombination between P1 palindromes and subsequently underwent mosaic loss. The patient's phenotype was attributable to deletion of some Y chromosomal genes and/or mosaic loss of chromosome Y (mLOY). The results suggest that idic(Y) can originate in postzygotic cells via palindrome-mediated crossovers. Moreover, our data indicate that somatically acquired idic(Y) can trigger mLOY, which usually appears as an aging-related phenomenon in elderly men. © 2018 S. Karger AG, Basel.

Spectrum of phenotypic anomalies in four families with deletion of the SHOX enhancer region.

PubMed

Gatta, Valentina; Palka, Chiara; Chiavaroli, Valentina; Franchi, Sara; Cannataro, Giovanni; Savastano, Massimo; Cotroneo, Antonio Raffaele; Chiarelli, Francesco; Mohn, Angelika; Stuppia, Liborio

2014-07-23

SHOX alterations have been reported in 67% of patients affected by Léri-Weill dyschondrosteosis (LWD), with a larger prevalence of gene deletions than point mutations. It has been recently demonstrated that these deletions can involve the SHOX enhancer region, rather that the coding region, with variable phenotype of the affected patients.Here, we report a SHOX gene analysis carried out by MLPA in 14 LWD patients from 4 families with variable phenotype. All patients presented a SHOX enhancer deletion. In particular, a patient with a severe bilateral Madelung deformity without short stature showed a homozygous alteration identical to the recently described 47.5 kb PAR1 deletion. Moreover, we identified, for the first time, in three related patients with a severe bilateral Madelung deformity, a smaller deletion than the 47.5 kb PAR1 deletion encompassing the same enhancer region (ECR1/CNE7). Data reported in this study provide new information about the spectrum of phenotypic alterations showed by LWD patients with different deletions of the SHOX enhancer region.

Spectrum of phenotypic anomalies in four families with deletion of the SHOX enhancer region

PubMed Central

2014-01-01

Background SHOX alterations have been reported in 67% of patients affected by Léri-Weill dyschondrosteosis (LWD), with a larger prevalence of gene deletions than point mutations. It has been recently demonstrated that these deletions can involve the SHOX enhancer region, rather that the coding region, with variable phenotype of the affected patients. Here, we report a SHOX gene analysis carried out by MLPA in 14 LWD patients from 4 families with variable phenotype. Case presentation All patients presented a SHOX enhancer deletion. In particular, a patient with a severe bilateral Madelung deformity without short stature showed a homozygous alteration identical to the recently described 47.5 kb PAR1 deletion. Moreover, we identified, for the first time, in three related patients with a severe bilateral Madelung deformity, a smaller deletion than the 47.5 kb PAR1 deletion encompassing the same enhancer region (ECR1/CNE7). Conclusions Data reported in this study provide new information about the spectrum of phenotypic alterations showed by LWD patients with different deletions of the SHOX enhancer region. PMID:25056248

Hybridization chain reaction: a versatile molecular tool for biosensing, bioimaging, and biomedicine.

PubMed

Bi, Sai; Yue, Shuzhen; Zhang, Shusheng

2017-07-17

Developing powerful, simple and low-cost DNA amplification techniques is of great significance to bioanalysis and biomedical research. Thus far, many signal amplification strategies have been developed, such as polymerase chain reaction (PCR), rolling circle amplification (RCA), and DNA strand displacement amplification (SDA). In particular, hybridization chain reaction (HCR), a type of toehold-mediated strand displacement (TMSD) reaction, has attracted great interest because of its enzyme-free nature, isothermal conditions, simple protocols, and excellent amplification efficiency. In a typical HCR, an analyte initiates the cross-opening of two DNA hairpins, yielding nicked double helices that are analogous to alternating copolymers. As an efficient amplification platform, HCR has been utilized for the sensitive detection of a wide variety of analytes, including nucleic acids, proteins, small molecules, and cells. In recent years, more complicated sets of monomers have been designed to develop nonlinear HCR, such as branched HCR and even dendritic systems, achieving quadratic and exponential growth mechanisms. In addition, HCR has attracted enormous attention in the fields of bioimaging and biomedicine, including applications in fluorescence in situ hybridization (FISH) imaging, live cell imaging, and targeted drug delivery. In this review, we introduce the fundamentals of HCR and examine the visualization and analysis techniques for HCR products in detail. The most recent HCR developments in biosensing, bioimaging, and biomedicine are subsequently discussed with selected examples. Finally, the review provides insight into the challenges and future perspectives of HCR.

An integrated microfluidic sensor for real-time detection of RNA in seawater using preserved reagents

NASA Astrophysics Data System (ADS)

Tsaloglou, M.-N.; Loukas, C. M.; Ruano-López, J. M.; Morgan, H.; Mowlem, M. C.

2012-04-01

Quantitation of RNA sequences coding either for key metabolic proteins or highly conserved ribosomal subunits can provide insight on cell abundance, speciation and viability. Nucleic sequence-based amplification (NASBA) is an isothermal alternative to traditional nucleic acid amplification methods, such as quantitative PCR. We present here an integrated microfluidic sensor for cell concentration and lysis, RNA extraction/purification and quantitative RNA detection for environmental applications. The portable system uses pre-loaded reagents, stored as a gel on a disposable microfluidic cartridge, which is manufactured using low-cost injection moulding. The NASBA reaction is monitored real-time using a bespoke control unit which includes: an external fluorescence detector, three peristaltic micro-pumps, two heaters and temperature sensors, a battery, seven pin actuated micro-motors (or valve actuators), and an automatic cartridge insertion mechanism. The system has USB connectivity and none of the expensive components require replacing between reactions. Long-term storage of reagents is critically important for any diagnostic tool that will be used in the field, whether for medical or environmental analysis and has not been previously demonstrated for NASBA reagents on-chip. We have shown effective amplification, for as little as 500 cells of the toxic microalga Karenia brevis using reagents which had been preserved as a gel for 45 days. This is the first reported real-time isothermal RNA amplification using with on-chip preservation. Annealing of primers, amplification at 41 °C and real-time fluorescence detection using, also for the first time, an internal control and sequence-specific molecular beacons was all performed on our microfluidic sensor. Our results show excellent promise as a future quantitative tool of in situ phytoplankton analysis and other environmental applications, where long-term reagent storage and low power consumption is essential.

Identification and Characterization of Genomic Amplifications in Ovarian Serous Carcinoma

DTIC Science & Technology

2009-07-01

oncogenes, Rsf1 and Notch3, which were up-regulated in both genomic DNA and transcript levels in ovarian cancer. In a large- scale FISH analysis, Rsf1...associated with worse disease outcome, suggesting that Rsf1 could be potentially used as a prognostic marker in the future (Appendix #1). For the...over- expressed in a recurrent carcinoma. Although the follow-up study in a larger- scale sample size did not demonstrate clear amplification in NAC1

Amplification of trace amounts of nucleic acids

DOEpatents

Church, George M [Brookline, MA; Zhang, Kun [Brighton, MA

2008-06-17

Methods of reducing background during amplification of small amounts of nucleic acids employ careful analysis of sources of low level contamination. Ultraviolet light can be used to reduce nucleic acid contaminants in reagents and equipment. "Primer-dimer" background can be reduced by judicious design of primers. We have shown clean signal-to-noise with as little as starting material as one single human cell (.about.6 picogram), E. coli cell (.about.5 femtogram) or Prochlorococcus cell (.about.3 femtogram).

Diagnostic accuracy of nucleic acid amplification tests (NAATs) in urine for genitourinary tuberculosis: a systematic review and meta-analysis.

PubMed

Altez-Fernandez, Carlos; Ortiz, Victor; Mirzazadeh, Majid; Zegarra, Luis; Seas, Carlos; Ugarte-Gil, Cesar

2017-06-05

Genitourinary tuberculosis is the third most common form of extrapulmonary tuberculosis. Diagnosis is difficult because of unspecific clinical manifestations and low accuracy of conventional tests. Unfortunately, the delayed diagnosis impacts the urinary tract severely. Nucleic acid amplification tests yield fast results, and among these, new technologies can also detect drug resistance. There is lack of consensus regarding the use of these tests in genitourinary tuberculosis; we therefore aimed to assess the accuracy of nucleic acid amplification tests in the diagnosis of genitourinary tuberculosis and to evaluate the heterogeneity between studies. We did a systematic review and meta-analysis of research articles comparing the accuracy of a reference standard and a nucleic acid amplification test for diagnosis of urinary tract tuberculosis. We searched Medline, EMBASE, Web of Science, LILACS, Cochrane Library, and Scopus for articles published between Jan 1, 1990, and Apr 14, 2016. Two investigators identified eligible articles and extracted data for individual study sites. We analyzed data in groups with the same index test. Then, we generated pooled summary estimates (95% CIs) for sensitivity and specificity by use of random-effects meta-analysis when studies were not heterogeneous. We identified eleven relevant studies from ten articles, giving information on PCR, LCR and Xpert MTB/RIF tests. All PCR studies were "in-house" tests, with different gene targets and had several quality concerns therefore we did not proceed with a pooled analysis. Only one study used LCR. Xpert studies were of good quality and not heterogeneous, pooled sensitivity was 0·87 (0·66-0·96) and specificity was 0·91 (0·84-0·95). PCR studies were highly heterogeneous. Among Xpert MTB/RIF studies, specificity was favorable with an acceptable confidence interval, however new studies can update meta-analysis and get more precise estimates. Further high-quality studies are urgently needed to improve diagnosis of genitourinary tuberculosis. PROSPERO CRD42016039020.

Digital LAMP in a sample self-digitization (SD) chip

PubMed Central

Herrick, Alison M.; Dimov, Ivan K.; Lee, Luke P.; Chiu, Daniel T.

2012-01-01

This paper describes the realization of digital loop-mediated DNA amplification (dLAMP) in a sample self-digitization (SD) chip. Digital DNA amplification has become an attractive technique to quantify absolute concentrations of DNA in a sample. While digital polymerase chain reaction is still the most widespread implementation, its use in resource—limited settings is impeded by the need for thermal cycling and robust temperature control. In such situations, isothermal protocols that can amplify DNA or RNA without thermal cycling are of great interest. Here, we showed the successful amplification of single DNA molecules in a stationary droplet array using isothermal digital loop-mediated DNA amplification. Unlike most (if not all) existing methods for sample discretization, our design allows for automated, loss-less digitization of sample volumes on-chip. We demonstrated accurate quantification of relative and absolute DNA concentrations with sample volumes of less than 2 μl. We assessed the homogeneity of droplet size during sample self-digitization in our device, and verified that the size variation was small enough such that straightforward counting of LAMP-active droplets sufficed for data analysis. We anticipate that the simplicity and robustness of our SD chip make it attractive as an inexpensive and easy-to-operate device for DNA amplification, for example in point-of-care settings. PMID:22399016

Graphene Nanoprobes for Real-Time Monitoring of Isothermal Nucleic Acid Amplification.

