Three molecular pathways model colorectal carcinogenesis in Lynch syndrome.

PubMed

Ahadova, Aysel; Gallon, Richard; Gebert, Johannes; Ballhausen, Alexej; Endris, Volker; Kirchner, Martina; Stenzinger, Albrecht; Burn, John; von Knebel Doeberitz, Magnus; Bläker, Hendrik; Kloor, Matthias

2018-07-01

Lynch syndrome is caused by germline mutations of DNA mismatch repair (MMR) genes. MMR deficiency has long been regarded as a secondary event in the pathogenesis of Lynch syndrome colorectal cancers. Recently, this concept has been challenged by the discovery of MMR-deficient crypt foci in the normal mucosa. We aimed to reconstruct colorectal carcinogenesis in Lynch syndrome by collecting molecular and histology evidence from Lynch syndrome adenomas and carcinomas. We determined the frequency of MMR deficiency in adenomas from Lynch syndrome mutation carriers by immunohistochemistry and by systematic literature analysis. To trace back the pathways of pathogenesis, histological growth patterns and mutational signatures were analyzed in Lynch syndrome colorectal cancers. Literature and immunohistochemistry analysis demonstrated MMR deficiency in 491 (76.7%) out of 640 adenomas (95% CI: 73.3% to 79.8%) from Lynch syndrome mutation carriers. Histologically normal MMR-deficient crypts were found directly adjacent to dysplastic adenoma tissue, proving their role as tumor precursors in Lynch syndrome. Accordingly, mutation signature analysis in Lynch colorectal cancers revealed that KRAS and APC mutations commonly occur after the onset of MMR deficiency. Tumors lacking evidence of polypous growth frequently presented with CTNNB1 and TP53 mutations. Our findings demonstrate that Lynch syndrome colorectal cancers can develop through three pathways, with MMR deficiency commonly representing an early and possibly initiating event. This underlines that targeting MMR-deficient cells by chemoprevention or vaccines against MMR deficiency-induced frameshift peptide neoantigens holds promise for tumor prevention in Lynch syndrome. © 2018 UICC.

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.

Somatic mosaicism containing double mutations in PTCH1 revealed by generation of induced pluripotent stem cells from nevoid basal cell carcinoma syndrome.

PubMed

Ikemoto, Yu; Takayama, Yoshinaga; Fujii, Katsunori; Masuda, Mokuri; Kato, Chise; Hatsuse, Hiromi; Fujitani, Kazuko; Nagao, Kazuaki; Kameyama, Kohzoh; Ikehara, Hajime; Toyoda, Masashi; Umezawa, Akihiro; Miyashita, Toshiyuki

2017-08-01

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterised by developmental defects and tumorigenesis, such as medulloblastomas and basal cell carcinomas, caused by mutations of the patched-1 ( PTCH1 ) gene. In this article, we seek to demonstrate a mosaicism containing double mutations in PTCH1 in an individual with NBCCS. A de novo germline mutation of PTCH1 (c.272delG) was detected in a 31-year-old woman with NBCCS. Gene analysis of two out of four induced pluripotent stem cell (iPSC) clones established from the patient unexpectedly revealed an additional mutation, c.274delT. Deep sequencing confirmed a low-prevalence somatic mutation (5.5%-15.6% depending on the tissue) identical to the one found in iPSC clones. This is the first case of mosaicism unequivocally demonstrated in NBCCS. Furthermore, the mosaicism is unique in that the patient carries one normal and two mutant alleles. Because these mutations are located in close proximity, reversion error is likely to be involved in this event rather than a spontaneous mutation. In addition, this study indicates that gene analysis of iPSC clones can contribute to the detection of mosaicism containing a minor population carrying a second mutation. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Mutations in the interleukin receptor IL11RA cause autosomal recessive Crouzon-like craniosynostosis

PubMed Central

Keupp, Katharina; Li, Yun; Vargel, Ibrahim; Hoischen, Alexander; Richardson, Rebecca; Neveling, Kornelia; Alanay, Yasemin; Uz, Elif; Elcioğlu, Nursel; Rachwalski, Martin; Kamaci, Soner; Tunçbilek, Gökhan; Akin, Burcu; Grötzinger, Joachim; Konas, Ersoy; Mavili, Emin; Müller-Newen, Gerhard; Collmann, Hartmut; Roscioli, Tony; Buckley, Michael F; Yigit, Gökhan; Gilissen, Christian; Kress, Wolfram; Veltman, Joris; Hammerschmidt, Matthias; Akarsu, Nurten A; Wollnik, Bernd

2013-01-01

We have characterized a novel autosomal recessive Crouzon-like craniosynostosis syndrome in a 12-affected member family from Antakya, Turkey, the presenting features of which include: multiple suture synostosis, midface hypoplasia, variable degree of exophthalmos, relative prognathism, a beaked nose, and conductive hearing loss. Homozygosity mapping followed by targeted next-generation sequencing identified a c.479+6T>G mutation in the interleukin 11 receptor alpha gene (IL11RA) on chromosome 9p21. This donor splice-site mutation leads to a high percentage of aberrant IL11RA mRNA transcripts in an affected individual and altered mRNA splicing determined by in vitro exon trapping. An extended IL11RA mutation screen was performed in a cohort of 79 patients with an initial clinical diagnosis of Crouzon syndrome, pansynostosis, or unclassified syndromic craniosynostosis. We identified mutations segregating with the disease in five families: a German patient of Turkish origin and a Turkish family with three affected sibs all of whom were homozygous for the previously identified IL11RA c.479+6T>G mutation; a family with pansynostosis with compound heterozygous missense mutations, p.Pro200Thr and p.Arg237Pro; and two further Turkish families with Crouzon-like syndrome carrying the homozygous nonsense mutations p.Tyr232* and p.Arg292*. Using transient coexpression in HEK293T and COS7 cells, we demonstrated dramatically reduced IL11-mediated STAT3 phosphorylation for all mutations. Immunofluorescence analysis of mouse Il11ra demonstrated specific protein expression in cranial mesenchyme which was localized around the coronal suture tips and in the lambdoidal suture. In situ hybridization analysis of adult zebrafish also detected zfil11ra expression in the coronal suture between the overlapping frontal and parietal plates. This study demonstrates that mutations in the IL11RA gene cause an autosomal recessive Crouzon-like craniosynostosis. PMID:24498618

Hyperinsulinism–hyperammonaemia syndrome: novel mutations in the GLUD1 gene and genotype–phenotype correlations

PubMed Central

Kapoor, Ritika R; Flanagan, Sarah E; Fulton, Piers; Chakrapani, Anupam; Chadefaux, Bernadette; Ben-Omran, Tawfeg; Banerjee, Indraneel; Shield, Julian P; Ellard, Sian; Hussain, Khalid

2009-01-01

Background Activating mutations in the GLUD1 gene (which encodes for the intra-mitochondrial enzyme glutamate dehydrogenase, GDH) cause the hyperinsulinism–hyperammonaemia (HI/HA) syndrome. Patients present with HA and leucine-sensitive hypoglycaemia. GDH is regulated by another intra-mitochondrial enzyme sirtuin 4 (SIRT4). Sirt4 knockout mice demonstrate activation of GDH with increased amino acid-stimulated insulin secretion. Objectives To study the genotype–phenotype correlations in patients with GLUD1 mutations. To report the phenotype and functional analysis of a novel mutation (P436L) in the GLUD1 gene associated with the absence of HA. Patients and methods Twenty patients with HI from 16 families had mutational analysis of the GLUD1 gene in view of HA (n=19) or leucine sensitivity (n=1). Patients negative for a GLUD1 mutation had sequence analysis of the SIRT4 gene. Functional analysis of the novel P436L GLUD1 mutation was performed. Results Heterozygous missense mutations were detected in 15 patients with HI/HA, 2 of which are novel (N410D and D451V). In addition, a patient with a normal serum ammonia concentration (21 μmol/l) was heterozygous for a novel missense mutation P436L. Functional analysis of this mutation confirms that it is associated with a loss of GTP inhibition. Seizure disorder was common (43%) in our cohort of patients with a GLUD1 mutation. No mutations in the SIRT4 gene were identified. Conclusion Patients with HI due to mutations in the GLUD1 gene may have normal serum ammonia concentrations. Hence, GLUD1 mutational analysis may be indicated in patients with leucine sensitivity; even in the absence of HA. A high frequency of epilepsy (43%) was observed in our patients with GLUD1 mutations. PMID:19690084

A KCNH2 branch point mutation causing aberrant splicing contributes to an explanation of genotype-negative long QT syndrome.

PubMed

Crotti, Lia; Lewandowska, Marzena A; Schwartz, Peter J; Insolia, Roberto; Pedrazzini, Matteo; Bussani, Erica; Dagradi, Federica; George, Alfred L; Pagani, Franco

2009-02-01

Genetic screening of long QT syndrome (LQTS) fails to identify disease-causing mutations in about 30% of patients. So far, molecular screening has focused mainly on coding sequence mutations or on substitutions at canonical splice sites. The purpose of this study was to explore the possibility that intronic variants not at canonical splice sites might affect splicing regulatory elements, lead to aberrant transcripts, and cause LQTS. Molecular screening was performed through DHPLC and sequence analysis. The role of the intronic mutation identified was assessed with a hybrid minigene splicing assay. A three-generation LQTS family was investigated. Molecular screening failed to identify an obvious disease-causing mutation in the coding sequences of the major LQTS genes but revealed an intronic A-to-G substitution in KCNH2 (IVS9-28A/G) cosegregating with the clinical phenotype in family members. In vitro analysis proved that the mutation disrupts the acceptor splice site definition by affecting the branch point (BP) sequence and promoting intron retention. We further demonstrated a tight functional relationship between the BP and the polypyrimidine tract, whose weakness is responsible for the pathological effect of the IVS9-28A/G mutation. We identified a novel BP mutation in KCNH2 that disrupts the intron 9 acceptor splice site definition and causes LQT2. The present finding demonstrates that intronic mutations affecting pre-mRNA processing may contribute to the failure of traditional molecular screening in identifying disease-causing mutations in LQTS subjects and offers a rationale strategy for the reduction of genotype-negative cases.

DHPLC-based mutation analysis of ENG and ALK-1 genes in HHT Italian population.

PubMed

Lenato, Gennaro M; Lastella, Patrizia; Di Giacomo, Marilena C; Resta, Nicoletta; Suppressa, Patrizia; Pasculli, Giovanna; Sabbà, Carlo; Guanti, Ginevra

2006-02-01

Hereditary haemorrhagic telangiectasia (HHT or Rendu-Osler-Weber syndrome) is an autosomal dominant disorder characterized by localized angiodysplasia due to mutations in endoglin, ALK-1 gene, and a still unidentified locus. The lack of highly recurrent mutations, locus heterogeneity, and the presence of mutations in almost all coding exons of the two genes makes the screening for mutations time-consuming and costly. In the present study, we developed a DHPLC-based protocol for mutation detection in ALK1 and ENG genes through retrospective analysis of known sequence variants, 20 causative mutations and 11 polymorphisms, and a prospective analysis on 47 probands with unknown mutation. Overall DHPLC analysis identified the causative mutation in 61 out 66 DNA samples (92.4%). We found 31 different mutations in the ALK1 gene, of which 15 are novel, and 20, of which 12 are novel, in the ENG gene, thus providing for the first time the mutational spectrum in a cohort of Italian HHT patients. In addition, we characterized the splicing pattern of ALK1 gene in lymphoblastoid cells, both in normal controls and in two individuals carrying a mutation in the non-invariant -3 position of the acceptor splice site upstream exon 6 (c.626-3C>G). Functional essay demonstrated the existence, also in normal individuals, of a small proportion of ALK1 alternative splicing, due to exon 5 skipping, and the presence of further aberrant splicing isoforms in the individuals carrying the c.626-3C>G mutation. 2006 Wiley-Liss, Inc.