PubMed

Li, Fan; Liu, Xiaoguo; Zhao, Bin; Yan, Juan; Li, Qian; Aldalbahi, Ali; Shi, Jiye; Song, Shiping; Fan, Chunhai; Wang, Lihua

2017-05-10

Isothermal amplification is an efficient way to amplify DNA with high accuracy; however, the real-time monitoring for quantification analysis mostly relied on expensive and precisely designed probes. In the present study, a graphene oxide (GO)-based nanoprobe was used to real-time monitor the isothermal amplification process. The interaction between GO and different DNA structures was systematically investigated, including single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), DNA 3-helix, and long rolling circle amplification (RCA) and hybridization chain reaction (HCR) products, which existed in one-, two-, and three-dimensional structures. It was found that the high rigid structures exhibited much lower affinity with GO than soft ssDNA, and generally the rigidity was dependent on the length of targets and the hybridization position with probe DNA. On the basis of these results, we successfully monitored HCR amplification process, RCA process, and the enzyme restriction of RCA products with GO nanoprobe; other applications including the detection of the assembly/disassembly of DNA 3-helix structures were also performed. Compared to the widely used end-point detection methods, the GO-based sensing platform is simple, sensitive, cost-effective, and especially in a real-time monitoring mode. We believe such studies can provide comprehensive understandings and evocation on design of GO-based biosensors for broad application in various fields.

Natural genomic amplification of cholinesterase genes in animals.

PubMed

Chatonnet, Arnaud; Lenfant, Nicolas; Marchot, Pascale; Selkirk, Murray E

2017-08-01

Tight control of the concentration of acetylcholine at cholinergic synapses requires precise regulation of the number and state of the acetylcholine receptors, and of the synthesis and degradation of the neurotransmitter. In particular, the cholinesterase activity has to be controlled exquisitely. In the genome of the first experimental models used (man, mouse, zebrafish and drosophila), there are only one or two genes coding for cholinesterases, whereas there are more genes for their closest relatives the carboxylesterases. Natural amplification of cholinesterase genes was first found to occur in some cancer cells and in insect species subjected to evolutionary pressure by insecticides. Analysis of the complete genome sequences of numerous representatives of the various metazoan phyla show that moderate amplification of cholinesterase genes is not uncommon in molluscs, echinoderms, hemichordates, prochordates or lepidosauria. Amplification of acetylcholinesterase genes is also a feature of parasitic nematodes or ticks. In these parasites, over-production of cholinesterase-like proteins in secreted products and the saliva are presumed to have effector roles related to host infection. These amplification events raise questions about the role of the amplified gene products, and the adaptation processes necessary to preserve efficient cholinergic transmission. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms. © 2017 International Society for Neurochemistry.

Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification.

PubMed

Ziesemer, Kirsten A; Mann, Allison E; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T; Brandt, Bernd W; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A; MacDonald, Sandy J; Thomas, Gavin H; Collins, Matthew J; Lewis, Cecil M; Hofman, Corinne; Warinner, Christina

2015-11-13

To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341-534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions.

Intrinsic challenges in ancient microbiome reconstruction using 16S rRNA gene amplification

PubMed Central

Ziesemer, Kirsten A.; Mann, Allison E.; Sankaranarayanan, Krithivasan; Schroeder, Hannes; Ozga, Andrew T.; Brandt, Bernd W.; Zaura, Egija; Waters-Rist, Andrea; Hoogland, Menno; Salazar-García, Domingo C.; Aldenderfer, Mark; Speller, Camilla; Hendy, Jessica; Weston, Darlene A.; MacDonald, Sandy J.; Thomas, Gavin H.; Collins, Matthew J.; Lewis, Cecil M.; Hofman, Corinne; Warinner, Christina

2015-01-01

To date, characterization of ancient oral (dental calculus) and gut (coprolite) microbiota has been primarily accomplished through a metataxonomic approach involving targeted amplification of one or more variable regions in the 16S rRNA gene. Specifically, the V3 region (E. coli 341–534) of this gene has been suggested as an excellent candidate for ancient DNA amplification and microbial community reconstruction. However, in practice this metataxonomic approach often produces highly skewed taxonomic frequency data. In this study, we use non-targeted (shotgun metagenomics) sequencing methods to better understand skewed microbial profiles observed in four ancient dental calculus specimens previously analyzed by amplicon sequencing. Through comparisons of microbial taxonomic counts from paired amplicon (V3 U341F/534R) and shotgun sequencing datasets, we demonstrate that extensive length polymorphisms in the V3 region are a consistent and major cause of differential amplification leading to taxonomic bias in ancient microbiome reconstructions based on amplicon sequencing. We conclude that systematic amplification bias confounds attempts to accurately reconstruct microbiome taxonomic profiles from 16S rRNA V3 amplicon data generated using universal primers. Because in silico analysis indicates that alternative 16S rRNA hypervariable regions will present similar challenges, we advocate for the use of a shotgun metagenomics approach in ancient microbiome reconstructions. PMID:26563586

Label-free thioflavin T/G-quadruplex-based real-time strand displacement amplification for biosensing applications.

PubMed

Du, Yi-Chen; Zhu, Li-Na; Kong, De-Ming

2016-12-15

To promote application of strand-displacement amplification (SDA) techniques in biosensing, a label-free, real-time monitoring strategy for isothermal nucleic acid amplification reactions was designed. G-quadruplex structures were introduced into SDA products using specific recognition of G-quadruplexes by the fluorogenic dye thioflavin T. Performance was good for real-time monitoring of traditional SDA by a linear-amplification mechanism and for exponential cross-triggered SDA amplification. The strategy worked on a commercial real-time PCR instrument, making it suitable for biosensing platforms. As examples, two highly sensitive and specific biosensors were designed for analysis of the activity of uracil-DNA glycosylase (UDG) and the restriction endonuclease EcoRI. Detection limits were 6×10(-5)U/mL for UDG and 0.016U/mL for EcoRI. Detection of corresponding targets in complex matrices such as cell lysates or human serum was also demonstrated. Compared to traditional end-point detection methods, real-time SDA-based approaches have the advantages of simple, fast operation; high sensitivity; low risk of carryover contamination; and very high throughput. The introduction of real-time monitoring strategies may promote application of SDA reactions in biosensor design. Copyright © 2016 Elsevier B.V. All rights reserved.

Diverse Roles of Axonemal Dyneins in Drosophila Auditory Neuron Function and Mechanical Amplification in Hearing.

PubMed

Karak, Somdatta; Jacobs, Julie S; Kittelmann, Maike; Spalthoff, Christian; Katana, Radoslaw; Sivan-Loukianova, Elena; Schon, Michael A; Kernan, Maurice J; Eberl, Daniel F; Göpfert, Martin C

2015-11-26

Much like vertebrate hair cells, the chordotonal sensory neurons that mediate hearing in Drosophila are motile and amplify the mechanical input of the ear. Because the neurons bear mechanosensory primary cilia whose microtubule axonemes display dynein arms, we hypothesized that their motility is powered by dyneins. Here, we describe two axonemal dynein proteins that are required for Drosophila auditory neuron function, localize to their primary cilia, and differently contribute to mechanical amplification in hearing. Promoter fusions revealed that the two axonemal dynein genes Dmdnah3 (=CG17150) and Dmdnai2 (=CG6053) are expressed in chordotonal neurons, including the auditory ones in the fly's ear. Null alleles of both dyneins equally abolished electrical auditory neuron responses, yet whereas mutations in Dmdnah3 facilitated mechanical amplification, amplification was abolished by mutations in Dmdnai2. Epistasis analysis revealed that Dmdnah3 acts downstream of Nan-Iav channels in controlling the amplificatory gain. Dmdnai2, in addition to being required for amplification, was essential for outer dynein arms in auditory neuron cilia. This establishes diverse roles of axonemal dyneins in Drosophila auditory neuron function and links auditory neuron motility to primary cilia and axonemal dyneins. Mutant defects in sperm competition suggest that both dyneins also function in sperm motility.

New Fpg probe chemistry for direct detection of recombinase polymerase amplification on lateral flow strips.

PubMed

Powell, Michael L; Bowler, Frank R; Martinez, Aurore J; Greenwood, Catherine J; Armes, Niall; Piepenburg, Olaf

2018-02-15

Rapid, cost-effective and sensitive detection of nucleic acids has the ability to improve upon current practices employed for pathogen detection in diagnosis of infectious disease and food testing. Furthermore, if assay complexity can be reduced, nucleic acid amplification tests could be deployed in resource-limited and home use scenarios. In this study, we developed a novel Fpg (Formamidopyrimidine DNA glycosylase) probe chemistry, which allows lateral flow detection of amplification in undiluted recombinase polymerase amplification (RPA) reactions. The prototype nucleic acid lateral flow chemistry was applied to a human genomic target (rs1207445), Campylobacter jejuni 16S rDNA and two genetic markers of the important food pathogen E. coli O157:H7. All four assays have an analytical sensitivity between 10 and 100 copies DNA per amplification. Furthermore, the assay is performed with fewer hands-on steps than using the current RPA Nfo lateral flow method as dilution of amplicon is not required for lateral flow analysis. Due to the simplicity of the workflow, we believe that the lateral flow chemistry for direct detection could be readily adapted to a cost-effective single-use consumable, ideal for use in non-laboratory settings. Copyright © 2017. Published by Elsevier Inc.

Enhanced solid-phase recombinase polymerase amplification and electrochemical detection.

PubMed

Del Río, Jonathan Sabaté; Lobato, Ivan Magriñà; Mayboroda, Olena; Katakis, Ioanis; O'Sullivan, Ciara K

2017-05-01

Recombinase polymerase amplification (RPA) is an elegant method for the rapid, isothermal amplification of nucleic acids. Here, we elucidate the optimal surface chemistry for rapid and efficient solid-phase RPA, which was fine-tuned in order to obtain a maximum signal-to-noise ratio, defining the optimal DNA probe density, probe-to-lateral spacer ratio (1:0, 1:1, 1:10 and 1:100) and length of a vertical spacer of the probe as well as investigating the effect of different types of lateral spacers. The use of different labelling strategies was also examined in order to reduce the number of steps required for the analysis, using biotin or horseradish peroxidase-labelled reverse primers. Optimisation of the amplification temperature used and the use of surface blocking agents were also pursued. The combination of these changes facilitated a significantly more rapid amplification and detection protocol, with a lowered limit of detection (LOD) of 1 · 10 -15 M. The optimised protocol was applied to the detection of Francisella tularensis in real samples from hares and a clear correlation with PCR and qPCR results observed and the solid-phase RPA demonstrated to be capable of detecting 500 fM target DNA in real samples. Graphical abstract Relative size of thiolated lateral spacers tested versus the primer and the uvsx recombinase protein.

Copy number variation in the region harboring SOX9 gene in dogs with testicular/ovotesticular disorder of sex development (78,XX; SRY-negative).