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.

Mutational analysis of BRAF and KRAS in ovarian serous borderline (atypical proliferative) tumours and associated peritoneal implants

PubMed Central

Ardighieri, Laura; Zeppernick, Felix; Hannibal, Charlotte G; Vang, Russell; Cope, Leslie; Junge, Jette; Kjaer, Susanne K; Kurman, Robert J; Shih, Ie-Ming

2014-01-01

There is debate as to whether peritoneal implants associated with serous borderline tumours/atypical proliferative serous tumours (SBT/APSTs) of the ovary are derived from the primary ovarian tumour or arise independently in the peritoneum. We analysed 57 SBT/APSTs from 45 patients with advanced-stage disease identified from a nation-wide tumour registry in Denmark. Mutational analysis for hotspots in KRAS and BRAF was successful in 55 APSTs and demonstrated KRAS mutations in 34 (61.8%) and BRAF mutations in eight (14.5%). Mutational analysis was successful in 56 peritoneal implants and revealed KRAS mutations in 34 (60.7%) and BRAF mutations in seven (12.5%). Mutational analysis could not be performed in two primary tumours and in nine implants, either because DNA amplification failed or because there was insufficient tissue for mutational analysis. For these specimens we performed VE1 immunohistochemistry, which was shown to be a specific and sensitive surrogate marker for a V600E BRAF mutation. VE1 staining was positive in one of two APSTs and seven of nine implants. Thus, among 63 implants for which mutation status was known (either by direct mutational analysis or by VE1 immunohistochemistry), 34 (53.9%) had KRAS mutations and 14 (22%) had BRAF mutations, of which identical KRAS mutations were found in 34 (91%) of 37 SBT/APST–implant pairs and identical BRAF mutations in 14 (100%) of 14 SBT/APST–implant pairs. Wild-type KRAS and BRAF (at the loci investigated) were found in 11 (100%) of 11 SBT/APST–implant pairs. Overall concordance of KRAS and BRAF mutations was 95% in 59 of 62 SBT/APST–implant (non-invasive and invasive) pairs (p < 0.00001). This study provides cogent evidence that the vast majority of peritoneal implants, non-invasive and invasive, harbour the identical KRAS or BRAF mutations that are present in the associated SBT/APST, supporting the view that peritoneal implants are derived from the primary ovarian tumour. PMID:24307542

[Analysis of H63D mutation in hemochromatosis (HFE) gene in populations of central Eurasia].

PubMed

Khusainova, R I; Khusnutdinova, N N; Litvinov, S S; Khusnutdinova, E K

2013-02-01

An analysis of the frequency of H63D (c. 187C>G) mutations in the HFEgene in 19 populations from Central Eurasia demonstrated that the distribution of the mutation in the region of interest was not uniform and that there were the areas of H63D accumulation. The investigation of three polymorphic variants, c.340+4T>C (rs2071303, IVS2(+4)T>C), c.893-44T>C (rs1800708, IVS4(-44)T>C), and c.1007-47G>A (rs1572982, IVS5(-47)A>G), in the HFE gene in individuals homozygous for H63D mutations in the HFE gene revealed the linkage of H63D with three haplotypes, *CTA, *TG, and *TTA. These findings indicated the partial spread of the mutation in Central Eurasia from Western Europe, as well as the possible repeated appearance of the mutation on the territory on interest.

Mutational Signature Mark Cancer’s Smoking Gun

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

Alexandrov, Ludmil

A broad computational study of cancer genome sequences by Los Alamos National Laboratory with the UK’s Wellcome Trust Sanger Institute and other collaborators identifies telltale mutational signatures associated with smoking tobacco. The research demonstrates, for the first time, that smoking increases cancer risk by causing somatic mutations in tissues directly and indirectly exposed to tobacco smoke. The international study was published in the November 4 issue of Science. The analysis shows that tobacco smoking causes mutations leading to cancer by multiple distinct mechanisms, including by damaging DNA in organs and by speeding up a mutational cellular clock.

Mutational Signature Mark Cancer’s Smoking Gun

ScienceCinema

Alexandrov, Ludmil

2018-06-13

A broad computational study of cancer genome sequences by Los Alamos National Laboratory with the UK’s Wellcome Trust Sanger Institute and other collaborators identifies telltale mutational signatures associated with smoking tobacco. The research demonstrates, for the first time, that smoking increases cancer risk by causing somatic mutations in tissues directly and indirectly exposed to tobacco smoke. The international study was published in the November 4 issue of Science. The analysis shows that tobacco smoking causes mutations leading to cancer by multiple distinct mechanisms, including by damaging DNA in organs and by speeding up a mutational cellular clock.

Analysis of ESR1 and PIK3CA mutations in plasma cell-free DNA from ER-positive breast cancer patients.

PubMed

Takeshita, Takashi; Yamamoto, Yutaka; Yamamoto-Ibusuki, Mutsuko; Tomiguchi, Mai; Sueta, Aiko; Murakami, Keiichi; Omoto, Yoko; Iwase, Hirotaka

2017-08-08

The measurement of ESR1 and PIK3CA mutations in plasma cell-free DNA (cfDNA) has been studied as a non-invasive method to quickly assess and monitor endocrine therapy (ET) resistant metastatic breast cancer (MBC) patients. The subjects of this retrospective study were a total of 185 plasma samples from 86 estrogen receptor-positive BC patients, of which 151 plasma samples were from 69 MBC patients and 34 plasma samples were from 17 primary BC (PBC) patients. We developed multiplex droplet digital PCR assays to verify the clinical significance of ESR1 and PIK3CA mutations both in a snapshot and serially in these patients. cfDNA ESR1 and PIK3CA mutations were found in 28.9% and 24.6 % of MBC patients, respectively. The relation between ESR1 or PIK3CA mutations and clinical features showed that ESR1 mutations occurred mostly in patients previously treated by ET, which was not the case for PIK3CA mutations. The analysis of the clinical impact of those mutations on subsequent lines of treatment for the 69 MBC patients revealed that both ESR1 and PIK3CA mutations detection were related to a shorter duration of ET effectiveness in univariate analysis but only for ESR1 mutations in multivariate analysis. The monitoring of cfDNA in a subset of 52 patients showed that loss of ESR1 mutations was related to a longer duration of response, which was not the case for PIK3CA mutations. We have demonstrated the clinical significance of on-treatment ESR1 mutations both in a snapshot and serially in comparison with PIK3CA mutations.

Analysis of ESR1 and PIK3CA mutations in plasma cell-free DNA from ER-positive breast cancer patients

PubMed Central

Takeshita, Takashi; Yamamoto, Yutaka; Yamamoto-Ibusuki, Mutsuko; Tomiguchi, Mai; Sueta, Aiko; Murakami, Keiichi; Omoto, Yoko; Iwase, Hirotaka

2017-01-01

Background The measurement of ESR1 and PIK3CA mutations in plasma cell-free DNA (cfDNA) has been studied as a non-invasive method to quickly assess and monitor endocrine therapy (ET) resistant metastatic breast cancer (MBC) patients. Methods The subjects of this retrospective study were a total of 185 plasma samples from 86 estrogen receptor-positive BC patients, of which 151 plasma samples were from 69 MBC patients and 34 plasma samples were from 17 primary BC (PBC) patients. We developed multiplex droplet digital PCR assays to verify the clinical significance of ESR1 and PIK3CA mutations both in a snapshot and serially in these patients. Results cfDNA ESR1 and PIK3CA mutations were found in 28.9% and 24.6 % of MBC patients, respectively. The relation between ESR1 or PIK3CA mutations and clinical features showed that ESR1 mutations occurred mostly in patients previously treated by ET, which was not the case for PIK3CA mutations. The analysis of the clinical impact of those mutations on subsequent lines of treatment for the 69 MBC patients revealed that both ESR1 and PIK3CA mutations detection were related to a shorter duration of ET effectiveness in univariate analysis but only for ESR1 mutations in multivariate analysis. The monitoring of cfDNA in a subset of 52 patients showed that loss of ESR1 mutations was related to a longer duration of response, which was not the case for PIK3CA mutations. Conclusions We have demonstrated the clinical significance of on-treatment ESR1 mutations both in a snapshot and serially in comparison with PIK3CA mutations. PMID:28881720

Novel USH2A compound heterozygous mutations cause RP/USH2 in a Chinese family.

PubMed

Liu, Xiaowen; Tang, Zhaohui; Li, Chang; Yang, Kangjuan; Gan, Guanqi; Zhang, Zibo; Liu, Jingyu; Jiang, Fagang; Wang, Qing; Liu, Mugen

2010-03-17

To identify the disease-causing gene in a four-generation Chinese family affected with retinitis pigmentosa (RP). Linkage analysis was performed with a panel of microsatellite markers flanking the candidate genetic loci of RP. These loci included 38 known RP genes. The complete coding region and exon-intron boundaries of Usher syndrome 2A (USH2A) were sequenced with the proband DNA to screen the disease-causing gene mutation. Restriction fragment length polymorphism (RFLP) analysis and direct DNA sequence analysis were done to demonstrate co-segregation of the USH2A mutations with the family disease. One hundred normal controls were used without the mutations. The disease-causing gene in this Chinese family was linked to the USH2A locus on chromosome 1q41. Direct DNA sequence analysis of USH2A identified two novel mutations in the patients: one missense mutation p.G1734R in exon 26 and a splice site mutation, IVS32+1G>A, which was found in the donor site of intron 32 of USH2A. Neither the p.G1734R nor the IVS32+1G>A mutation was found in the unaffected family members or the 100 normal controls. One patient with a homozygous mutation displayed only RP symptoms until now, while three patients with compound heterozygous mutations in the family of study showed both RP and hearing impairment. This study identified two novel mutations: p.G1734R and IVS32+1G>A of USH2A in a four-generation Chinese RP family. In this study, the heterozygous mutation and the homozygous mutation in USH2A may cause Usher syndrome Type II or RP, respectively. These two mutations expand the mutant spectrum of USH2A.