PubMed

Marcinkowska-Swojak, Malgorzata; Szczerbal, Izabela; Pausch, Hubert; Nowacka-Woszuk, Joanna; Flisikowski, Krzysztof; Dzimira, Stanislaw; Nizanski, Wojciech; Payan-Carreira, Rita; Fries, Ruedi; Kozlowski, Piotr; Switonski, Marek

2015-10-01

Although the disorder of sex development in dogs with female karyotype (XX DSD) is quite common, its molecular basis is still unclear. Among mutations underlying XX DSD in mammals are duplication of a long sequence upstream of the SOX9 gene (RevSex) and duplication of the SOX9 gene (also observed in dogs). We performed a comparative analysis of 16 XX DSD and 30 control female dogs, using FISH and MLPA approaches. Our study was focused on a region harboring SOX9 and a region orthologous to the human RevSex (CanRevSex), which was located by in silico analysis downstream of SOX9. Two highly polymorphic copy number variable regions (CNVRs): CNVR1 upstream of SOX9 and CNVR2 encompassing CanRevSex were identified. Although none of the detected copy number variants were specific to either affected or control animals, we observed that the average number of copies in CNVR1 was higher in XX DSD. No copy variation of SOX9 was observed. Our extensive studies have excluded duplication of SOX9 as the common cause of XX DSD in analyzed samples. However, it remains possible that the causative mutation is hidden in highly polymorphic CNVR1.

Copy number variation in the region harboring SOX9 gene in dogs with testicular/ovotesticular disorder of sex development (78,XX; SRY-negative)

PubMed Central

Marcinkowska-Swojak, Malgorzata; Szczerbal, Izabela; Pausch, Hubert; Nowacka-Woszuk, Joanna; Flisikowski, Krzysztof; Dzimira, Stanislaw; Nizanski, Wojciech; Payan-Carreira, Rita; Fries, Ruedi; Kozlowski, Piotr; Switonski, Marek

2015-01-01

Although the disorder of sex development in dogs with female karyotype (XX DSD) is quite common, its molecular basis is still unclear. Among mutations underlying XX DSD in mammals are duplication of a long sequence upstream of the SOX9 gene (RevSex) and duplication of the SOX9 gene (also observed in dogs). We performed a comparative analysis of 16 XX DSD and 30 control female dogs, using FISH and MLPA approaches. Our study was focused on a region harboring SOX9 and a region orthologous to the human RevSex (CanRevSex), which was located by in silico analysis downstream of SOX9. Two highly polymorphic copy number variable regions (CNVRs): CNVR1 upstream of SOX9 and CNVR2 encompassing CanRevSex were identified. Although none of the detected copy number variants were specific to either affected or control animals, we observed that the average number of copies in CNVR1 was higher in XX DSD. No copy variation of SOX9 was observed. Our extensive studies have excluded duplication of SOX9 as the common cause of XX DSD in analyzed samples. However, it remains possible that the causative mutation is hidden in highly polymorphic CNVR1. PMID:26423656

Microfluidic "Pouch" Chips for Immunoassays and Nucleic Acid Amplification Tests.

PubMed

Mauk, Michael G; Liu, Changchun; Qiu, Xianbo; Chen, Dafeng; Song, Jinzhao; Bau, Haim H

2017-01-01

Microfluidic cassettes ("chips") for processing and analysis of clinical specimens and other sample types facilitate point-of-care (POC) immunoassays and nucleic acid based amplification tests. These single-use test chips can be self-contained and made amenable to autonomous operation-reducing or eliminating supporting instrumentation-by incorporating laminated, pliable "pouch" and membrane structures for fluid storage, pumping, mixing, and flow control. Materials and methods for integrating flexible pouch compartments and diaphragm valves into hard plastic (e.g., acrylic and polycarbonate) microfluidic "chips" for reagent storage, fluid actuation, and flow control are described. We review several versions of these pouch chips for immunoassay and nucleic acid amplification tests, and describe related fabrication techniques. These protocols thus offer a "toolbox" of methods for storage, pumping, and flow control functions in microfluidic devices.

Digital Microfluidics for Nucleic Acid Amplification

PubMed Central

Veigas, Bruno; Fortunato, Elvira; Martins, Rodrigo; Águas, Hugo; Igreja, Rui; Baptista, Pedro V.

2017-01-01

Digital Microfluidics (DMF) has emerged as a disruptive methodology for the control and manipulation of low volume droplets. In DMF, each droplet acts as a single reactor, which allows for extensive multiparallelization of biological and chemical reactions at a much smaller scale. DMF devices open entirely new and promising pathways for multiplex analysis and reaction occurring in a miniaturized format, thus allowing for healthcare decentralization from major laboratories to point-of-care with accurate, robust and inexpensive molecular diagnostics. Here, we shall focus on DMF platforms specifically designed for nucleic acid amplification, which is key for molecular diagnostics of several diseases and conditions, from pathogen identification to cancer mutations detection. Particular attention will be given to the device architecture, materials and nucleic acid amplification applications in validated settings. PMID:28672827

Extrachromosomal oncogene amplification drives tumor evolution and genetic heterogeneity

PubMed Central

Turner, Kristen M.; Deshpande, Viraj; Beyter, Doruk; Koga, Tomoyuki; Rusert, Jessica; Lee, Catherine; Li, Bin; Arden, Karen; Ren, Bing; Nathanson, David A.; Kornblum, Harley I.; Taylor, Michael D.; Kaushal, Sharmeela; Cavenee, Webster K.; Wechsler-Reya, Robert; Furnari, Frank B.; Vandenberg, Scott R.; Rao, P. Nagesh; Wahl, Geoffrey M.; Bafna, Vineet; Mischel, Paul S.

2017-01-01

Human cells have twenty-three pairs of chromosomes but in cancer, genes can be amplified in chromosomes or in circular extrachromosomal DNA (ECDNA), whose frequency and functional significance are not understood1–4. We performed whole genome sequencing, structural modeling and cytogenetic analyses of 17 different cancer types, including 2572 metaphases, and developed ECdetect to conduct unbiased integrated ECDNA detection and analysis. ECDNA was found in nearly half of human cancers varying by tumor type, but almost never in normal cells. Driver oncogenes were amplified most commonly on ECDNA, elevating transcript level. Mathematical modeling predicted that ECDNA amplification elevates oncogene copy number and increases intratumoral heterogeneity more effectively than chromosomal amplification, which we validated by quantitative analyses of cancer samples. These results suggest that ECDNA contributes to accelerated evolution in cancer. PMID:28178237

Associations between arsenic (+3 oxidation state) methyltransferase (AS3MT) and N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) polymorphisms, arsenic metabolism, and cancer risk in a chilean population.

PubMed

de la Rosa, Rosemarie; Steinmaus, Craig; Akers, Nicholas K; Conde, Lucia; Ferreccio, Catterina; Kalman, David; Zhang, Kevin R; Skibola, Christine F; Smith, Allan H; Zhang, Luoping; Smith, Martyn T

2017-07-01

Inter-individual differences in arsenic metabolism have been linked to arsenic-related disease risks. Arsenic (+3) methyltransferase (AS3MT) is the primary enzyme involved in arsenic metabolism, and we previously demonstrated in vitro that N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) also methylates the toxic inorganic arsenic (iAs) metabolite, monomethylarsonous acid (MMA), to the less toxic dimethylarsonic acid (DMA). Here, we evaluated whether AS3MT and N6AMT1 gene polymorphisms alter arsenic methylation and impact iAs-related cancer risks. We assessed AS3MT and N6AMT1 polymorphisms and urinary arsenic metabolites (%iAs, %MMA, %DMA) in 722 subjects from an arsenic-cancer case-control study in a uniquely exposed area in northern Chile. Polymorphisms were genotyped using a custom designed multiplex, ligation-dependent probe amplification (MLPA) assay for 6 AS3MT SNPs and 14 tag SNPs in the N6AMT1 gene. We found several AS3MT polymorphisms associated with both urinary arsenic metabolite profiles and cancer risk. For example, compared to wildtypes, individuals carrying minor alleles in AS3MT rs3740393 had lower %MMA (mean difference = -1.9%, 95% CI: -3.3, -0.4), higher %DMA (mean difference = 4.0%, 95% CI: 1.5, 6.5), and lower odds ratios for bladder (OR = 0.3; 95% CI: 0.1-0.6) and lung cancer (OR = 0.6; 95% CI: 0.2-1.1). Evidence of interaction was also observed for both lung and bladder cancer between these polymorphisms and elevated historical arsenic exposures. Clear associations were not seen for N6AMT1. These results are the first to demonstrate a direct association between AS3MT polymorphisms and arsenic-related internal cancer risk. This research could help identify subpopulations that are particularly vulnerable to arsenic-related disease. Environ. Mol. Mutagen. 58:411-422, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Molecular subtypes in stage II-III colon cancer defined by genomic instability: early recurrence-risk associated with a high copy-number variation and loss of RUNX3 and CDKN2A.

PubMed

Berg, Marianne; Nordgaard, Oddmund; Kørner, Hartwig; Oltedal, Satu; Smaaland, Rune; Søreide, Jon Arne; Søreide, Kjetil

2015-01-01

We sought to investigate various molecular subtypes defined by genomic instability that may be related to early death and recurrence in colon cancer. We sought to investigate various molecular subtypes defined by instability at microsatellites (MSI), changes in methylation patterns (CpG island methylator phenotype, CIMP) or copy number variation (CNV) in 8 genes. Stage II-III colon cancers (n = 64) were investigated by methylation-specific multiplex ligated probe amplification (MS-MLPA). Correlation of CNV, CIMP and MSI, with mutations in KRAS and BRAFV600E were assessed for overlap in molecular subtypes and early recurrence risk by uni- and multivariate regression. The CIMP phenotype occurred in 34% (22/64) and MSI in 27% (16/60) of the tumors, with noted CIMP/MSI overlap. Among the molecular subtypes, a high CNV phenotype had an associated odds ratio (OR) for recurrence of 3.2 (95% CI 1.1-9.3; P = 0.026). Losses of CACNA1G (OR of 2.9, 95% CI 1.4-6.0; P = 0.001), IGF2 (OR of 4.3, 95% CI 1.1-15.8; P = 0.007), CDKN2A (p16) (OR of 2.0, 95% CI 1.1-3.6; P = 0.024), and RUNX3 (OR of 3.4, 95% CI 1.3-8.7; P = 0.002) were associated with early recurrence, while MSI, CIMP, KRAS or BRAF V600E mutations were not. The CNV was significantly higher in deceased patients (CNV in 6 of 8) compared to survivors (CNV in 3 of 8). Only stage and loss of RUNX3 and CDKN2A were significant in the multivariable risk-model for early recurrence. A high copy number variation phenotype is a strong predictor of early recurrence and death, and may indicate a dose-dependent relationship between genetic instability and outcome. Loss of tumor suppressors RUNX3 and CDKN2A were related to recurrence-risk and warrants further investigation.

An improved strategy and a useful housekeeping gene for RNA analysis from formalin-fixed, paraffin-embedded tissues by PCR.

PubMed

Finke, J; Fritzen, R; Ternes, P; Lange, W; Dölken, G

1993-03-01

Specific amplification of nucleic acid sequences by PCR has been extensively used for the detection of gene rearrangements and gene expression. Although successful amplification of DNA sequences has been carried out with DNA prepared from formalin-fixed, paraffin-embedded (FFPE) tissues, there are only a few reports regarding RNA analysis in this kind of material. We describe a procedure for RNA extraction from different types of FFPE tissues, involving digestion with proteinase K followed by guanidinium-thiocyanate acid phenol extraction and DNase I digestion. These RNA preparations are suitable for PCR analysis of mRNA and even of intronless genes. Furthermore, the universally expressed porphobilinogen deaminase mRNA proved to be useful as a positive control because of the lack of pseudogenes.

Quantitative high-resolution genomic analysis of single cancer cells.

PubMed

Hannemann, Juliane; Meyer-Staeckling, Sönke; Kemming, Dirk; Alpers, Iris; Joosse, Simon A; Pospisil, Heike; Kurtz, Stefan; Görndt, Jennifer; Püschel, Klaus; Riethdorf, Sabine; Pantel, Klaus; Brandt, Burkhard

2011-01-01

During cancer progression, specific genomic aberrations arise that can determine the scope of the disease and can be used as predictive or prognostic markers. The detection of specific gene amplifications or deletions in single blood-borne or disseminated tumour cells that may give rise to the development of metastases is of great clinical interest but technically challenging. In this study, we present a method for quantitative high-resolution genomic analysis of single cells. Cells were isolated under permanent microscopic control followed by high-fidelity whole genome amplification and subsequent analyses by fine tiling array-CGH and qPCR. The assay was applied to single breast cancer cells to analyze the chromosomal region centred by the therapeutical relevant EGFR gene. This method allows precise quantitative analysis of copy number variations in single cell diagnostics.

Parametric amplification in quasi-PT symmetric coupled waveguide structures

NASA Astrophysics Data System (ADS)

Zhong, Q.; Ahmed, A.; Dadap, J. I.; Osgood, R. M., Jr.; El-Ganainy, R.

2016-12-01

The concept of non-Hermitian parametric amplification was recently proposed as a means to achieve an efficient energy conversion throughout the process of nonlinear three wave mixing in the absence of phase matching. Here we investigate this effect in a waveguide coupler arrangement whose characteristics are tailored to introduce passive PT symmetry only for the idler component. By means of analytical solutions and numerical analysis, we demonstrate the utility of these novel schemes and obtain the optimal design conditions for these devices.

Unknown sequence amplification: Application to in vitro genome walking in Chlamydia trachomatis L2

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

Copley, C.G.; Boot, C.; Bundell, K.

1991-01-01

A recently described technique, Chemical Genetics' unknown sequence amplification method, which requires only one specific oligonucleotide, has broadened the applicability of the polymerase chain reaction to DNA of unknown sequence. The authors have adapted this technique to the study of the genome of Chlamydia trachomatis, an obligate intracellular bacterium, and describe modifications that significantly improve the utility of this approach. These techniques allow for rapid genomic analysis entirely in vitro, using DNA of limited quantity of purity.

A simple analytical model for signal amplification by reversible exchange (SABRE) process.

PubMed

Barskiy, Danila A; Pravdivtsev, Andrey N; Ivanov, Konstantin L; Kovtunov, Kirill V; Koptyug, Igor V

2016-01-07

We demonstrate an analytical model for the description of the signal amplification by reversible exchange (SABRE) process. The model relies on a combined analysis of chemical kinetics and the evolution of the nuclear spin system during the hyperpolarization process. The presented model for the first time provides rationale for deciding which system parameters (i.e. J-couplings, relaxation rates, reaction rate constants) have to be optimized in order to achieve higher signal enhancement for a substrate of interest in SABRE experiments.

High-Resolution Mapping of Structural Mutations in Prostate Cancer with Single Nucleotide Polymorphism Arrays

DTIC Science & Technology

2006-11-01

study of the NCI60 panel of cancer cell lines [39]. More recently, amplifications of NOTCH3 were noted in ovarian tumors by an SNP array analysis...and the functional role of NOTCH3 was suggested by the ability to suppress cell proliferation by inhibiting NOTCH3 [40]. Allele-specific copy...Identified and functionally validated the oncogene MITF. 40 Park JT, Li M, Nakayama K, et al. Notch3 gene amplification in ovarian cancer. Cancer Res

Discovery of natural gain amplification in the 10 muon m CO2 laser bands on Mars: The first definite natural laser

NASA Technical Reports Server (NTRS)

Mumma, M.; Buhl, D.; Chin, G.; Deming, D.; Espenak, F.; Kostiuk, T.; Zipoy, D.

1980-01-01

Fully resolved intensity profiles of various lines in the CO2 bands at 9.4 micrometers and 10.4 micrometers were measured on Mars using an infrared heterodyne spectrometer. Analysis of the line shapes shows that the Mars atmosphere exhibits positive gain on these lines, providing the first definite detection of natural optical gain amplification and enabling identification of these lines as the first definite natural laser ever discovered.

Stress-induced rearrangement of Fusarium retrotransposon sequences.

PubMed

Anaya, N; Roncero, M I

1996-11-27

Rearrangement of fusarium oxysporum retrotransposon skippy was induced by growth in the presence of potassium chlorate. Three fungal strains, one sensitive to chlorate (Co60) and two resistant to chlorate and deficient for nitrate reductase (Co65 and Co94), were studied by Southern analysis of their genomic DNA. Polymorphism was detected in their hybridization banding pattern, relative to the wild type grown in the absence of chlorate, using various enzymes with or without restriction sites within the retrotransposon. Results were consistent with the assumption that three different events had occurred in strain Co60: genomic amplification of skippy yielding tandem arrays of the element, generation of new skippy sequences, and deletion of skippy sequences. Amplification of Co60 genomic DNA using the polymerase chain reaction and divergent primers derived from the retrotransposon generated a new band, corresponding to one long terminal repeat plus flanking sequences, that was not present in the wild-type strain. Molecular analysis of nitrate reductase-deficient mutants showed that generation and deletion of skippy sequences, but not genomic amplification in tandem repeats, had occurred in their genomes.

A Multiplex Snapback Primer System for the Enrichment and Detection of JAK2 V617F and MPL W515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

PubMed Central

Zhang, Yunqing; Zhang, Xinju; Xu, Xiao; Kang, Zhihua; Li, Shibao; Zhang, Chen; Su, Bing

2014-01-01

A multiplex snapback primer system was developed for the simultaneous detection of JAK2 V617F and MPL W515L/K mutations in Philadelphia chromosome- (Ph-) negative myeloproliferative neoplasms (MPNs). The multiplex system comprises two snapback versus limiting primer sets for JAK2 and MPL mutation enrichment and detection, respectively. Linear-After exponential (LATE) PCR strategy was employed for the primer design to maximize the amplification efficiency of the system. Low ionic strength buffer and rapid PCR protocol allowed for selective amplification of the mutant alleles. Amplification products were analyzed by melting curve analysis for mutation identification. The multiplex system archived 0.1% mutation load sensitivity and <5% coefficient of variation inter-/intra-assay reproducibility. 120 clinical samples were tested by the multiplex snapback primer assay, and verified with amplification refractory system (ARMS), quantitative PCR (qPCR) and Sanger sequencing method. The multiplex system, with a favored versatility, provided the molecular diagnosis of Ph-negative MPNs with a suitable implement and simplified the genetic test process. PMID:24729973

Recombinase-based isothermal amplification of nucleic acids with self-avoiding molecular recognition systems (SAMRS).

PubMed

Sharma, Nidhi; Hoshika, Shuichi; Hutter, Daniel; Bradley, Kevin M; Benner, Steven A

2014-10-13

Recombinase polymerase amplification (RPA) is an isothermal method to amplify nucleic acid sequences without the temperature cycling that classical PCR uses. Instead of using heat to denature the DNA duplex, RPA uses recombination enzymes to swap single-stranded primers into the duplex DNA product; these are then extended using a strand-displacing polymerase to complete the cycle. Because RPA runs at low temperatures, it never forces the system to recreate base-pairs following Watson-Crick rules, and therefore it produces undesired products that impede the amplification of the desired product, complicating downstream analysis. Herein, we show that most of these undesired side products can be avoided if the primers contain components of a self-avoiding molecular recognition system (SAMRS). Given the precision that is necessary in the recombination systems for them to function biologically, it is surprising that they accept SAMRS. SAMRS-RPA is expected to be a powerful tool within the range of amplification techniques available to scientists. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

An exo probe-based recombinase polymerase amplification assay for the rapid detection of porcine parvovirus.

PubMed

Wang, Jian-Chang; Liu, Li-Bing; Han, Qing-An; Wang, Jin-Feng; Yuan, Wan-Zhe

2017-10-01

Recombinase polymerase amplification (RPA), an isothermal amplification technology, has been developed as an alternative to PCR in pathogen detection. A real-time RPA assay (rt-RPA) was developed to detect the porcine parvovirus (PPV) using primers and exo probe specific for the VP2 gene. The amplification was performed at 39°C for 20min. There was no cross-reaction with other pathogens tested. Using the recombinant plasmid pPPV-VP2 as template, the analytical sensitivity was 103 copies. The assay performance was evaluated by testing 115 field samples by rt-RPA and a real-time PCR assay. The diagnostic agreement between assays was 100%, and PPV DNA was detected in 94 samples. The R 2 value of rt-RPA and real-time PCR was 0.909 by linear regression analysis. The developed rt-RPA assay provides a useful alternative tool for rapid, simple and reliable detection of PPV in diagnostic laboratories and at point-of-care, especially in remote and rural areas. Copyright © 2017 Elsevier B.V. All rights reserved.

Exponential isothermal amplification of nucleic acids and amplified assays for proteins, cells, and enzyme activities.

PubMed

Reid, Michael S; Le, X Chris; Zhang, Hongquan

2018-04-27

Isothermal exponential amplification techniques, such as strand-displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA), and recombinase polymerase amplification (RPA), have great potential for on-site, point-of-care, and in-situ assay applications. These amplification techniques eliminate the need for temperature cycling required for polymerase chain reaction (PCR) while achieving comparable amplification yield. We highlight here recent advances in exponential amplification reaction (EXPAR) for the detection of nucleic acids, proteins, enzyme activities, cells, and metal ions. We discuss design strategies, enzyme reactions, detection techniques, and key features. Incorporation of fluorescence, colorimetric, chemiluminescence, Raman, and electrochemical approaches enables highly sensitive detection of a variety of targets. Remaining issues, such as undesirable background amplification resulting from non-specific template interactions, must be addressed to further improve isothermal and exponential amplification techniques. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fluidics platform and method for sample preparation and analysis

DOEpatents

Benner, W. Henry; Dzenitis, John M.; Bennet, William J.; Baker, Brian R.

2014-08-19

Herein provided are fluidics platform and method for sample preparation and analysis. The fluidics platform is capable of analyzing DNA from blood samples using amplification assays such as polymerase-chain-reaction assays and loop-mediated-isothermal-amplification assays. The fluidics platform can also be used for other types of assays and analyzes. In some embodiments, a sample in a sealed tube can be inserted directly. The following isolation, detection, and analyzes can be performed without a user's intervention. The disclosed platform may also comprises a sample preparation system with a magnetic actuator, a heater, and an air-drying mechanism, and fluid manipulation processes for extraction, washing, elution, assay assembly, assay detection, and cleaning after reactions and between samples.

Clinical significance of ESR1 gene copy number changes in breast cancer as measured by fluorescence in situ hybridisation.