Genetic Basis of Glycogen Storage Disease Type 1a: Prevalent Mutations at the Glucose-6-Phosphatase Locus

PubMed Central

Lei, Ke-Jian; Chen, Yuan-Tsong; Chen, Hungwen; Wong, Lee-Jun C.; Liu, Ji-Lan; McConkie-Rosell, Allyn; Van Hove, Johan L. K.; Ou, Henry C.-Y.; Yeh, Nan Jung; Pan, Lorraine Y.; Chou, Janice Yang

1995-01-01

Diagnosis of glycogen storage disease (GSD) type 1a currently is established by demonstrating the lack of glucose-6-phosphatase (G6Pase) activity in the patient's biopsied liver specimen. Recent cloning of the G6Pase gene and identification of mutations within the gene that causes GSD type 1a allow for the development of a DNA-based diagnostic method. Using SSCP analysis and DNA sequencing, we characterized the G6Pase gene of 70 unrelated patients with enzymatically confirmed diagnosis of GSD type 1a and detected mutations in all except 17 alleles (88%). Sixteen mutations were uncovered that were shown by expression to abolish or greatly reduce G6Pase activity and that therefore are responsible for the GSD type 1a disorder. R83C and Q347X are the most prevalent mutations found in Caucasians, 130X and R83C are most prevalent in Hispanics, and R83H is most prevalent in Chinese. The Q347X mutation has thus far been identified only in Caucasian patients, and the 130X mutation has been identified only in Hispanic patients. Our results demonstrate that the DNA-based analysis can accurately, rapidly, and noninvasively detect the majority of mutations in GSD type 1a. This DNA-based diagnosis now permits prenatal diagnosis among at-risk patients and serves as a database in screening and counseling patients clinically suspected of having this disease. ImagesFigure 1Figure 2 PMID:7573034

Impacts of the Callipyge Mutation on Ovine Plasma Metabolites and Muscle Fibre Type

PubMed Central

Li, Juan; Greenwood, Paul L.; Cockett, Noelle E.; Hadfield, Tracy S.; Vuocolo, Tony; Byrne, Keren; White, Jason D.; Tellam, Ross L.; Schirra, Horst Joachim

2014-01-01

The ovine Callipyge mutation causes postnatal muscle hypertrophy localized to the pelvic limbs and torso, as well as body leanness. The mechanism underpinning enhanced muscle mass is unclear, as is the systemic impact of the mutation. Using muscle fibre typing immunohistochemistry, we confirmed muscle specific effects and demonstrated that affected muscles had greater prevalence and hypertrophy of type 2X fast twitch glycolytic fibres and decreased representation of types 1, 2C, 2A and/or 2AX fibres. To investigate potential systemic effects of the mutation, proton NMR spectra of plasma taken from lambs at 8 and 12 weeks of age were measured. Multivariate statistical analysis of plasma metabolite profiles demonstrated effects of development and genotype but not gender. Plasma from Callipyge lambs at 12 weeks of age, but not 8 weeks, was characterized by a metabolic profile consistent with contributions from the affected hypertrophic fast twitch glycolytic muscle fibres. Microarray analysis of the perirenal adipose tissue depot did not reveal a transcriptional effect of the mutation in this tissue. We conclude that there is an indirect systemic effect of the Callipyge mutation in skeletal muscle in the form of changes of blood metabolites, which may contribute to secondary phenotypes such as body leanness. PMID:24937646

Impacts of the Callipyge mutation on ovine plasma metabolites and muscle fibre type.

PubMed

Li, Juan; Greenwood, Paul L; Cockett, Noelle E; Hadfield, Tracy S; Vuocolo, Tony; Byrne, Keren; White, Jason D; Tellam, Ross L; Schirra, Horst Joachim

2014-01-01

The ovine Callipyge mutation causes postnatal muscle hypertrophy localized to the pelvic limbs and torso, as well as body leanness. The mechanism underpinning enhanced muscle mass is unclear, as is the systemic impact of the mutation. Using muscle fibre typing immunohistochemistry, we confirmed muscle specific effects and demonstrated that affected muscles had greater prevalence and hypertrophy of type 2X fast twitch glycolytic fibres and decreased representation of types 1, 2C, 2A and/or 2AX fibres. To investigate potential systemic effects of the mutation, proton NMR spectra of plasma taken from lambs at 8 and 12 weeks of age were measured. Multivariate statistical analysis of plasma metabolite profiles demonstrated effects of development and genotype but not gender. Plasma from Callipyge lambs at 12 weeks of age, but not 8 weeks, was characterized by a metabolic profile consistent with contributions from the affected hypertrophic fast twitch glycolytic muscle fibres. Microarray analysis of the perirenal adipose tissue depot did not reveal a transcriptional effect of the mutation in this tissue. We conclude that there is an indirect systemic effect of the Callipyge mutation in skeletal muscle in the form of changes of blood metabolites, which may contribute to secondary phenotypes such as body leanness.

Development and validation of a whole-exome sequencing test for simultaneous detection of point mutations, indels and copy-number alterations for precision cancer care

PubMed Central

Rennert, Hanna; Eng, Kenneth; Zhang, Tuo; Tan, Adrian; Xiang, Jenny; Romanel, Alessandro; Kim, Robert; Tam, Wayne; Liu, Yen-Chun; Bhinder, Bhavneet; Cyrta, Joanna; Beltran, Himisha; Robinson, Brian; Mosquera, Juan Miguel; Fernandes, Helen; Demichelis, Francesca; Sboner, Andrea; Kluk, Michael; Rubin, Mark A; Elemento, Olivier

2016-01-01

We describe Exome Cancer Test v1.0 (EXaCT-1), the first New York State-Department of Health-approved whole-exome sequencing (WES)-based test for precision cancer care. EXaCT-1 uses HaloPlex (Agilent) target enrichment followed by next-generation sequencing (Illumina) of tumour and matched constitutional control DNA. We present a detailed clinical development and validation pipeline suitable for simultaneous detection of somatic point/indel mutations and copy-number alterations (CNAs). A computational framework for data analysis, reporting and sign-out is also presented. For the validation, we tested EXaCT-1 on 57 tumours covering five distinct clinically relevant mutations. Results demonstrated elevated and uniform coverage compatible with clinical testing as well as complete concordance in variant quality metrics between formalin-fixed paraffin embedded and fresh-frozen tumours. Extensive sensitivity studies identified limits of detection threshold for point/indel mutations and CNAs. Prospective analysis of 337 cancer cases revealed mutations in clinically relevant genes in 82% of tumours, demonstrating that EXaCT-1 is an accurate and sensitive method for identifying actionable mutations, with reasonable costs and time, greatly expanding its utility for advanced cancer care. PMID:28781886

RHO Mutations (p.W126L and p.A346P) in Two Japanese Families with Autosomal Dominant Retinitis Pigmentosa

PubMed Central

Akahori, Masakazu; Itabashi, Takeshi; Nishino, Jo; Yoshitake, Kazutoshi; Ikeo, Kazuho; Tsuneoka, Hiroshi

2014-01-01

Purpose. To investigate genetic and clinical features of patients with rhodopsin (RHO) mutations in two Japanese families with autosomal dominant retinitis pigmentosa (adRP). Methods. Whole-exome sequence analysis was performed in ten adRP families. Identified RHO mutations for the cosegregation analysis were confirmed by Sanger sequencing. Ophthalmic examinations were performed to evaluate the RP phenotypes. The impact of the RHO mutation on the rhodopsin conformation was examined by molecular modeling analysis. Results. In two adRP families, we identified two RHO mutations (c.377G>T (p.W126L) and c.1036G>C (p.A346P)), one of which was novel. Complete cosegregation was confirmed for each mutation exhibiting the RP phenotype in both families. Molecular modeling predicted that the novel mutation (p.W126L) might impair rhodopsin function by affecting its conformational transition in the light-adapted form. Clinical phenotypes showed that patients with p.W126L exhibited sector RP, whereas patients with p.A346P exhibited classic RP. Conclusions. Our findings demonstrated that the novel mutation (p.W126L) may be associated with the phenotype of sector RP. Identification of RHO mutations is a very useful tool for predicting disease severity and providing precise genetic counseling. PMID:25485142

Germ line mutation analysis in families with multiple endocrine neoplasia type 2A or familial medullary thyroid carcinoma.

PubMed

Karga, H J; Karayianni, M K; Linos, D A; Tseleni, S C; Karaiskos, K D; Papapetrou, P D

1998-10-01

The RET proto-oncogene has been identified as the multiple endocrine neoplasia type 2 disease gene. An association between specific RET mutation and disease phenotype has been reported. We present the phenotype-genotype of 12 Greek families with multiple endocrine neoplasia type 2A (MEN 2A) or familial medullary thyroid carcinoma (FMTC). Seventy members were studied and DNA analysis for RET mutations was performed in fifty-eight of them. Exons 10, 11, 13, 14 and 16 of the RET proto-oncogene were analyzed by single strand conformation polymorphism analysis, direct DNA sequencing and/or restriction enzyme analysis. No mutations of the RET proto-oncogene were identified in 1 of 9 families with MEN 2A and in the 3 families with FMTC. In 7 MEN 2A families, the mutation was demonstrated in codon 634 and in 1 family it was demonstrated in codon 620. There was a low frequency, about 8%, of hyperparathyroidism associated with MEN 2A. The specific causative mutations for pararthyroid disease were C634R or C634Y. Among the MEN 2A individuals there was one case with de novo C634R mutation and one case, C634Y, with cutaneous lichen amyloidosis which predated by 24 years the diagnosis of MEN 2A. In 2 children who were MEN 2A gene carriers, microscopic medullary thyroid carcinomas were found. These data show a low frequency of hyperparathyroidism in our cases and provide further evidence that individuals with C634R as well as with C634Y mutations of the RET proto-oncogene could be at risk for parathyroid disease. Cutaneous lichen amyloidosis could be an early feature of MEN 2A. Additionally, direct DNA testing provided an opportunity to resect medullary thyroid carcinoma at an early stage.

Familial Mediterranean fever with a single MEFV mutation: where is the second hit?

PubMed

Booty, Matthew G; Chae, Jae Jin; Masters, Seth L; Remmers, Elaine F; Barham, Beverly; Le, Julie M; Barron, Karyl S; Holland, Steve M; Kastner, Daniel L; Aksentijevich, Ivona

2009-06-01

Familial Mediterranean fever (FMF) has traditionally been considered an autosomal-recessive disease; however, it has been observed that a substantial number of patients with clinical FMF possess only 1 demonstrable MEFV mutation. The purpose of this study was to perform an extensive search for a second MEFV mutation in 46 patients diagnosed clinically as having FMF and carrying only 1 high-penetrance FMF mutation. MEFV and other candidate genes were sequenced by standard capillary electrophoresis. In 10 patients, the entire 15-kb MEFV genomic region was resequenced using hybridization-based chip technology. MEFV gene expression levels were determined by quantitative reverse transcription-polymerase chain reaction. Pyrin protein levels were examined by Western blotting. A second MEFV mutation was not identified in any of the patients who were screened. Haplotype analysis did not identify a common haplotype that might be associated with the transmission of a second FMF allele. Western blots did not demonstrate a significant difference in pyrin levels between patients with a single mutation and those with a double mutation; however, FMF patients of both types showed higher protein expression as compared with controls and with non-FMF patients with active inflammation. Screening of genes encoding pyrin-interacting proteins identified rare mutations in a small number of patients, suggesting the possibility of digenic inheritance. Our data underscore the existence of a significant subset of FMF patients who are carriers of only 1 MEFV mutation and demonstrate that complete MEFV sequencing is not likely to yield a second mutation. Screening for the set of the most common mutations and detection of a single mutation appears to be sufficient in the presence of clinical symptoms for the diagnosis of FMF and the initiation of a trial of colchicine.

Mutations in extracellular matrix genes NID1 and LAMC1 cause autosomal dominant Dandy-Walker malformation and occipital cephaloceles

PubMed Central

Darbro, Benjamin W.; Mahajan, Vinit B.; Gakhar, Lokesh; Skeie, Jessica M.; Campbell, Elizabeth; Wu, Shu; Bing, Xinyu; Millen, Kathleen J.; Dobyns, William B.; Kessler, John A.; Jalali, Ali; Cremer, James; Segre, Alberto; Manak, J. Robert; Aldinger, Kimerbly A.; Suzuki, Satoshi; Natsume, Nagato; Ono, Maya; Hai, Huynh Dai; Viet, Le Thi; Loddo, Sara; Valente, Enza M.; Bernardini, Laura; Ghonge, Nitin; Ferguson, Polly J.; Bassuk, Alexander G.