PubMed

Lin, Ching-Hung; Liu, Jacqueline M; Lu, Yen-Shen; Lan, Chieh; Lee, Wei-Chung; Kuo, Kuan-Ting; Wang, Chung-Chieh; Chang, Dwan-Ying; Huang, Chiun-Sheng; Cheng, Ann-Lii

2013-02-01

The ESR1 gene encodes for oestrogen receptor (ER) α, which plays a crucial role in mammary carcinogenesis and clinical outcome in patients with breast cancer. However, the clinical significance of the ESR1 gene copy number change for breast cancer has not been clarified. ESR1 gene copy number was determined by fluorescence in situ hybridisation (FISH) on tissue sections. A minimum of 20 tumour cells were counted per section, and a FISH ratio of ESR1 gene to CEP6 ≥ 2.0 was considered ESR1 amplification. A ratio >1.2 but <2.0 was considered ESR1 gain. The ESR1 copy number was further measured by quantitative real-time PCR (Q-PCR) with ASXL2 as a reference. FISH revealed ESR1 amplification in six cases (4.0%) and ESR1 gain in 13 cases (8.7%) from a total of 150 cases. ESR1 gain and amplification were more common in older patients (p<0.001), and correlated well with ER protein expression (p=0.03) measured by immunohistochemistry, and ESR1 copy number (p<0.001) measured by Q-PCR. Furthermore, the multivariate analysis revealed that ESR1 amplification was associated with a shorter disease-free survival (HR=5.56, p=0.03) and a shorter overall survival (HR=5.11, p=0.04). In general, the frequency of ESR1 amplification in breast cancer is low when measured by FISH in large sections. ESR1 gain and amplification in breast cancer may be associated with older age and poorer outcomes.

Frequent amplification of PTP1B is associated with poor survival of gastric cancer patients.

PubMed

Wang, Na; She, Junjun; Liu, Wei; Shi, Jing; Yang, Qi; Shi, Bingyin; Hou, Peng

2015-01-01

The protein tyrosine phosphatase 1B (PTP1B), a non-transmembrane protein tyrosine phosphatase, has been implicated in gastric pathogenesis. Several lines of recent evidences have shown that PTP1B is highly amplified in breast and prostate cancers. The aim of this study was to investigate PTP1B amplification in gastric cancer and its association with poor prognosis of gastric cancer patients, and further determine the role of PTP1B in gastric tumorigenesis. Our data demonstrated that PTP1B was significantly up-regulated in gastric cancer tissues as compared with matched normal gastric tissues by using quantitative RT-PCR (qRT-PCR) assay. In addition, copy number analysis showed that PTP1B was amplified in 68/131 (51.9%) gastric cancer cases, whereas no amplification was found in the control subjects. Notably, PTP1B amplification was positively associated with its protein expression, and was significantly related to poor survival of gastric cancer patients. Knocking down PTP1B expression in gastric cancer cells significantly inhibited cell proliferation, colony formation, migration and invasion, and induced cell cycle arrested and apoptosis. Mechanically, PTP1B promotes gastric cancer cell proliferation, survival and invasiveness through modulating Src-related signaling pathways, such as Src/Ras/MAPK and Src/phosphatidylinositol-3-kinase (PI3K)/Akt pathways. Collectively, our data demonstrated frequent overexpression and amplification PTP1B in gastric cancer, and further determined the oncogenic role of PTP1B in gastric carcinogenesis. Importantly, PTP1B amplification predicts poor survival of gastric cancer patients.

Highly sensitive and selective microRNA detection based on DNA-bio-bar-code and enzyme-assisted strand cycle exponential signal amplification.

PubMed

Dong, Haifeng; Meng, Xiangdan; Dai, Wenhao; Cao, Yu; Lu, Huiting; Zhou, Shufeng; Zhang, Xueji

2015-04-21

Herein, a highly sensitive and selective microRNA (miRNA) detection strategy using DNA-bio-bar-code amplification (BCA) and Nb·BbvCI nicking enzyme-assisted strand cycle for exponential signal amplification was designed. The DNA-BCA system contains a locked nucleic acid (LNA) modified DNA probe for improving hybridization efficiency, while a signal reported molecular beacon (MB) with an endonuclease recognition site was designed for strand cycle amplification. In the presence of target miRNA, the oligonucleotides functionalized magnetic nanoprobe (MNP-DNA) and gold nanoprobe (AuNP-DNA) with numerous reported probes (RP) can hybridize with target miRNA, respectively, to form a sandwich structure. After sandwich structures were separated from the solution by the magnetic field, the RP were released under high temperature to recognize the MB and cleaved the hairpin DNA to induce the dissociation of RP. The dissociated RP then triggered the next strand cycle to produce exponential fluorescent signal amplification for miRNA detection. Under optimized conditions, the exponential signal amplification system shows a good linear range of 6 orders of magnitude (from 0.3 pM to 3 aM) with limit of detection (LOD) down to 52.5 zM, while the sandwich structure renders the system with high selectivity. Meanwhile, the feasibility of the proposed strategy for cell miRNA detection was confirmed by analyzing miRNA-21 in HeLa lysates. Given the high-performance for miRNA analysis, the strategy has a promising application in biological detection and in clinical diagnosis.

Combining active and passive seismic methods for the characterization of urban sites in Cairo, Egypt

NASA Astrophysics Data System (ADS)

Adly, Ashraf; Poggi, Valerio; Fäh, Donat; Hassoup, Awad; Omran, Awad

2017-07-01

The geology at Kottamiya, Rehab City and Zahraa-Madinat-Nasr to the East of Cairo (Egypt) is composed of low-velocity sediments on top of a rigid rock basement. Such sediments include the loose sands of the Gebel Ahmar formation, marl and shales of Maadi formation, in addition to sparse quaternary soil covers. Due to the contrast of the seismic impedance with the underlying bedrock, these soft sediments have the potential of considerably amplifying the ground motion during an earthquake. For the evaluation of site-specific seismic hazard, we computed the seismic site response in these areas by developing 1-D velocity models and derived average seismic velocities, including Vs30. To do that, we applied different active and passive source techniques, including the horizontal to vertical Fourier spectral ratio of ambient vibration recordings and multichannel analysis of artificially generated surface waves. A set of models representing the velocity structure of the site is then obtained by combined inversion of Rayleigh wave dispersion curves and ellipticity functions. While dispersion curves are used to constrain the uppermost low-velocity part of the soil profile, ellipticity helps in resolving the structure at the depth of the sediment-bedrock interface. From the retrieved velocity models, numerical ground-motion amplification is finally derived using 1-D SH-wave transfer function. We account for uncertainty in amplification by using a statistical model that accounts for the misfit of all the inverted velocity profiles. The study reveals that the different sites experience an important frequency-dependent amplification, with largest amplification occurring at the resonance frequencies of the sites. Amplification up to a factor of 5 is found, with some variability depending on the soil type (Vs30 ranges between 340 and 415 m s-2). Moreover, amplification is expected in the frequency range that is important for buildings (0.8-10 Hz), which is additional confirmation for the need of microzonation analysis of the area. The obtained results will be used for the development of a new seismic hazard model.

Seismic loading due to mining: Wave amplification and vibration of structures

NASA Astrophysics Data System (ADS)

Lokmane, N.; Semblat, J.-F.; Bonnet, G.; Driad, L.; Duval, A.-M.

2003-04-01

A vibration induced by the ground motion, whatever its source is, can in certain cases damage surface structures. The scientific works allowing the analysis of this phenomenon are numerous and well established. However, they generally concern dynamic motion from real earthquakes. The goal of this work is to analyse the impact of shaking induced by mining on the structures located on the surface. The methods allowing to assess the consequences of earthquakes of strong amplitude are well established, when the methodology to estimate the consequences of moderate but frequent dynamic loadings is not well defined. The mining such as the "Houillères de Bassin du Centre et du Midi" (HBCM) involves vibrations which are regularly felt on the surface. An extracting work of coal generates shaking similar to those caused by earthquakes (standard waves and laws of propagation) but of rather low magnitude. On the other hand, their recurrent feature makes the vibrations more harmful. A three-dimensional modeling of standard structure of the site was carried out. The first results show that the fundamental frequencies of this structure are compatible with the amplification measurements carried out on site. The motion amplification in the surface soil layers is then analyzed. The modeling works are performed on the surface soil layers of Gardanne (Provence), where measurements of microtremors were performed. The analysis of H/V spectral ratio (horizontal on vertical component) indeed makes it possible to characterize the fundamental frequencies of the surface soil layers. This experiment also allows to characterize local evolution of amplification induced by the topmost soil layers. The numerical methods we consider to model seismic wave propagation and amplification in the site, is the Boundary Element Methode (BEM) The main advantage of the boundary element method is to get rid of artificial truncations of the mesh (as in Finite Element Method) in the case of infinite medium. For dynamic problems, these truncations lead to spurious wave reflections giving a numerical error in the solution. The experimental and numerical (BEM) results on surface motion amplification are then compared in terms of both amplitude and frequency range.

Evaluation of quantification methods for real-time PCR minor groove binding hybridization probe assays.

PubMed

Durtschi, Jacob D; Stevenson, Jeffery; Hymas, Weston; Voelkerding, Karl V

2007-02-01

Real-time PCR data analysis for quantification has been the subject of many studies aimed at the identification of new and improved quantification methods. Several analysis methods have been proposed as superior alternatives to the common variations of the threshold crossing method. Notably, sigmoidal and exponential curve fit methods have been proposed. However, these studies have primarily analyzed real-time PCR with intercalating dyes such as SYBR Green. Clinical real-time PCR assays, in contrast, often employ fluorescent probes whose real-time amplification fluorescence curves differ from those of intercalating dyes. In the current study, we compared four analysis methods related to recent literature: two versions of the threshold crossing method, a second derivative maximum method, and a sigmoidal curve fit method. These methods were applied to a clinically relevant real-time human herpes virus type 6 (HHV6) PCR assay that used a minor groove binding (MGB) Eclipse hybridization probe as well as an Epstein-Barr virus (EBV) PCR assay that used an MGB Pleiades hybridization probe. We found that the crossing threshold method yielded more precise results when analyzing the HHV6 assay, which was characterized by lower signal/noise and less developed amplification curve plateaus. In contrast, the EBV assay, characterized by greater signal/noise and amplification curves with plateau regions similar to those observed with intercalating dyes, gave results with statistically similar precision by all four analysis methods.

Strong ground motion in Port-au-Prince, Haiti, during the M7.0 12 January 2010 Haiti earthquake

USGS Publications Warehouse

Hough, Susan E; Given, Doug; Taniguchi, Tomoyo; Altidor, J.R.; Anglade, Dieuseul; Mildor, S-L.

2011-01-01

No strong motion records are available for the 12 January 2010 M7.0 Haiti earthquake. We use aftershock recordings as well as detailed considerations of damage to estimate the severity and distribution of mainshock shaking in Port-au-Prince. Relative to ground motions at a hard - rock reference site, peak accelerations are amplified by a factor of approximately 2 at sites on low-lying deposits in central Port-au-Prince and by a factor of 2.5 - 3.5 on a steep foothill ridge in the southern Port-au-Prince metropolitan region. The observed amplification along the ridge cannot be explained by sediment - induced amplification , but is consistent with predicted topographic amplification by a steep, narrow ridge. Although damage was largely a consequence of poor construction , the damage pattern inferred from analysis of remote sensing imagery provides evidence for a correspondence between small-scale (0.1 - 1.0 km) topographic relief and high damage. Mainshock shaking intensity can be estimated crudely from a consideration of macroseismic effects . We further present detailed, quantitative analysis of the marks left on a tile floor by an industrial battery rack displaced during the mainshock, at the location where we observed the highest weak motion amplifications. Results of this analysis indicate that mainshock shaking was significantly higher at this location (~0.5 g , MMI VIII) relative to the shaking in parts of Port-au-Prince that experienced light damage. Our results further illustrate how observations of rigid body horizontal displacement during earthquakes can be used to estimate peak ground accelerations in the absence of instrumental data .