2013-01-01

We performed whole-exome sequencing of a family with autosomal dominant Dandy-Walker malformation and occipital cephaloceles (ADDWOC) and detected a mutation in the extracellular matrix protein encoding gene NID1. In a second family, protein interaction network analysis identified a mutation in LAMC1, which encodes a NID1 binding partner. Structural modeling the NID1-LAMC1 complex demonstrated that each mutation disrupts the interaction. These findings implicate the extracellular matrix in the pathogenesis of Dandy-Walker spectrum disorders. PMID:23674478

Clinical and mutation analysis of 51 probands with anophthalmia and/or severe microphthalmia from a single center

PubMed Central

Gerth-Kahlert, Christina; Williamson, Kathleen; Ansari, Morad; Rainger, Jacqueline K; Hingst, Volker; Zimmermann, Theodor; Tech, Stefani; Guthoff, Rudolf F; van Heyningen, Veronica; FitzPatrick, David R

2013-01-01

Clinical evaluation and mutation analysis was performed in 51 consecutive probands with severe eye malformations – anophthalmia and/or severe microphthalmia – seen in a single specialist ophthalmology center. The mutation analysis consisted of bidirectional sequencing of the coding regions of SOX2, OTX2, PAX6 (paired domain), STRA6, BMP4, SMOC1, FOXE3, and RAX, and genome-wide array-based copy number assessment. Fifteen (29.4%) of the 51 probands had likely causative mutations affecting SOX2 (9/51), OTX2 (5/51), and STRA6 (1/51). Of the cases with bilateral anophthalmia, 9/12 (75%) were found to be mutation positive. Three of these mutations were large genomic deletions encompassing SOX2 (one case) or OTX2 (two cases). Familial inheritance of three intragenic, plausibly pathogenic, and heterozygous mutations was observed. An unaffected carrier parent of an affected child with an identified OTX2 mutation confirmed the previously reported nonpenetrance for this disorder. Two families with SOX2 mutations demonstrated a parent and child both with significant but highly variable eye malformations. Heterozygous loss-of-function mutations in SOX2 and OTX2 are the most common genetic pathology associated with severe eye malformations and bi-allelic loss-of-function in STRA6 is confirmed as an emerging cause of nonsyndromal eye malformations. PMID:24498598

iMARS--mutation analysis reporting software: an analysis of spontaneous cII mutation spectra.

PubMed

Morgan, Claire; Lewis, Paul D

2006-01-31

The sensitivity of any mutational assay is determined by the level at which spontaneous mutations occur in the corresponding untreated controls. Establishing the type and frequency at which mutations occur naturally within a test system is essential if one is to draw scientifically sound conclusions regarding chemically induced mutations. Currently, mutation-spectra analysis is laborious and time-consuming. Thus, we have developed iMARS, a comprehensive mutation-spectrum analysis package that utilises routinely used methodologies and visualisation tools. To demonstrate the use and capabilities of iMARS, we have analysed the distribution, types and sequence context of spontaneous base substitutions derived from the cII gene mutation assay in transgenic animals. Analysis of spontaneous mutation spectra revealed variation both within and between the transgenic rodent test systems Big Blue Mouse, MutaMouse and Big Blue Rat. The most common spontaneous base substitutions were G:C-->A:T transitions and G:C-->T:A transversions. All Big Blue Mouse spectra were significantly different from each other by distribution and nearly all by mutation type, whereas the converse was true for the other test systems. Twenty-eight mutation hotspots were observed across all spectra generally occurring in CG, GA/TC, GG and GC dinucleotides. A mutation hotspot at nucleotide 212 occurred at a higher frequency in MutaMouse and Big Blue Rat. In addition, CG dinucleotides were the most mutable in all spectra except two Big Blue Mouse spectra. Thus, spontaneous base-substitution spectra showed more variation in distribution, type and sequence context in Big Blue Mouse relative to spectra derived from MutaMouse and Big Blue Rat. The results of our analysis provide a baseline reference for mutation studies utilising the cII gene in transgenic rodent models. The potential differences in spontaneous base-substitution spectra should be considered when making comparisons between these test systems. The ease at which iMARS has allowed us to carry out an exhaustive investigation to assess mutation distribution, mutation type, strand bias, target sequences and motifs, as well as predict mutation hotspots provides us with a valuable tool in helping to distinguish true chemically induced hotspots from background mutations and gives a true reflection of mutation frequency.

Clinical mutational profiling of 1006 lung cancers by next generation sequencing

PubMed Central

Illei, Peter B.; Belchis, Deborah; Tseng, Li-Hui; Nguyen, Doreen; De Marchi, Federico; Haley, Lisa; Riel, Stacy; Beierl, Katie; Zheng, Gang; Brahmer, Julie R.; Askin, Frederic B.; Gocke, Christopher D.; Eshleman, James R.; Forde, Patrick M.; Lin, Ming-Tseh

2017-01-01

Analysis of lung adenocarcinomas for actionable mutations has become standard of care. Here, we report our experience using next generation sequencing (NGS) to examine AKT1, BRAF, EGFR, ERBB2, KRAS, NRAS, and PIK3CA genes in 1006 non-small cell lung cancers in a clinical diagnostic setting. NGS demonstrated high sensitivity. Among 760 mutations detected, the variant allele frequency (VAF) was 2–5% in 33 (4.3%) mutations and 2–10% in 101 (13%) mutations. A single bioinformatics pipeline using Torrent Variant Caller, however, missed a variety of EGFR mutations. Mutations were detected in KRAS (36% of tumors), EGFR (19%) including 8 (0.8%) within the extracellular domain (4 at codons 108 and 4 at codon 289), BRAF (6.3%), and PIK3CA (3.7%). With a broader reportable range, exon 19 deletion and p.L858R accounted for only 36% and 26% of EGFR mutations and p.V600E accounted for only 24% of BRAF mutations. NGS provided accurate sequencing of complex mutations seen in 19% of EGFR exon 19 deletion mutations. Doublet (compound) EGFR mutations were observed in 29 (16%) of 187 EGFR-mutated tumors, including 69% with two non-p.L858R missense mutations and 24% with p.L858 and non-p.L858R missense mutations. Concordant VAFs suggests doublet EGFR mutations were present in a dominant clone and cooperated in oncogenesis. Mutants with predicted impaired kinase, observed in 25% of BRAF-mutated tumors, were associated with a higher incidence of concomitant activating KRAS mutations. NGS demonstrates high analytic sensitivity, broad reportable range, quantitative VAF measurement, single molecule sequencing to resolve complex deletion mutations, and simultaneous detection of concomitant mutations. PMID:29228562

Genetic analysis of leukemic transformation of chronic myeloproliferative neoplasms

PubMed Central

Abdel-Wahab, Omar; Manshouri, Taghi; Patel, Jay; Harris, Kelly; Yao, JinJuan; Hedvat, Cyrus; Heguy, Adriana; Bueso-Ramos, Carlos; Kantarjian, Hagop; Levine, Ross L.; Verstovsek, Srdan

2009-01-01

The genetic events which contribute to transformation of myeloproliferative neoplasms (MPN) to acute myeloid leukemia (AML) are not well characterized. We investigated the role of JAK2, TET2, ASXL1, and IDH1 mutations in leukemic transformation of MPNs through mutational analysis of 63 patients with AML secondary to a preexisting MPN (sAML). We identified frequent TET2 (26.3%), ASXL1 (19.3%), IDH1 (9.5%), and JAK2 (36.8%) mutations in sAML; all possible mutational combinations of these genes were observed. Analysis of 14 patients for which paired samples from MPN and sAML were available demonstrated TET2 mutations were frequently acquired at leukemic transformation (6/14=43%). In contrast, ASXL1 mutations were almost always detected in both the MPN and AML clones from individual patients. A case was also observed where TET2 and ASXL1 mutations were found before the patient acquired a JAK2 mutation or developed clinical evidence of MPN. We conclude that mutations in TET2, ASXL1, and IDH1 are common in sAML derived from a pre-existing MPN. Although TET2/ASXL1 mutations may precede acquisition of JAK2 mutations by the MPN clone, mutations in TET2, but not ASXL1, are commonly acquired at the time of leukemic transformation. These data suggest the mutational order of events in MPN and sAML varies in different patients, and that TET2 and ASXL1 mutations have distinct roles in MPN pathogenesis and leukemic transformation. The presence of sAML with no pre-existing JAK2/TET2/ASXL1/IDH1 mutations indicates the existence of other mutations necessary for leukemic transformation. PMID:20068184

Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients.

PubMed

Taly, Valerie; Pekin, Deniz; Benhaim, Leonor; Kotsopoulos, Steve K; Le Corre, Delphine; Li, Xinyu; Atochin, Ivan; Link, Darren R; Griffiths, Andrew D; Pallier, Karine; Blons, Hélène; Bouché, Olivier; Landi, Bruno; Hutchison, J Brian; Laurent-Puig, Pierre

2013-12-01

Multiplex digital PCR (dPCR) enables noninvasive and sensitive detection of circulating tumor DNA with performance unachievable by current molecular-detection approaches. Furthermore, picodroplet dPCR facilitates simultaneous screening for multiple mutations from the same sample. We investigated the utility of multiplex dPCR to screen for the 7 most common mutations in codons 12 and 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) oncogene from plasma samples of patients with metastatic colorectal cancer. Fifty plasma samples were tested from patients for whom the primary tumor biopsy tissue DNA had been characterized by quantitative PCR. Tumor characterization revealed that 19 patient tumors had KRAS mutations. Multiplex dPCR analysis of the plasma DNA prepared from these samples identified 14 samples that matched the mutation identified in the tumor, 1 sample contained a different KRAS mutation, and 4 samples had no detectable mutation. Among the tumor samples that were wild type for KRAS, 2 KRAS mutations were identified in the corresponding plasma samples. Duplex dPCR (i.e., wild-type and single-mutation assay) was also used to analyze plasma samples from patients with KRAS-mutated tumors and 5 samples expected to contain the BRAF (v-raf murine sarcoma viral oncogene homolog B) V600E mutation. The results for the duplex analysis matched those for the multiplex analysis for KRAS-mutated samples and, owing to its higher sensitivity, enabled detection of 2 additional samples with low levels of KRAS-mutated DNA. All 5 samples with BRAF mutations were detected. This work demonstrates the clinical utility of multiplex dPCR to screen for multiple mutations simultaneously with a sensitivity sufficient to detect mutations in circulating DNA obtained by noninvasive blood collection.

The origin of the p.E180 growth hormone receptor gene mutation.

PubMed

Ostrer, Harry

2016-06-01

Laron syndrome, an autosomal recessive condition of extreme short stature, is caused by the absence or dysfunction of the growth hormone receptor. A recurrent mutation in the GHR gene, p.E180, did not alter the encoded amino acid, but activated a cryptic splice acceptor resulting in a receptor protein with an 8-amino acid deletion in the extracellular domain. This mutation has been observed among Sephardic Jews and among individuals in Ecuador, Brazil and Chile, most notably in a large genetic isolate in Loja, Ecuador. A common origin has been postulated based on a shared genetic background of markers flanking this mutation, suggesting that the Lojanos (and others) may have Sephardic (Converso) Jewish ancestry. Analysis of the population structure of Lojanos based on genome-wide analysis demonstrated European, Sephardic Jewish and Native American ancestry in this group. X-autosomal comparison and monoallelic Y chromosomal and mitochondrial genetic analysis demonstrated gender-biased admixture between Native American women and European and Sephardic Jewish men. These findings are compatible with the co-occurrence of the Inquisition and the colonization of the Americas, including Converso Jews escaping the Inquisition in the Iberian Peninsula. Although not found among Lojanos, Converso Jews also brought founder mutations to contemporary Hispanic and Latino populations in the BRCA1 (c.68_69delAG) and BLM (c.2207_2212delATCTGAinsTAGATTC) genes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Novel compound heterozygous SPTA1 mutations in a patient with hereditary elliptocytosis

PubMed Central

Ma, Shiyue; Qin, Jinqiu; Wei, Aiqiu; Li, Xiaohong; Qin, Yuanyuan; Liao, Lin; Lin, Faquan

2018-01-01

Hereditaryelliptocytosis (HE) is a hereditary hemolytic disease, characterized by the presence of many elliptical erythrocytes in the peripheral blood that is caused by abnormal cytoskeletal proteins in the erythrocyte membrane. In the present study, a novel, causal HE mutation was reported. Routine blood examinations were performed on the proband and their family, and the fluorescence intensity of eosin-5-maleimide (EMA)-labeled erythrocytes was determined via flow cytometry. Subsequently, DNA was extracted from the peripheral blood of the proband and their family members, and amplified by quantitative polymerase chain reaction. The Sanger sequencing approach was used to determine and identify gene mutations, which were verified by matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry. To exclude genetic polymorphisms, newly identified mutations were subjected to large-scale gene screening using high-resolution melt analysis. Protein expression levels in the erythrocyte membrane of the proband were determined via SDS-PAGE, which demonstrated that, compared with healthy controls, the proband exhibited a reduction in EMA-labeled erythrocytes. In addition, DNA analysis demonstrated that the proband carried three mutations in the spectrin α chain erythrocytic 1 (SPTA1) gene: c.161A>C, c.5572C>G and 6531-12C>T. The corresponding mutant polypeptides were also analyzed by MALDI-TOF mass spectroscopy. SDS-PAGE analysis indicated that the proband exhibited normal levels of erythrocyte membrane proteins. In the present study, a novel HE case with a His54Pro mutation in the SPTA1 gene was reported. The results suggested that the His54Pro mutation influenced the role of erythrocyte membrane proteins without reducing its level of expression. PMID:29484404

Further Insights into the Allan-Herndon-Dudley Syndrome: Clinical and Functional Characterization of a Novel MCT8 Mutation.

PubMed

Armour, Christine M; Kersseboom, Simone; Yoon, Grace; Visser, Theo J

2015-01-01

Mutations in the thyroid hormone (TH) transporter MCT8 have been identified as the cause for Allan-Herndon-Dudley Syndrome (AHDS), characterized by severe psychomotor retardation and altered TH serum levels. Here we report a novel MCT8 mutation identified in 4 generations of one family, and its functional characterization. Proband and family members were screened for 60 genes involved in X-linked cognitive impairment and the MCT8 mutation was confirmed. Functional consequences of MCT8 mutations were studied by analysis of [125I]TH transport in fibroblasts and transiently transfected JEG3 and COS1 cells, and by subcellular localization of the transporter. The proband and a male cousin demonstrated clinical findings characteristic of AHDS. Serum analysis showed high T3, low rT3, and normal T4 and TSH levels in the proband. A MCT8 mutation (c.869C>T; p.S290F) was identified in the proband, his cousin, and several female carriers. Functional analysis of the S290F mutant showed decreased TH transport, metabolism and protein expression in the three cell types, whereas the S290A mutation had no effect. Interestingly, both uptake and efflux of T3 and T4 was impaired in fibroblasts of the proband, compared to his healthy brother. However, no effect of the S290F mutation was observed on TH efflux from COS1 and JEG3 cells. Immunocytochemistry showed plasma membrane localization of wild-type MCT8 and the S290A and S290F mutants in JEG3 cells. We describe a novel MCT8 mutation (S290F) in 4 generations of a family with Allan-Herndon-Dudley Syndrome. Functional analysis demonstrates loss-of-function of the MCT8 transporter. Furthermore, our results indicate that the function of the S290F mutant is dependent on cell context. Comparison of the S290F and S290A mutants indicates that it is not the loss of Ser but its substitution with Phe, which leads to S290F dysfunction.

Further Insights into the Allan-Herndon-Dudley Syndrome: Clinical and Functional Characterization of a Novel MCT8 Mutation

PubMed Central

Yoon, Grace; Visser, Theo J.

2015-01-01

Background Mutations in the thyroid hormone (TH) transporter MCT8 have been identified as the cause for Allan-Herndon-Dudley Syndrome (AHDS), characterized by severe psychomotor retardation and altered TH serum levels. Here we report a novel MCT8 mutation identified in 4 generations of one family, and its functional characterization. Methods Proband and family members were screened for 60 genes involved in X-linked cognitive impairment and the MCT8 mutation was confirmed. Functional consequences of MCT8 mutations were studied by analysis of [125I]TH transport in fibroblasts and transiently transfected JEG3 and COS1 cells, and by subcellular localization of the transporter. Results The proband and a male cousin demonstrated clinical findings characteristic of AHDS. Serum analysis showed high T3, low rT3, and normal T4 and TSH levels in the proband. A MCT8 mutation (c.869C>T; p.S290F) was identified in the proband, his cousin, and several female carriers. Functional analysis of the S290F mutant showed decreased TH transport, metabolism and protein expression in the three cell types, whereas the S290A mutation had no effect. Interestingly, both uptake and efflux of T3 and T4 was impaired in fibroblasts of the proband, compared to his healthy brother. However, no effect of the S290F mutation was observed on TH efflux from COS1 and JEG3 cells. Immunocytochemistry showed plasma membrane localization of wild-type MCT8 and the S290A and S290F mutants in JEG3 cells. Conclusions We describe a novel MCT8 mutation (S290F) in 4 generations of a family with Allan-Herndon-Dudley Syndrome. Functional analysis demonstrates loss-of-function of the MCT8 transporter. Furthermore, our results indicate that the function of the S290F mutant is dependent on cell context. Comparison of the S290F and S290A mutants indicates that it is not the loss of Ser but its substitution with Phe, which leads to S290F dysfunction. PMID:26426690

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

PubMed

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

2015-11-14

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

Genetic basis of glycogen storage disease type 1a: Prevalent mutations at the glucose-6-phosphatase locus

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

Ke-Jian Lei; Hungwen Chen; Ji-Lan Liu

Diagnosis of glycogen storage disease (GSD) type 1a currently is established by demonstrating the lack of glucose-6-phosphatase (G6Pase) activity in the patient`s biopsied liver specimen. Recent cloning of the G6Pase gene and identification of mutations within the gene that causes GSD type 1a allow for the development of a DNA-based diagnostic method. Using SSCP analysis and DNA sequencing, we characterized the G6Pase gene of 70 unrelated patients with enzymatically confirmed diagnosis of GSD type 1a and detected mutations in all except 17 alleles (88%). Sixteen mutations were uncovered that were shown by expression to abolish or greatly reduce G6Pase activitymore » and that therefore are responsible for the GSD type la disorder. R83C and Q347X are the most prevalent mutations found in Caucasians, 130X and R83C are most prevalent in Hispanics, and R83H is most prevalent in Chinese. The Q347X mutation has thus far been identified only in Caucasian patients, and the 130X mutation has been identified only in Hispanic patients. Our results demonstrate that the DNA-based analysis can accurately, rapidly, and noninvasively detect the majority of mutations in GSD type 1a. This DNA-based diagnosis now permits prenatal diagnosis among at-risk patients and serves as a database in screening and counseling patients clinically suspected of having this disease. 22 refs., 2 figs., 4 tabs.« less

Impact of proto-oncogene mutation detection in cytological specimens from thyroid nodules improves the diagnostic accuracy of cytology.

PubMed

Cantara, Silvia; Capezzone, Marco; Marchisotta, Stefania; Capuano, Serena; Busonero, Giulia; Toti, Paolo; Di Santo, Andrea; Caruso, Giuseppe; Carli, Anton Ferdinando; Brilli, Lucia; Montanaro, Annalisa; Pacini, Furio

2010-03-01

Fine-needle aspiration cytology (FNAC) is the gold standard for the differential diagnosis of thyroid nodules but has the limitation of inadequate sampling or indeterminate lesions. We aimed to verify whether search of thyroid cancer-associated protooncogene mutations in cytological samples may improve the diagnostic accuracy of FNAC. One hundred seventy-four consecutive patients undergoing thyroid surgery were submitted to FNAC (on 235 thyroid nodules) that was used for cytology and molecular analysis of BRAF, RAS, RET, TRK, and PPRgamma mutations. At surgery these nodules were sampled to perform the same molecular testing. Mutations were found in 67 of 235 (28.5%) cytological samples. Of the 67 mutated samples, 23 (34.3%) were mutated by RAS, 33 (49.3%) by BRAF, and 11 (16.4%) by RET/PTC. In 88.2% of the cases, the mutation was confirmed in tissue sample. The presence of mutations at cytology was associated with cancer 91.1% of the times and follicular adenoma 8.9% of the time. BRAF or RET/PTC mutations were always associated with cancer, whereas RAS mutations were mainly associated with cancer (74%) but also follicular adenoma (26%). The diagnostic performance of molecular analysis was superior to that of traditional cytology, with better sensitivity and specificity, and the combination of the two techniques further contributed to improve the total accuracy (93.2%), compared with molecular analysis (90.2%) or traditional cytology (83.0%). Our findings demonstrate that molecular analysis of cytological specimens is feasible and that its results in combination with cytology improves the diagnostic performance of traditional cytology.

Genetic analysis of hispanic individuals with cystic fibrosis

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

Grebe, T.A.; Doane, W.W.; Norman, R.A.

1994-03-01

The authors have performed molecular genetic analysis of Hispanic individuals with cystic fibrosis (CF) in the southwestern United States. Of 129 CF chromosomes analyzed, oly 46% (59/129) carry [Delta]F508. The G542X mutation was found on 5% (7/129) of CF chromosomes. The 3849+10kbC[yields]T mutation, detected primarily in Ashkenazi Jews, was present on 2% (3/129). R1162X and R334W, mutations identified in Spain and Italy, each occurred on 1.6% (2/129) of CF chromosomes. W1282X and R553X were each detected once. G551D and N1303K were not found. Overall, screening for 22 or more mutations resulted in detection of only 58% of CF transmembrane conductancemore » regulator gene mutations among Hispanic individuals. Analysis of KM19/XV2c haplotypes revealed an unusual distribution. Although the majority of [Delta]508 mutations are on chromosomes of B haplotypes, the other CF mutations are on A and C haplotypes at higher-than-expected frequencies. These genetic analysis demonstrate significant differences between Hispanic individuals with CF and those of the general North American population. Assessment of carrier/affected risk in Hispanic CF individuals cannot, therefore, be based on the mutation frequencies found through studies of the general population but must be adjusted to better reflect the genetic makeup of this ethnic group. Further studies are necessary to identify the causative mutation(s) in this population and to better delineate genotype/phenotype correlations. These will enable counselors to provide more accurate genetic counseling. 22 refs., 2 tabs.« less

A protein domain-centric approach for the comparative analysis of human and yeast phenotypically relevant mutations

PubMed Central

2013-01-01

Background The body of disease mutations with known phenotypic relevance continues to increase and is expected to do so even faster with the advent of new experimental techniques such as whole-genome sequencing coupled with disease association studies. However, genomic association studies are limited by the molecular complexity of the phenotype being studied and the population size needed to have adequate statistical power. One way to circumvent this problem, which is critical for the study of rare diseases, is to study the molecular patterns emerging from functional studies of existing disease mutations. Current gene-centric analyses to study mutations in coding regions are limited by their inability to account for the functional modularity of the protein. Previous studies of the functional patterns of known human disease mutations have shown a significant tendency to cluster at protein domain positions, namely position-based domain hotspots of disease mutations. However, the limited number of known disease mutations remains the main factor hindering the advancement of mutation studies at a functional level. In this paper, we address this problem by incorporating mutations known to be disruptive of phenotypes in other species. Focusing on two evolutionarily distant organisms, human and yeast, we describe the first inter-species analysis of mutations of phenotypic relevance at the protein domain level. Results The results of this analysis reveal that phenotypic mutations from yeast cluster at specific positions on protein domains, a characteristic previously revealed to be displayed by human disease mutations. We found over one hundred domain hotspots in yeast with approximately 50% in the exact same domain position as known human disease mutations. Conclusions We describe an analysis using protein domains as a framework for transferring functional information by studying domain hotspots in human and yeast and relating phenotypic changes in yeast to diseases in human. This first-of-a-kind study of phenotypically relevant yeast mutations in relation to human disease mutations demonstrates the utility of a multi-species analysis for advancing the understanding of the relationship between genetic mutations and phenotypic changes at the organismal level. PMID:23819456

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

PubMed

Dong, Chongmei; Vincent, Kate; Sharp, Peter

2009-12-04

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

Microarray-based mutation analysis of the ABCA4 (ABCR) gene in autosomal recessive cone-rod dystrophy and retinitis pigmentosa.