Chronic low back pain and the transdiagnostic process: How do cognitive and emotional dysregulations contribute to the intensity of risk factors and pain?

PubMed

Le Borgne, Margaux; Boudoukha, Abdel Halim; Petit, Audrey; Roquelaure, Yves

2017-10-01

Based on a transdiagnostic approach, this study assesses the impact of cognitive and emotional processes (difficulties in emotional regulation, impulsiveness, rumination and somatosensory amplification) on the psychological risk factors of chronic low-back pain. The study was carried out with 256 patients with chronic low-back pain. All the variables were assessed through a booklet of 10 validated questionnaires. Multiple regression analysis and moderation analysis were performed. Predictors included in multiple regression models explain 3%-42% (adjusted R 2 ) of the variance in psychological risk factors. Moreover, analyses reveal a significant moderator effect of somatosensory amplification on the link between fear-avoidance beliefs linked to work and pain intensity (F (3;250) =12.33; p=.00), of somatosensory amplification and brooding on the link between depression and functional repercussions (FR) on everyday life (F (3;252) =13.36; p=.000; F (1;252) =12.42; p=.00), of the reflection dimension of rumination on the link between the helplessness dimension of catastrophizing and FRs on sociability (F (3;252) =37.02; p=.00). There is also a moderation analysis with a significant trend concerning the lack of emotional awareness and the difficulties in controlling impulsive behaviours. Our results indicate an important role of some dimensions of difficulties in emotional regulation, somatosensory amplification and rumination in the increase in negative affects and dysfunctional beliefs, and in the links between those psychological risk factors and pain/disability. This study identifies some cognitive and emotional dysregulations substantially involved in work-related chronic pain. This contribute to put in place psychotherapeutic protocols to tackle these deficits and dysregulations in a relevant way. Copyright © 2017 Scandinavian Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

Identification of the genetic and clinical characteristics of neuroblastomas using genome-wide analysis.

PubMed

Uryu, Kumiko; Nishimura, Riki; Kataoka, Keisuke; Sato, Yusuke; Nakazawa, Atsuko; Suzuki, Hiromichi; Yoshida, Kenichi; Seki, Masafumi; Hiwatari, Mitsuteru; Isobe, Tomoya; Shiraishi, Yuichi; Chiba, Kenichi; Tanaka, Hiroko; Miyano, Satoru; Koh, Katsuyoshi; Hanada, Ryoji; Oka, Akira; Hayashi, Yasuhide; Ohira, Miki; Kamijo, Takehiko; Nagase, Hiroki; Takimoto, Tetsuya; Tajiri, Tatsuro; Nakagawara, Akira; Ogawa, Seishi; Takita, Junko

2017-12-08

To provide better insight into the genetic signatures of neuroblastomas, we analyzed 500 neuroblastomas (included specimens from JNBSG) using targeted-deep sequencing for 10 neuroblastoma-related genes and SNP arrays analysis. ALK expression was evaluated using immunohistochemical analysis in 259 samples. Based on genetic alterations, the following 6 subgroups were identified: groups A ( ALK abnormalities), B (other gene mutations), C ( MYCN amplification), D (11q loss of heterozygosity [LOH]), E (at least 1 copy number variants), and F (no genetic changes). Groups A to D showed advanced disease and poor prognosis, whereas groups E and F showed excellent prognosis. Intriguingly, in group A, MYCN amplification was not a significant prognostic marker, while high ALK expression was a relevant indicator for prognosis ( P = 0.033). Notably, the co-existence of MYCN amplification and 1p LOH, and the co-deletion of 3p and 11q were significant predictors of relapse ( P = 0.043 and P = 0.040). Additionally, 6q/8p LOH and 17q gain were promising indicators of survival in patients older than 5 years, and 1p, 4p, and 11q LOH potentially contributed to outcome prediction in the intermediate-risk group. Our genetic overview clarifies the clinical impact of genetic signatures and aids in the better understanding of genetic basis of neuroblastoma.

EGFR gene amplification is relatively common and associates with outcome in intestinal adenocarcinoma of the stomach, gastro-oesophageal junction and distal oesophagus.

PubMed

Birkman, Eva-Maria; Ålgars, Annika; Lintunen, Minnamaija; Ristamäki, Raija; Sundström, Jari; Carpén, Olli

2016-07-07

Approximately 50 % of gastric adenocarcinomas belong to a molecular subgroup characterised by chromosomal instability and a strong association with the intestinal histological subtype. This subgroup typically contains alterations in the receptor tyrosine kinase-RAS pathway, for example EGFR or HER2 gene amplifications leading to protein overexpression. In clinical practice, HER2 overexpressing metastatic gastric cancer is known to respond to treatment with anti-HER2 antibodies. By contrast, anti-EGFR antibodies have not been able to provide survival benefit in clinical trials, which, however, have not included patient selection based on the histological subtype or EGFR gene copy number analysis of the tumours. To examine the role of EGFR as a potential biomarker, we studied the prevalence, clinicopathological associations as well as prognostic role of EGFR and HER2 expression and gene amplification in intestinal adenocarcinomas of the stomach, gastro-oesophageal junction and distal oesophagus. Tissue samples from 220 patients were analysed with EGFR and HER2 immunohistochemistry. Those samples with moderate/strong staining intensity were further analysed with silver in situ hybridization to quantify gene copy numbers. The results were associated with clinical patient characteristics and survival. Moderate/strong EGFR protein expression was found in 72/220 (32.7 %) and EGFR gene amplification in 31/220 (14.1 %) of the tumours, while moderate/strong HER2 protein expression was detected in 31/220 (14.1 %) and HER2 gene amplification in 29/220 (13.2 %) of the tumours. EGFR and HER2 genes were co-amplified in eight tumours (3.6 %). EGFR gene amplification was more common in tumours of distal oesophagus/gastro-oesophageal junction/cardia than in those of gastric corpus (p = 0.013). It was associated with shortened time to cancer recurrence (p = 0.026) and cancer specific survival (p = 0.033). EGFR gene amplification is relatively common in intestinal adenocarcinomas and associates with decreased survival. It is rarely concurrent with HER2 gene amplification, suggesting that anti-EGFR therapies might be applicable to some patients not eligible for anti-HER2 treatment. Analogous to HER2 testing, determination of EGFR gene amplification status in concert with immunohistochemistry could improve the specificity of patient selection when investigating the possible benefits of anti-EGFR therapies in the treatment of gastric adenocarcinomas.

A novel real time PCR assay using melt curve analysis for ivory identification.

PubMed

Kitpipit, Thitika; Penchart, Kitichaya; Ouithavon, Kanita; Satasook, Chutamas; Linacre, Adrian; Thanakiatkrai, Phuvadol

2016-10-01

Demand for ivory and expansion of human settlements have resulted in a rapid decline in the number of elephants. Enforcement of local and international laws and regulations requires identification of the species from which any ivory, or ivory products, originated. Further geographical assignment of the dead elephant from which the ivory was taken can assist in forensic investigations. In this study, a real-time PCR assay using melt curve analysis was developed and fully validated for forensic use. The presence or absence of three Elephantidae-specific and elephant species-specific melting peaks was used to identify the elephant species. Using 141 blood and ivory samples from the three extant elephant species, the assay demonstrated very high reproducibility and accuracy. The limit of detection was as low as 0.031ng of input DNA for conventional amplification and 0.002ng for nested amplification. Both DNA concentrations are typically encountered in forensic casework, especially for degraded samples. No cross-reactivity was observed for non-target species. Evaluation of direct amplification and nested amplification demonstrated the assay's flexibility and capability of analyzing low-template DNA samples and aged samples. Additionally, blind trial testing showed the assay's suitability application in real casework. In conclusion, wildlife forensic laboratories could use this novel, quick, and low-cost assay to help combat the continuing poaching crises leading to the collapse of elephant numbers in the wild. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A Paper-Based Device for Performing Loop-Mediated Isothermal Amplification with Real-Time Simultaneous Detection of Multiple DNA Targets.

PubMed

Seok, Youngung; Joung, Hyou-Arm; Byun, Ju-Young; Jeon, Hyo-Sung; Shin, Su Jeong; Kim, Sanghyo; Shin, Young-Beom; Han, Hyung Soo; Kim, Min-Gon

2017-01-01

Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 10 2 -10 5 copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing.

A Paper-Based Device for Performing Loop-Mediated Isothermal Amplification with Real-Time Simultaneous Detection of Multiple DNA Targets

PubMed Central

Seok, Youngung; Joung, Hyou-Arm; Byun, Ju-Young; Jeon, Hyo-Sung; Shin, Su Jeong; Kim, Sanghyo; Shin, Young-Beom; Han, Hyung Soo; Kim, Min-Gon

2017-01-01

Paper-based diagnostic devices have many advantages as a one of the multiple diagnostic test platforms for point-of-care (POC) testing because they have simplicity, portability, and cost-effectiveness. However, despite high sensitivity and specificity of nucleic acid testing (NAT), the development of NAT based on a paper platform has not progressed as much as the others because various specific conditions for nucleic acid amplification reactions such as pH, buffer components, and temperature, inhibitions from technical differences of paper-based device. Here, we propose a paper-based device for performing loop-mediated isothermal amplification (LAMP) with real-time simultaneous detection of multiple DNA targets. We determined the optimal chemical components to enable dry conditions for the LAMP reaction without lyophilization or other techniques. We also devised the simple paper device structure by sequentially stacking functional layers, and employed a newly discovered property of hydroxynaphthol blue fluorescence to analyze real-time LAMP signals in the paper device. This proposed platform allowed analysis of three different meningitis DNA samples in a single device with single-step operation. This LAMP-based multiple diagnostic device has potential for real-time analysis with quantitative detection of 102-105 copies of genomic DNA. Furthermore, we propose the transformation of DNA amplification devices to a simple and affordable paper system approach with great potential for realizing a paper-based NAT system for POC testing. PMID:28740546

A novel CMOS image sensor system for quantitative loop-mediated isothermal amplification assays to detect food-borne pathogens.