PubMed

Klevering, B Jeroen; Yzer, Suzanne; Rohrschneider, Klaus; Zonneveld, Marijke; Allikmets, Rando; van den Born, L Ingeborgh; Maugeri, Alessandra; Hoyng, Carel B; Cremers, Frans P M

2004-12-01

Mutations in the ABCA4 gene have been associated with autosomal recessive Stargardt disease (STGD1), cone-rod dystrophy (CRD), and retinitis pigmentosa (RP). We employed a recently developed genotyping microarray, the ABCR400-chip, to search for known ABCA4 mutations in patients with isolated or autosomal recessive CRD (54 cases) or RP (90 cases). We performed detailed ophthalmologic examinations and identified at least one ABCA4 mutation in 18 patients (33%) with CRD and in five patients (5.6%) with RP. Single-strand conformation polymorphism (SSCP) analysis and subsequent DNA sequencing revealed four novel missense mutations (R24C, E161K, P597S, G618E) and a novel 1-bp deletion (5888delG). Ophthalmoscopic abnormalities in CRD patients ranged from minor granular pigmentary changes in the posterior pole to widespread atrophy. In 12 patients with recordable electroretinogram (ERG) tracings, a cone-rod pattern was detected. Three patients demonstrated progression from a retinal dystrophy resembling STGD1 to a more widespread degeneration, and were subsequently diagnosed as CRD. In addition to a variable degree of atrophy, all RP patients displayed ophthalmologic characteristics of classic RP. When detectable, ERG recordings in these patients demonstrated rod-cone patterns of photoreceptor degeneration. In conclusion, in this study, we show that the ABCA4 mutation chip is an efficient first screening tool for arCRD.

Novel HSF4 mutation causes congenital total white cataract in a Chinese family.

PubMed

Ke, Tie; Wang, Qing K; Ji, Binchu; Wang, Xu; Liu, Ping; Zhang, Xianqin; Tang, Zhaohui; Ren, Xiang; Liu, Mugen

2006-08-01

To identify the disease-causing gene (mutation) in a Chinese family affected with autosomal dominant congenital total white cataract. Observational case series. Genotyping and linkage analyses were used to identify the linkage of the disease-causing gene in the Chinese family to the HSF4 gene encoding a member of the family of heat shock transcription factors (HSFs). Direct DNA sequence analysis was used to identify the disease-causing mutation. Polymerase chain reaction/restriction fragment length polymorphism analysis was used to demonstrate cosegregation of the HSF4 mutation with the cataract and the absence of the mutation in the normal controls. The cataract gene in the Chinese family was linked to marker D16S3043, and further haplotype analysis defined the causative gene between D16S515 and D16S415 within which HSF4 is located. A novel mutation c.221G>A was identified in HSF4, which results in substitution of a highly conserved arginine residue by histidine at codon 74 (p.R74H). The R74H mutation cosegregated with the affected individuals in the family and did not exist in unaffected family members and 150 unrelated normal controls. These results identified a novel missense mutation R74H in the transcription factor gene HSF4 in a Chinese cataract family and expand the spectrum of HSF4 mutations causing cataract.

Molecular analysis of the AGXT gene in Italian patients with primary hyperoxaluria type 1 (PH1).

PubMed

Ferrettini, C; Pirulli, D; Cosseddu, D; Marangella, M; Petrarulo, M; Mazzola, G; Vatta, S; Amoroso, A

1998-01-01

Specimens were collected from 22 Italian patients with primary hyperoxaluria type 1 (PH1). Ten of them had already been analyzed by molecular biology. To clarify the molecular characteristics of the AGXT gene disease responsible for PH1, DNA samples were examined for known mutations by hybridisation of PCR products with Sequence Specific Oligonucleotides (PCR-SSO). We planned to identify new mutations of the AGXT gene by heteroduplex analysis followed by direct sequencing. We had already standardized a) the conditions for the amplification of the 11 exons of AGXT, b) the PCR-SSO technique and c) the heteroduplex analysis of amplified products. Preliminary results demonstrated that the AGXT mutations described in previous studies were found only in 40% of the examined Italian patients with PH1. The remaining 60% of mutations should be characterised in future studies.

Label-free and high-sensitive detection for genetic point mutation based on hyperspectral interferometry

NASA Astrophysics Data System (ADS)

Fu, Rongxin; Li, Qi; Zhang, Junqi; Wang, Ruliang; Lin, Xue; Xue, Ning; Su, Ya; Jiang, Kai; Huang, Guoliang

2016-10-01

Label free point mutation detection is particularly momentous in the area of biomedical research and clinical diagnosis since gene mutations naturally occur and bring about highly fatal diseases. In this paper, a label free and high sensitive approach is proposed for point mutation detection based on hyperspectral interferometry. A hybridization strategy is designed to discriminate a single-base substitution with sequence-specific DNA ligase. Double-strand structures will take place only if added oligonucleotides are perfectly paired to the probe sequence. The proposed approach takes full use of the inherent conformation of double-strand DNA molecules on the substrate and a spectrum analysis method is established to point out the sub-nanoscale thickness variation, which benefits to high sensitive mutation detection. The limit of detection reach 4pg/mm2 according to the experimental result. A lung cancer gene point mutation was demonstrated, proving the high selectivity and multiplex analysis capability of the proposed biosensor.

Prolonged Response to Trastuzumab in a Patient With HER2-Nonamplified Breast Cancer With Elevated HER2 Dimerization Harboring an ERBB2 S310F Mutation.

PubMed

Chumsri, Saranya; Weidler, Jodi; Ali, Siraj; Balasubramanian, Sohail; Wallweber, Gerald; DeFazio-Eli, Lisa; Chenna, Ahmed; Huang, Weidong; DeRidder, Angela; Goicocheal, Lindsay; Perez, Edith A

2015-09-01

In the current genomic era, increasing evidence demonstrates that approximately 2% of HER2-negative breast cancers, by current standard testings, harbor activating mutations of ERBB2. However, whether patients with HER2-negative breast cancer with activating mutations of ERBB2 also experience response to anti-HER2 therapies remains unclear. This case report describes a patient with HER2-nonamplified heavily pretreated breast cancer who experienced prolonged response to trastuzumab in combination with pertuzumab and fulvestrant. Further molecular analysis demonstrated that her tumors had an elevated HER2 dimerization that corresponded to ERBB2 S310F mutation. Located in the extracellular domain of the HER2 protein, this mutation was reported to promote noncovalent dimerization that results in the activation of the downstream signaling pathways. This case highlights the fact that HER2-targeted therapy may be valuable in patients harboring an ERBB2 S310F mutation. Copyright © 2015 by the National Comprehensive Cancer Network.

Mutations in extracellular matrix genes NID1 and LAMC1 cause autosomal dominant Dandy-Walker malformation and occipital cephaloceles.

PubMed

Darbro, Benjamin W; Mahajan, Vinit B; Gakhar, Lokesh; Skeie, Jessica M; Campbell, Elizabeth; Wu, Shu; Bing, Xinyu; Millen, Kathleen J; Dobyns, William B; Kessler, John A; Jalali, Ali; Cremer, James; Segre, Alberto; Manak, J Robert; Aldinger, Kimerbly A; Suzuki, Satoshi; Natsume, Nagato; Ono, Maya; Hai, Huynh Dai; Viet, Le Thi; Loddo, Sara; Valente, Enza M; Bernardini, Laura; Ghonge, Nitin; Ferguson, Polly J; Bassuk, Alexander G

2013-08-01

We performed whole-exome sequencing of a family with autosomal dominant Dandy-Walker malformation and occipital cephaloceles and detected a mutation in the extracellular matrix (ECM) protein-encoding gene NID1. In a second family, protein interaction network analysis identified a mutation in LAMC1, which encodes a NID1-binding partner. Structural modeling of the NID1-LAMC1 complex demonstrated that each mutation disrupts the interaction. These findings implicate the ECM in the pathogenesis of Dandy-Walker spectrum disorders. © 2013 WILEY PERIODICALS, INC.

Calreticulin mutation-specific immunostaining in myeloproliferative neoplasms: pathogenetic insight and diagnostic value

PubMed Central

Vannucchi, A M; Rotunno, G; Bartalucci, N; Raugei, G; Carrai, V; Balliu, M; Mannarelli, C; Pacilli, A; Calabresi, L; Fjerza, R; Pieri, L; Bosi, A; Manfredini, R; Guglielmelli, P

2014-01-01

Mutations in the gene calreticulin (CALR) occur in the majority of JAK2- and MPL-unmutated patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF); identifying CALR mutations contributes to the diagnostic pathway of ET and PMF. CALR mutations are heterogeneous spanning over the exon 9, but all result in a novel common protein C terminus. We developed a polyclonal antibody against a 17-amino-acid peptide derived from mutated calreticulin that was used for immunostaining of bone marrow biopsies. We show that this antibody specifically recognized patients harboring different types of CALR mutation with no staining in healthy controls and JAK2- or MPL-mutated ET and PMF. The labeling was mostly localized in megakaryocytes, whereas myeloid and erythroid cells showed faint staining, suggesting a preferential expression of calreticulin in megakaryocytes. Megakaryocytic-restricted expression of calreticulin was also demonstrated using an antibody against wild-type calreticulin and by measuring the levels of calreticulin RNA by gene expression analysis. Immunostaining using an antibody specific for mutated calreticulin may become a rapid, simple and cost-effective method for identifying CALR-mutated patients complementing molecular analysis; furthermore, the labeling pattern supports the preferential expansion of megakaryocytic cell lineage as a result of CALR mutation in an immature hematopoietic stem cell. PMID:24618731

Secondary structure prediction and structure-specific sequence analysis of single-stranded DNA.

PubMed

Dong, F; Allawi, H T; Anderson, T; Neri, B P; Lyamichev, V I

2001-08-01

DNA sequence analysis by oligonucleotide binding is often affected by interference with the secondary structure of the target DNA. Here we describe an approach that improves DNA secondary structure prediction by combining enzymatic probing of DNA by structure-specific 5'-nucleases with an energy minimization algorithm that utilizes the 5'-nuclease cleavage sites as constraints. The method can identify structural differences between two DNA molecules caused by minor sequence variations such as a single nucleotide mutation. It also demonstrates the existence of long-range interactions between DNA regions separated by >300 nt and the formation of multiple alternative structures by a 244 nt DNA molecule. The differences in the secondary structure of DNA molecules revealed by 5'-nuclease probing were used to design structure-specific probes for mutation discrimination that target the regions of structural, rather than sequence, differences. We also demonstrate the performance of structure-specific 'bridge' probes complementary to non-contiguous regions of the target molecule. The structure-specific probes do not require the high stringency binding conditions necessary for methods based on mismatch formation and permit mutation detection at temperatures from 4 to 37 degrees C. Structure-specific sequence analysis is applied for mutation detection in the Mycobacterium tuberculosis katG gene and for genotyping of the hepatitis C virus.

Mutational Analysis of Cell Types in Tuberous Sclerosis Complex (TSC)

DTIC Science & Technology

2009-01-01

from mutations in the TSC1 or TSC2 genes that is associated with epilepsy, cognitive disability, and autism . TSC1/TSC2 gene mutations lead to...gene inactivation and leads to activation of the mTOR cascade as evidenced by phosphorylation of ribosomal S6 protein (P-S6). We demonstrate that...phosphorylation of the ribosomal S6 protein (phospho-S6 or P-S6), a marker for enhanced mTOR signaling. We find P-S6 expression in cortex as well as

An N-terminal glycine to cysteine mutation in the collagen COL1A1 gene produces moderately severe osteogenesis imperfecta

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

Wilcox, W.; Scott, L.; Cohn, D.

Osteogenesis imperfecta (OI) is usually due to mutations in the type I procollagen genes COL1A1 and COL1A2. Point mutations close to the N-terminus are generally milder than those near the C-terminus of the molecule (the gradient hypothesis of collagen mutations). We describe a patient with moderately severe OI due to a mutation in the N-terminal portion of the triple helical domain of the {alpha}1(I) chain. Electrophoretic analysis of collagen isolated from fibroblast cultures suggested the abnormal presence of a cysteine in the N-terminal portion of the {alpha}1(I) chain. Five overlapping DNA fragments amplified from fibroblast RNA were screened for mutationsmore » using single strand conformational polymorphism (SSCP) and heteroduplex analyses. Direct DNA sequence analysis of the single positive fragment demonstrated a G to T transversion, corresponding to a glycine to cysteine substitution at position 226 of the triple helical domain of the {alpha}1(I) chain. The mutation was confirmed by restriction enzyme analysis of amplified genomic DNA. The mutation was not present in fibroblasts from either phenotypically normal parent. Combining this mutation with other reported mutations, glycine to cysteine substitutions at positions 205, 211, 223, and 226 produce a moderately severe phenotype whereas flanking mutations at positions 175 and 382 produce a mild phenotype. This data supports a regional rather than a gradient model of the relationship between the nature and location of type I collagen mutations and OI phenotype.« less

Origin and migration of an Afrikaner founder mutation FHAfrikaner-2 (V408M) causing familial hypercholesterolemia.

PubMed

Defesche, J C; Van Diermen, D E; Hayden, M R; Kastelein, J P

1996-04-01

Of the three major Afrikaner founder mutations, responsible for more than 95% of Familial Hypercholesterolemia cases among South African Afrikaners, one mutation called V408M or FHAfrikaner-2 was identified in the Netherlands. Subsequent analysis of a group of Canadian patients of Dutch origin with Familial Hypercholesterolemia revealed the presence of this mutation in western Canada. The founder of the Canadian family, suffering from Familial Hypercholesterolemia caused by V408M, was traced back to Andijk, a small village in the northwestern part of the Netherlands, a region from where the first settlers to South Africa departed in the 17th and 18th century. Further genealogical investigation demonstrated that the mutation must have been introduced in the Netherlands by an individual from northern Germany. Haplotype analysis resulted in the identification of the common haplotypes TaqI-, StuI+, AvaII+, NcoI+ in Canadian as well as Dutch patients with V408M. The results of this study further support the hypothesis that Dutch settlers introduced this Afrikaner founder mutation in the Afrikaner population in South Africa. After a recombinational event in the mutated gene, the mutation was also introduced in western Canada.

Two novel compound heterozygous mutations in the BCKDHB gene that cause the intermittent form of maple syrup urine disease.

PubMed

Guo, Yi; Liming, Liu; Jiang, Li

2015-12-01

Intermittent maple syrup urine disease (MSUD) is a potentially life-threatening metabolic disorder caused by a deficiency of branched chain α-ketoacid dehydrogenase (BCKD) complex. In contrast to classic MSUD, children with the intermittent form usually have an atypical clinical manifestation. Here, we describe the presenting symptoms and clinical course of a Chinese boy with intermittent MSUD. Mutation analysis identified two previously unreported mutations in exon 7 of the BCKDHB gene: c.767A > G (p.Y256C) and c.768C > G (p.Y256X); the parents were each heterozygous for one of these mutations. In silico analysis predicted Y256C probably affects protein structure; Y256X leads to a premature stop codon. This case demonstrates intermittent MSUD should be suspected in cases with symptoms of recurrent encephalopathy, especially ataxia or marked drowsiness, which usually present after the neonatal period and in conjunction with infection. symmetrical basal ganglia damage but normal myelination in the posterior limb will assist differential diagnosis; alloisoleucine is a useful diagnostic marker and mutation analysis may be of prognostic value. These novel mutations Y256C and Y256X result in the clinical manifestation of a variant form of MSUD, expanding the mutation spectrum of this disease.

Genetic epidemiology of amyotrophic lateral sclerosis: a systematic review and meta-analysis.

PubMed

Zou, Zhang-Yu; Zhou, Zhi-Rui; Che, Chun-Hui; Liu, Chang-Yun; He, Rao-Li; Huang, Hua-Pin

2017-07-01

Genetic studies have shown that C9orf72 , SOD1 , TARDBP and FUS are the most common mutated genes in amyotrophic lateral sclerosis (ALS). Here, we performed a meta-analysis to determine the mutation frequencies of these major ALS-related genes in patients with ALS. We performed an extensive literature research to identify all original articles reporting frequencies of C9orf72 , SOD1 , TARDBP and FUS mutations in ALS. The mutation frequency and effect size of each study were combined. Possible sources of heterogeneity across studies were determined by meta-regression, sensitivity analysis and subgroup analysis. 111 studies were included in the meta-analysis. The overall pooled mutation frequencies of these major ALS-related genes were 47.7% in familial amyotrophic lateral sclerosis (FALS) and 5.2% in sporadic ALS (SALS). A significant difference was identified regarding the frequencies of mutations in major ALS genes between European and Asian patients. In European populations, the most common mutations were the C9orf72 repeat expansions (FALS 33.7%, SALS 5.1%), followed by SOD1 (FALS 14.8%, SALS 1.2%), TARDBP (FALS 4.2%, SALS 0.8%) and FUS mutations (FALS 2.8%, SALS 0.3%), while in Asian populations the most common mutations were SOD1 mutations (FALS 30.0%, SALS 1.5%), followed by FUS (FALS 6.4%, SALS 0.9%), C9orf72 (FALS 2.3%, SALS 0.3%) and TARDBP (FALS 1.5%, SALS 0.2%) mutations. These findings demonstrated that the genetic architecture of ALS in Asian populations is distinct from that in European populations, which need to be given appropriate consideration when performing genetic testing of patients with ALS. 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/.

Mutational signature analysis identifies MUTYH deficiency in colorectal cancers and adrenocortical carcinomas: Mutational signature associated with MUTYH deficiency in cancers

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

Pilati, Camilla; Shinde, Jayendra; Alexandrov, Ludmil B.

Germline alterations in DNA repair genes are implicated in cancer predisposition and can result in characteristic mutational signatures. However, specific mutational signatures associated with base excision repair (BER) defects remain to be characterized. Here, by analysing a series of colorectal cancers (CRCs) using exome sequencing, we identified a particular spectrum of somatic mutations characterized by an enrichment of C > A transversions in NpCpA or NpCpT contexts in three tumours from a MUTYH-associated polyposis (MAP) patient and in two cases harbouring pathogenic germline MUTYH mutations. In two series of adrenocortical carcinomas (ACCs), we identified four tumours with a similar signaturemore » also presenting germline MUTYH mutations. Altogether, these findings demonstrate that MUTYH inactivation results in a particular mutational signature, which may serve as a useful marker of BER-related genomic instability in new cancer types.« less

Mutational signature analysis identifies MUTYH deficiency in colorectal cancers and adrenocortical carcinomas: Mutational signature associated with MUTYH deficiency in cancers

DOE PAGES

Pilati, Camilla; Shinde, Jayendra; Alexandrov, Ludmil B.; ...

2017-03-29

Germline alterations in DNA repair genes are implicated in cancer predisposition and can result in characteristic mutational signatures. However, specific mutational signatures associated with base excision repair (BER) defects remain to be characterized. Here, by analysing a series of colorectal cancers (CRCs) using exome sequencing, we identified a particular spectrum of somatic mutations characterized by an enrichment of C > A transversions in NpCpA or NpCpT contexts in three tumours from a MUTYH-associated polyposis (MAP) patient and in two cases harbouring pathogenic germline MUTYH mutations. In two series of adrenocortical carcinomas (ACCs), we identified four tumours with a similar signaturemore » also presenting germline MUTYH mutations. Altogether, these findings demonstrate that MUTYH inactivation results in a particular mutational signature, which may serve as a useful marker of BER-related genomic instability in new cancer types.« less

New insights into genotype-phenotype correlations for the doublecortin-related lissencephaly spectrum.

PubMed

Bahi-Buisson, Nadia; Souville, Isabelle; Fourniol, Franck J; Toussaint, Aurelie; Moores, Carolyn A; Houdusse, Anne; Lemaitre, Jean Yves; Poirier, Karine; Khalaf-Nazzal, Reham; Hully, Marie; Leger, Pierre Louis; Elie, Caroline; Boddaert, Nathalie; Beldjord, Cherif; Chelly, Jamel; Francis, Fiona

2013-01-01

X-linked isolated lissencephaly sequence and subcortical band heterotopia are allelic human disorders associated with mutations of doublecortin (DCX), giving both familial and sporadic forms. DCX encodes a microtubule-associated protein involved in neuronal migration during brain development. Structural data show that mutations can fall either in surface residues, likely to impair partner interactions, or in buried residues, likely to impair protein stability. Despite the progress in understanding the molecular basis of these disorders, the prognosis value of the location and impact of individual DCX mutations has largely remained unclear. To clarify this point, we investigated a cohort of 180 patients who were referred with the agyria-pachygyria subcortical band heterotopia spectrum. DCX mutations were identified in 136 individuals. Analysis of the parents' DNA revealed the de novo occurrence of DCX mutations in 76 cases [62 of 70 females screened (88.5%) and 14 of 60 males screened (23%)], whereas in the remaining cases, mutations were inherited from asymptomatic (n = 14) or symptomatic mothers (n = 11). This represents 100% of families screened. Female patients with DCX mutation demonstrated three degrees of clinical-radiological severity: a severe form with a thick band (n = 54), a milder form (n = 24) with either an anterior thin or an intermediate thickness band and asymptomatic carrier females (n = 14) with normal magnetic resonance imaging results. A higher proportion of nonsense and frameshift mutations were identified in patients with de novo mutations. An analysis of predicted effects of missense mutations showed that those destabilizing the structure of the protein were often associated with more severe phenotypes. We identified several severe- and mild-effect mutations affecting surface residues and observed that the substituted amino acid is also critical in determining severity. Recurrent mutations representing 34.5% of all DCX mutations often lead to similar phenotypes, for example, either severe in sporadic subcortical band heterotopia owing to Arg186 mutations or milder in familial cases owing to Arg196 mutations. Taken as a whole, these observations demonstrate that DCX-related disorders are clinically heterogeneous, with severe sporadic and milder familial subcortical band heterotopia, each associated with specific DCX mutations. There is a clear influence of the individual mutated residue and the substituted amino acid in determining phenotype severity.

Pitfalls in genetic analysis of pheochromocytomas/paragangliomas-case report.