PubMed

Wang, Tiantian; Kim, Sanghyo; An, Jeong Ho

2017-02-01

Loop-mediated isothermal amplification (LAMP) is considered as one of the alternatives to the conventional PCR and it is an inexpensive portable diagnostic system with minimal power consumption. The present work describes the application of LAMP in real-time photon detection and quantitative analysis of nucleic acids integrated with a disposable complementary-metal-oxide semiconductor (CMOS) image sensor. This novel system works as an amplification-coupled detection platform, relying on a CMOS image sensor, with the aid of a computerized circuitry controller for the temperature and light sources. The CMOS image sensor captures the light which is passing through the sensor surface and converts into digital units using an analog-to-digital converter (ADC). This new system monitors the real-time photon variation, caused by the color changes during amplification. Escherichia coli O157 was used as a proof-of-concept target for quantitative analysis, and compared with the results for Staphylococcus aureus and Salmonella enterica to confirm the efficiency of the system. The system detected various DNA concentrations of E. coli O157 in a short time (45min), with a detection limit of 10fg/μL. The low-cost, simple, and compact design, with low power consumption, represents a significant advance in the development of a portable, sensitive, user-friendly, real-time, and quantitative analytic tools for point-of-care diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Dual-colour HER2/chromosome 17 chromogenic in situ hybridisation enables accurate assessment of HER2 genomic status in ovarian tumours.

PubMed

Yan, Benedict; Choo, Shoa Nian; Mulyadi, Patricia; Srivastava, Supriya; Ong, Chee Wee; Yong, Kol Jia; Putti, Thomas; Salto-Tellez, Manuel; Lim, Gkeok Stzuan Diana

2011-12-01

Ovarian cancer is a leading cause of gynaecological cancer-related morbidity and mortality. There has been increasing interest in the potential utility of anti-human epidermal growth factor receptor 2 (anti-HER2) agents in the treatment of this disease, with the attendant need to identify suitable predictive biomarkers of response to treatment. The authors studied the prevalence of HER2 genomic amplification and overexpression in 85 ovarian tumours in the local patient cohort of this study, as well as the concordance rate between immunohistochemistry, fluorescent in situ hybridisation (FISH) and a dual-colour HER2/chromosome 17 centromere chromogenic in situ hybridisation (CISH) assay. The authors identified HER2 genomic amplification and protein overexpression in 35.3% (6/17) and 29.4% (5/17), respectively, of primary ovarian mucinous carcinomas. No other cancer subtypes displayed HER2 amplification or protein overexpression. The authors also found a perfect concordance between FISH and dual-colour CISH analysis (κ coefficient 1.0, p<0.001). The results of this study support existing reports that HER2 genomic amplification and protein overexpression are predominantly found in primary ovarian mucinous carcinomas. Given the perfect concordance between the FISH and dual-colour CISH assays and the advantages of CISH over FISH analysis, future clinical trials investigating the use of anti-HER2 therapeutics in ovarian carcinomas should incorporate dual-colour CISH as part of the HER2 status assessment algorithm.

Miniaturized devices towards an integrated lab-on-a-chip platform for DNA diagnostics

NASA Astrophysics Data System (ADS)

Kaprou, G.; Papadakis, G.; Kokkoris, G.; Papadopoulos, V.; Kefala, I.; Papageorgiou, D.; Gizeli, E.; Tserepi, A.

2015-06-01

Microfluidics is an emerging technology enabling the development of Lab-on-a-chip (LOC) systems for clinical diagnostics, drug discovery and screening, food safety and environmental analysis. LOC systems integrate and scale down one or several laboratory functions on a single chip of a few mm2 to cm2 in size, and account for many advantages on biochemical analyses, such as low sample and reagent consumption, low cost, reduced analysis time, portability and point-of-need compatibility. Currently, available nucleic acid diagnostic tests take advantage of Polymerase Chain Reaction (PCR) that allows exponential amplification of portions of nucleic acid sequences that can be used as indicators for the identification of various diseases. Here, we present a comparison between static chamber and continuous flow miniaturized PCR devices, in terms of energy consumption for devices fabricated on the same material stack, with identical sample volume and channel dimensions. The comparison is implemented by a computational study coupling heat transfer in both solid and fluid, mass conservation of species, and joule heating. Based on the conclusions of this study, we develop low-cost and fast DNA amplification devices for both PCR and isothermal amplification, and we implement them in the detection of mutations related to breast cancer. The devices are fabricated by mass production amenable technologies on printed circuit board (PCB) substrates, where copper facilitates the incorporation of on-chip microheaters, defining the thermal zones necessary for PCR or isothermal amplification methods.

Compaction of rolling circle amplification products increases signal integrity and signal-to-noise ratio

PubMed Central

Clausson, Carl-Magnus; Arngården, Linda; Ishaq, Omer; Klaesson, Axel; Kühnemund, Malte; Grannas, Karin; Koos, Björn; Qian, Xiaoyan; Ranefall, Petter; Krzywkowski, Tomasz; Brismar, Hjalmar; Nilsson, Mats; Wählby, Carolina; Söderberg, Ola

2015-01-01

Rolling circle amplification (RCA) for generation of distinct fluorescent signals in situ relies upon the self-collapsing properties of single-stranded DNA in commonly used RCA-based methods. By introducing a cross-hybridizing DNA oligonucleotide during rolling circle amplification, we demonstrate that the fluorophore-labeled RCA products (RCPs) become smaller. The reduced size of RCPs increases the local concentration of fluorophores and as a result, the signal intensity increases together with the signal-to-noise ratio. Furthermore, we have found that RCPs sometimes tend to disintegrate and may be recorded as several RCPs, a trait that is prevented with our cross-hybridizing DNA oligonucleotide. These effects generated by compaction of RCPs improve accuracy of visual as well as automated in situ analysis for RCA based methods, such as proximity ligation assays (PLA) and padlock probes. PMID:26202090

Molecular barcodes detect redundancy and contamination in hairpin-bisulfite PCR

PubMed Central

Miner, Brooks E.; Stöger, Reinhard J.; Burden, Alice F.; Laird, Charles D.; Hansen, R. Scott

2004-01-01

PCR amplification of limited amounts of DNA template carries an increased risk of product redundancy and contamination. We use molecular barcoding to label each genomic DNA template with an individual sequence tag prior to PCR amplification. In addition, we include molecular ‘batch-stamps’ that effectively label each genomic template with a sample ID and analysis date. This highly sensitive method identifies redundant and contaminant sequences and serves as a reliable method for positive identification of desired sequences; we can therefore capture accurately the genomic template diversity in the sample analyzed. Although our application described here involves the use of hairpin-bisulfite PCR for amplification of double-stranded DNA, the method can readily be adapted to single-strand PCR. Useful applications will include analyses of limited template DNA for biomedical, ancient DNA and forensic purposes. PMID:15459281

Application of heteroduplex analysis for detecting variation within the growth hormone 2 gene in Salmo trutta L. (brown trout).

PubMed

Gross, R; Nilsson, J

1995-03-01

A new method to detect variation at a single copy nuclear gene in brown trout, Salmo trutta L., is provided. The technique entails (i) selective gene amplification by the polymerase chain reaction (PCR), (ii) digestion of amplification products by restriction endonucleases to obtain fragments of suitable size, (iii) hybridization with heterologous DNA followed by denaturation and reannealing to obtain heteroduplex molecules, and (iv) screening for variation in polyacrylamide gels. Variation was studied within a growth hormone 2 gene 1489 bp segment and polymorphism was detected in two HinfI-digested fragments. Formation of different heteroduplex patterns in experimental mixtures of digested amplification products from brown trout and Atlantic salmon, Salmo salar L., allowed us to determine the genotype of the brown trout. Polymorphism was observed in four out of six studied populations.

Detection and quantitation of HER-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization.

PubMed

Pauletti, G; Godolphin, W; Press, M F; Slamon, D J

1996-07-04

Amplification and overexpression of the HER-2/neu gene occurs in 25-30% of human breast cancers. This genetic alteration is associated with a poor clinical prognosis in women with either node negative or node positive breast cancers. The initial studies testing this association were somewhat controversial and this controversy was due in large part to significant heterogeneity in both the methods and/or reagents used in testing archival material for the presence of the alteration. These methods included a number of solid matrix blotting techniques for DNA, RNA and protein as well as immunohistochemistry. Fluorescence in situ hybridization (FISH) represents the newest methodologic approach for testing for this genetic alteration. In this study, FISH is compared to Southern, Northern and Western blot analyses as well as immunohistochemistry in a large cohort of archival human breast cancer specimens. FISH was found to be superior to all other methodologies tested in assessing formalin fixed, paraffin embedded material for HER-2/neu amplification. The results from this study also confirm that overexpression of HER-2/neu rarely occurs in the absence of gene amplification in breast cancer (approximately 3% of cases). This method of analysis is rapid, reproducible and extremely reliable in detecting presence of HER-2/neu gene amplification and should have clinical utility.

Multiple displacement amplification on single cell and possible PGD applications.

PubMed

Hellani, Ali; Coskun, Serdar; Benkhalifa, Moncef; Tbakhi, Abelghani; Sakati, Nadia; Al-Odaib, Ali; Ozand, Pinar

2004-11-01

Multiple displacement amplification (MDA) is a technique used in the amplification of very low amounts of DNA and reported to yield large quantities of high-quality DNA. We used MDA to amplify the whole genome directly from a single cell. The most common techniques used in PGD are PCR and fluorescent in-situ hybridization (FISH). There are many limitations to these techniques including, the number of chromosomes diagnosed for FISH or the quality of DNA issued from a single cell PCR. This report shows, for the first time, use of MDA for single cell whole genome amplification. A total of 16 short tandem repeats (STRs) were amplified successfully with a similar pattern to the genomic DNA. Furthermore, allelic drop out (ADO) derived from MDA was assessed in 40 single cells by analysing (i) heterozygosity for a known beta globin mutation (IVSI-5 C-G) and by studying (ii) the heterozygous loci present in the STRs. ADO turned out to be 10.25% for the beta globin gene sequencing and 5% for the fluorescent PCR analysis of STRs. Moreover, the amplification accuracy of MDA permitted the detection of trisomy 21 on a single cell using comparative genome hybridization-array. Altogether, these data suggest that MDA can be used for single cell molecular karyotyping and the diagnosis of any single gene disorder in PGD.

Molecular Technique to Understand Deep Microbial Diversity

NASA Technical Reports Server (NTRS)

Vaishampayan, Parag A.; Venkateswaran, Kasthuri J.

2012-01-01

Current sequencing-based and DNA microarray techniques to study microbial diversity are based on an initial PCR (polymerase chain reaction) amplification step. However, a number of factors are known to bias PCR amplification and jeopardize the true representation of bacterial diversity. PCR amplification of the minor template appears to be suppressed by the exponential amplification of the more abundant template. It is widely acknowledged among environmental molecular microbiologists that genetic biosignatures identified from an environment only represent the most dominant populations. The technological bottleneck has overlooked the presence of the less abundant minority population, and underestimated their role in the ecosystem maintenance. To generate PCR amplicons for subsequent diversity analysis, bacterial l6S rRNA genes are amplified by PCR using universal primers. Two distinct PCR regimes are employed in parallel: one using normal and the other using biotinlabeled universal primers. PCR products obtained with biotin-labeled primers are mixed with streptavidin-labeled magnetic beads and selectively captured in the presence of a magnetic field. Less-abundant DNA templates that fail to amplify in this first round of PCR amplification are subjected to a second round of PCR using normal universal primers. These PCR products are then subjected to downstream diversity analyses such as conventional cloning and sequencing. A second round of PCR amplified the minority population and completed the deep diversity picture of the environmental sample.

Systematic evaluation of bias in microbial community profiles induced by whole genome amplification.

PubMed

Direito, Susana O L; Zaura, Egija; Little, Miranda; Ehrenfreund, Pascale; Röling, Wilfred F M

2014-03-01

Whole genome amplification methods facilitate the detection and characterization of microbial communities in low biomass environments. We examined the extent to which the actual community structure is reliably revealed and factors contributing to bias. One widely used [multiple displacement amplification (MDA)] and one new primer-free method [primase-based whole genome amplification (pWGA)] were compared using a polymerase chain reaction (PCR)-based method as control. Pyrosequencing of an environmental sample and principal component analysis revealed that MDA impacted community profiles more strongly than pWGA and indicated that this related to species GC content, although an influence of DNA integrity could not be excluded. Subsequently, biases by species GC content, DNA integrity and fragment size were separately analysed using defined mixtures of DNA from various species. We found significantly less amplification of species with the highest GC content for MDA-based templates and, to a lesser extent, for pWGA. DNA fragmentation also interfered severely: species with more fragmented DNA were less amplified with MDA and pWGA. pWGA was unable to amplify low molecular weight DNA (< 1.5 kb), whereas MDA was inefficient. We conclude that pWGA is the most promising method for characterization of microbial communities in low-biomass environments and for currently planned astrobiological missions to Mars. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

Non-normality and classification of amplification mechanisms in stability and resolvent analysis

NASA Astrophysics Data System (ADS)

Symon, Sean; Rosenberg, Kevin; Dawson, Scott T. M.; McKeon, Beverley J.

2018-05-01

Eigenspectra and pseudospectra of the mean-linearized Navier-Stokes operator are used to characterize amplification mechanisms in laminar and turbulent flows in which linear mechanisms are important. Success of mean flow (linear) stability analysis for a particular frequency is shown to depend on whether two scalar measures of non-normality agree: (1) the product between the resolvent norm and the distance from the imaginary axis to the closest eigenvalue and (2) the inverse of the inner product between the most amplified resolvent forcing and response modes. If they agree, the resolvent operator can be rewritten in its dyadic representation to reveal that the adjoint and forward stability modes are proportional to the forcing and response resolvent modes at that frequency. Hence the real parts of the eigenvalues are important since they are responsible for resonant amplification and the resolvent operator is low rank when the eigenvalues are sufficiently separated in the spectrum. If the amplification is pseudoresonant, then resolvent analysis is more suitable to understand the origin of observed flow structures. Two test cases are studied: low Reynolds number cylinder flow and turbulent channel flow. The first deals mainly with resonant mechanisms, hence the success of both classical and mean stability analysis with respect to predicting the critical Reynolds number and global frequency of the saturated flow. Both scalar measures of non-normality agree for the base and mean flows, and the region where the forcing and response modes overlap scales with the length of the recirculation bubble. In the case of turbulent channel flow, structures result from both resonant and pseudoresonant mechanisms, suggesting that both are necessary elements to sustain turbulence. Mean shear is exploited most efficiently by stationary disturbances while bounds on the pseudospectra illustrate how pseudoresonance is responsible for the most amplified disturbances at spatial wavenumbers and temporal frequencies corresponding to well-known turbulent structures. Some implications for flow control are discussed.

A Tale of Tails: Dissecting the Enhancing Effect of Tailed Primers in Real-Time PCR

PubMed Central

Vandenbussche, Frank; Mathijs, Elisabeth; Lefebvre, David; De Clercq, Kris; Van Borm, Steven

2016-01-01

Non-specific tail sequences are often added to the 5’-terminus of primers to improve the robustness and overall performance of diagnostic assays. Despite the widespread use of tailed primers, the underlying working mechanism is not well understood. To address this problem, we conducted a detailed in vitro and in silico analysis of the enhancing effect of primer tailing on 2 well-established foot-and-mouth disease virus (FMDV) RT-qPCR assays using an FMDV reference panel. Tailing of the panFMDV-5UTR primers mainly affected the shape of the amplification curves. Modelling of the raw fluorescence data suggested a reduction of the amplification efficiency due to the accumulation of inhibitors. In depth analysis of PCR products indeed revealed the rapid accumulation of forward-primer derived artefacts. More importantly, tailing of the forward primer delayed artefacts formation and concomitantly restored the sigmoidal shape of the amplification curves. Our analysis also showed that primer tailing can alter utilisation patterns of degenerate primers and increase the number of primer variants that are able to participate in the reaction. The impact of tailed primers was less pronounced in the panFMDV-3D assay with only 5 out of 50 isolates showing a clear shift in Cq values. Sequence analysis of the target region of these 5 isolates revealed several mutations in the inter-primer region that extend an existing hairpin structure immediately downstream of the forward primer binding site. Stabilisation of the forward primer with either a tail sequence or cationic spermine units restored the sensitivity of the assay, which suggests that the enhancing effect in the panFMDV-3D assay is due to a more efficient extension of the forward primer. ur results show that primer tailing can alter amplification through various mechanisms that are determined by both the assay and target region. These findings expand our understanding of primer tailing and should enable a more targeted and efficient use of tailed primers. PMID:27723800

Sensitive and inexpensive digital DNA analysis by microfluidic enrichment of rolling circle amplified single-molecules

PubMed Central

Kühnemund, Malte; Hernández-Neuta, Iván; Sharif, Mohd Istiaq; Cornaglia, Matteo; Gijs, Martin A.M.

2017-01-01

Abstract Single molecule quantification assays provide the ultimate sensitivity and precision for molecular analysis. However, most digital analysis techniques, i.e. droplet PCR, require sophisticated and expensive instrumentation for molecule compartmentalization, amplification and analysis. Rolling circle amplification (RCA) provides a simpler means for digital analysis. Nevertheless, the sensitivity of RCA assays has until now been limited by inefficient detection methods. We have developed a simple microfluidic strategy for enrichment of RCA products into a single field of view of a low magnification fluorescent sensor, enabling ultra-sensitive digital quantification of nucleic acids over a dynamic range from 1.2 aM to 190 fM. We prove the broad applicability of our analysis platform by demonstrating 5-plex detection of as little as ∼1 pg (∼300 genome copies) of pathogenic DNA with simultaneous antibiotic resistance marker detection, and the analysis of rare oncogene mutations. Our method is simpler, more cost-effective and faster than other digital analysis techniques and provides the means to implement digital analysis in any laboratory equipped with a standard fluorescent microscope. PMID:28077562

Synergistic Activation upon MET and ALK Coamplification Sustains Targeted Therapy in Sarcomatoid Carcinoma, a Deadly Subtype of Lung Cancer.

PubMed

Pelosi, Giuseppe; Gasparini, Patrizia; Conte, Davide; Fabbri, Alessandra; Perrone, Federica; Tamborini, Elena; Pupa, Serenella M; Ciravolo, Valentina; Caserini, Roberto; Rossi, Giulio; Cavazza, Alberto; Papotti, Mauro; Nakatani, Yukio; Maisonneuve, Patrick; Pastorino, Ugo; Sozzi, Gabriella

2016-05-01

Genetic alterations suitable for targeted therapy are poorly known issues in pulmonary sarcomatoid carcinoma (PSC), an uncommon and life-threatening family of non-small cell lung cancers. Ninety-eight PSCs were assessed for MNNG HOS Transforming gene (MET) and anaplastic lymphoma receptor tyrosine kinase gene (ALK) status by fluorescence in situ hybridization (FISH) and for relevant protein expression by immunohistochemical analysis, also taking advantage of phosphorylated (p-) antibodies. Moreover, levels of ALK and MET mRNA were also determined by real-time polymerase chain reaction and Western blot analysis for downstream activation pathways involving p-MET, p-protein kinase B, p-mitogen-activated protein kinase, p-SRC proto-oncogene tyrosine-protein kinase, and p-focal adhesion kinase (p-FAK). MET amplification was detected by FISH in 25 of 98 PSCs (25.6%) and ALK amplification (but not the relevant rearrangement) was found in 16 of 98 (16.3%), with all ALK-amplified tumors also showing MET amplification (p < 0.0001). Nine PSCs, however, showed MET amplification without any ALK gene alteration. ALK protein expression was always lacking, whereas MET and p-MET were confined to the relevant amplified tumors only. Increased levels of ALK and MET mRNA were detectable in tumors with no direct relationship between mRNA content, protein expression, or alterations detected by FISH. Western blot assays showed complete activation of downstream signal pathways up to p-SRC proto-oncogene tyrosine-protein kinase, and p-focal adhesion kinase recruitment in MET and ALK-coamplified tumors only, whereas isolated MET amplification, MET and ALK borderline amplification (5%-10% of tumor cells with ≥15 copies of the relevant gene), or negative tumors showing eusomy or chromosome polysomy were confined to p-mitogen-activated protein kinase, p-protein kinase B, and/or p-MET activation. Multivariate survival analysis pushed a higher percentage of MET altered cells or a higher value of MET copy gain per cell to marginally emerge for overall survival (p = 0.140) and disease-free survival (p = 0.060), respectively. ALK and MET seemed to act as synergistic, nonrandom coactivators of downstream signal when coamplified in a subset of patients with PSC, thus likely suggesting a combined mechanism of oncogene addiction. These alterations could be a suitable target for therapy based on specific inhibitors. Copyright © 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

HER2 Amplification and HER2 Mutation Are Distinct Molecular Targets in Lung Cancers.

PubMed

Li, Bob T; Ross, Dara S; Aisner, Dara L; Chaft, Jamie E; Hsu, Meier; Kako, Severine L; Kris, Mark G; Varella-Garcia, Marileila; Arcila, Maria E

2016-03-01

Human epidermal growth factor receptor 2 gene (HER2 [also known as ERBB2]) alterations have been identified as oncogenic drivers and potential therapeutic targets in lung cancers. The molecular associations of HER2 gene amplification, mutation, and HER2 protein overexpression in lung cancers have not been distinctly defined. To explore these associations, Memorial Sloan Kettering Cancer Center and the University of Colorado combined their data on HER2 alterations in lung cancers. Tumor specimens from 175 patients with lung adenocarcinomas and no prior targeted therapy were evaluated for the presence of HER2 amplification and mutation and HER2 protein overexpression. Amplification was assessed by fluorescence in situ hybridization (FISH) and defined as an HER2-to-chromosome enumeration probe 17 ratio of at least 2.0. Mutation was assessed by fragment analysis, mass spectrometry genotyping, and Sanger sequencing. Overexpression was assessed by immunohistochemical (IHC) staining. The frequencies of HER2 amplification and mutation and HER2 overexpression were calculated and their overlap examined. HER2 amplification was detected by FISH in 5 of 175 cases (3%). HER2 mutation was detected in 4 of 148 specimens (3%), including three identical 12-base pair insertions (p.A775_G776insYVMA) and a 9-base pair insertion, all in exon 20. None of the HER2-mutant cases was amplified. HER2 overexpression (2+ or 3+) on IHC staining was not detected in the 25 specimens available for testing, and negative IHC staining correlated with the negative results according to FISH. HER2 mutations are not associated with HER2 amplification, thus suggesting a distinct entity and therapeutic target. HER2-positive lung cancer may not be an adequate term, and patient cohorts for the study of HER2-targeted agents should be defined by the specific HER2 alteration present. Copyright © 2015 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.