PubMed

Canu, Letizia; Rapizzi, Elena; Zampetti, Benedetta; Fucci, Rossella; Nesi, Gabriella; Richter, Susan; Qin, Nan; Giachè, Valentino; Bergamini, Carlo; Parenti, Gabriele; Valeri, Andrea; Ercolino, Tonino; Eisenhofer, Graeme; Mannelli, Massimo

2014-07-01

About 35% of patients with pheochromocytoma/paraganglioma carry a germline mutation in one of the 10 main susceptibility genes. The recent introduction of next-generation sequencing will allow the analysis of all these genes in one run. When positive, the analysis is generally unequivocal due to the association between a germline mutation and a concordant clinical presentation or positive family history. When genetic analysis reveals a novel mutation with no clinical correlates, particularly in the presence of a missense variant, the question arises whether the mutation is pathogenic or a rare polymorphism. We report the case of a 35-year-old patient operated for a pheochromocytoma who turned out to be a carrier of a novel SDHD (succinate dehydrogenase subunit D) missense mutation. With no positive family history or clinical correlates, we decided to perform additional analyses to test the clinical significance of the mutation. We performed in silico analysis, tissue loss of heterozygosity analysis, immunohistochemistry, Western blot analysis, SDH enzymatic assay, and measurement of the succinate/fumarate concentration ratio in the tumor tissue by tandem mass spectrometry. Although the in silico analysis gave contradictory results according to the different methods, all the other tests demonstrated that the SDH complex was conserved and normally active. We therefore came to the conclusion that the variant was a nonpathogenic polymorphism. Advancements in technology facilitate genetic analysis of patients with pheochromocytoma but also offer new challenges to the clinician who, in some cases, needs clinical correlates and/or functional tests to give significance to the results of the genetic assay.

ITGB6 loss-of-function mutations cause autosomal recessive amelogenesis imperfecta

PubMed Central

Wang, Shih-Kai; Choi, Murim; Richardson, Amelia S.; Reid, Bryan M.; Lin, Brent P.; Wang, Susan J.; Kim, Jung-Wook; Simmer, James P.; Hu, Jan C.-C.

2014-01-01

Integrins are cell-surface adhesion receptors that bind to extracellular matrices (ECM) and mediate cell–ECM interactions. Some integrins are known to play critical roles in dental enamel formation. We recruited two Hispanic families with generalized hypoplastic amelogenesis imperfecta (AI). Analysis of whole-exome sequences identified three integrin beta 6 (ITGB6) mutations responsible for their enamel malformations. The female proband of Family 1 was a compound heterozygote with an ITGB6 transition mutation in Exon 4 (g.4545G > A c.427G > A p.Ala143Thr) and an ITGB6 transversion mutation in Exon 6 (g.27415T > A c.825T > A p.His275Gln). The male proband of Family 2 was homozygous for an ITGB6 transition mutation in Exon 11 (g.73664C > T c.1846C > T p.Arg616*) and hemizygous for a transition mutation in Exon 6 of Nance–Horan Syndrome (NHS Xp22.13; g.355444T > C c.1697T > C p.Met566Thr). These are the first disease-causing ITGB6 mutations to be reported. Immunohistochemistry of mouse mandibular incisors localized ITGB6 to the distal membrane of differentiating ameloblasts and pre-ameloblasts, and then ITGB6 appeared to be internalized by secretory stage ameloblasts. ITGB6 expression was strongest in the maturation stage and its localization was associated with ameloblast modulation. Our findings demonstrate that early and late amelogenesis depend upon cell–matrix interactions. Our approach (from knockout mouse phenotype to human disease) demonstrates the power of mouse reverse genetics in mutational analysis of human genetic disorders and attests to the need for a careful dental phenotyping in large-scale knockout mouse projects. PMID:24305999

Fatal mitochondrial encephalopathy caused by fumarase deficiency: A molecular-genetic study

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

Gellera, C.; Cavadini, P.; Baratta, S.

Fumarase deficiency is a rare autosomal recessive disorder of the citric acid cycle resulting in severe organic aciduria and encephalopathy. Mammalian cells contain two fumarase isoenzymes, one mitochondrial and one cytosolic. In rat, the two proteins are encoded by the same gene and are synthesized by alternative initiation of translation at two in-phase AUG codons. One single fumarase gene locus has been identified on human chromosome 1. In most of the patients so far described, the activities of both isozymes are severely affected, suggesting that mutations within a single gene may underlie the disease. Here, we report the molecular studymore » of fumarase deficiency in a patient exhibiting compound heterozygosity for two different allelic mutations affecting the amino acid composition of both isoforms. The proband, an Italian boy of nonconsanguineous parents, died at 7 months of age of a progressive encephalopathy. Immunoblot demonstrated absence of cross-reacting material in both cytosolic and mitochondrial fraction of all tissues examined. Molecular analysis of the patient`s fumarase cDNA amplified by RT-PCR showed the presence of two mutations affecting the amino acid composition of both isoforms, a missense mutation resulting in the nonconservative amino acid substitution at codon 190 (Arg190Cys) and an amino acid in-frame insertion at codon 434 (Lys434ins). SSCP analysis of genomic PCR fragments encompassing the mutations demonstrated that the patient was heterozygous for both mutations, having inherited the Arg-to-Cys substitution from the father and the in-frame insertion from the mother. Finally, the effects of the mutations on enzyme function were investigated by expressing both normal and mutated fumarase cDNAs in a fumarase-deficient ({delta}FUM1) S. cerevisiae strain.« less

ITGB6 loss-of-function mutations cause autosomal recessive amelogenesis imperfecta.

PubMed

Wang, Shih-Kai; Choi, Murim; Richardson, Amelia S; Reid, Bryan M; Lin, Brent P; Wang, Susan J; Kim, Jung-Wook; Simmer, James P; Hu, Jan C-C

2014-04-15

Integrins are cell-surface adhesion receptors that bind to extracellular matrices (ECM) and mediate cell-ECM interactions. Some integrins are known to play critical roles in dental enamel formation. We recruited two Hispanic families with generalized hypoplastic amelogenesis imperfecta (AI). Analysis of whole-exome sequences identified three integrin beta 6 (ITGB6) mutations responsible for their enamel malformations. The female proband of Family 1 was a compound heterozygote with an ITGB6 transition mutation in Exon 4 (g.4545G > A c.427G > A p.Ala143Thr) and an ITGB6 transversion mutation in Exon 6 (g.27415T > A c.825T > A p.His275Gln). The male proband of Family 2 was homozygous for an ITGB6 transition mutation in Exon 11 (g.73664C > T c.1846C > T p.Arg616*) and hemizygous for a transition mutation in Exon 6 of Nance-Horan Syndrome (NHS Xp22.13; g.355444T > C c.1697T > C p.Met566Thr). These are the first disease-causing ITGB6 mutations to be reported. Immunohistochemistry of mouse mandibular incisors localized ITGB6 to the distal membrane of differentiating ameloblasts and pre-ameloblasts, and then ITGB6 appeared to be internalized by secretory stage ameloblasts. ITGB6 expression was strongest in the maturation stage and its localization was associated with ameloblast modulation. Our findings demonstrate that early and late amelogenesis depend upon cell-matrix interactions. Our approach (from knockout mouse phenotype to human disease) demonstrates the power of mouse reverse genetics in mutational analysis of human genetic disorders and attests to the need for a careful dental phenotyping in large-scale knockout mouse projects.

Assessment of clinical analytical sensitivity and specificity of next-generation sequencing for detection of simple and complex mutations.

PubMed

Chin, Ephrem L H; da Silva, Cristina; Hegde, Madhuri

2013-02-19

Detecting mutations in disease genes by full gene sequence analysis is common in clinical diagnostic laboratories. Sanger dideoxy terminator sequencing allows for rapid development and implementation of sequencing assays in the clinical laboratory, but it has limited throughput, and due to cost constraints, only allows analysis of one or at most a few genes in a patient. Next-generation sequencing (NGS), on the other hand, has evolved rapidly, although to date it has mainly been used for large-scale genome sequencing projects and is beginning to be used in the clinical diagnostic testing. One advantage of NGS is that many genes can be analyzed easily at the same time, allowing for mutation detection when there are many possible causative genes for a specific phenotype. In addition, regions of a gene typically not tested for mutations, like deep intronic and promoter mutations, can also be detected. Here we use 20 previously characterized Sanger-sequenced positive controls in disease-causing genes to demonstrate the utility of NGS in a clinical setting using standard PCR based amplification to assess the analytical sensitivity and specificity of the technology for detecting all previously characterized changes (mutations and benign SNPs). The positive controls chosen for validation range from simple substitution mutations to complex deletion and insertion mutations occurring in autosomal dominant and recessive disorders. The NGS data was 100% concordant with the Sanger sequencing data identifying all 119 previously identified changes in the 20 samples. We have demonstrated that NGS technology is ready to be deployed in clinical laboratories. However, NGS and associated technologies are evolving, and clinical laboratories will need to invest significantly in staff and infrastructure to build the necessary foundation for success.

Familial Mediterranean fever with a single MEFV mutation: Where is the second hit?

PubMed Central

Booty, Matthew G.; Chae, Jae Jin; Masters, Seth L.; Remmers, Elaine F.; Barham, Beverly; Lee, Julie M.; Barron, Karyl S.; Holland, Steve; Kastner, Daniel L.; Aksentijevich, Ivona

2009-01-01

Objective FMF has traditionally been considered an autosomal recessive disease; however, it has been observed that a substantial number of patients with clinical FMF possess only one demonstrable MEFV mutation. Here, an extensive search for a second MEFV mutation was performed in 46 patients clinically diagnosed with FMF and carrying only one high-penetrance FMF mutation. Methods MEFV and other candidate genes were sequenced by standard capillary electrophoresis. The entire 15 kb MEFV genomic region was re-sequenced in 10 patients using a hybridization-based chip technology. MEFV gene expression levels were determined by qRT-PCR and pyrin protein levels were examined by Western blotting. Results A second MEFV mutation was not identified in any of the screened patients. Haplotype analysis did not identify a common haplotype that might be associated with the transmission of a second FMF allele. Western blots did not demonstrate a significant difference in pyrin levels between single and double variant patients; however, FMF patients of both types showed higher protein expression compared to controls and non-FMF patients with active inflammation. Screening of genes encoding pyrin-interacting proteins identified rare variants in a small number of patients, suggesting the possibility of digenic inheritance. Conclusion Our data underscore the existence of a significant subset of FMF patients who are carriers of only one MEFV mutation and demonstrate that complete MEFV sequencing is not likely to yield a second mutation. Screening for the set of most common mutations appears sufficient in the presence of clinical symptoms to diagnose FMF and initiate a trial of colchicine. PMID:19479870

Whole-exome sequencing identifies USH2A mutations in a pseudo-dominant Usher syndrome family.

PubMed

Zheng, Sui-Lian; Zhang, Hong-Liang; Lin, Zhen-Lang; Kang, Qian-Yan

2015-10-01

Usher syndrome (USH) is an autosomal recessive (AR) multi-sensory degenerative disorder leading to deaf-blindness. USH is clinically subdivided into three subclasses, and 10 genes have been identified thus far. Clinical and genetic heterogeneities in USH make a precise diagnosis difficult. A dominant‑like USH family in successive generations was identified, and the present study aimed to determine the genetic predisposition of this family. Whole‑exome sequencing was performed in two affected patients and an unaffected relative. Systematic data were analyzed by bioinformatic analysis to remove the candidate mutations via step‑wise filtering. Direct Sanger sequencing and co‑segregation analysis were performed in the pedigree. One novel and two known mutations in the USH2A gene were identified, and were further confirmed by direct sequencing and co‑segregation analysis. The affected mother carried compound mutations in the USH2A gene, while the unaffected father carried a heterozygous mutation. The present study demonstrates that whole‑exome sequencing is a robust approach for the molecular diagnosis of disorders with high levels of genetic heterogeneity.

A new nonsense mutation in the NF1 gene with neurofibromatosis-Noonan syndrome phenotype.

PubMed

Yimenicioğlu, Sevgi; Yakut, Ayten; Karaer, Kadri; Zenker, Martin; Ekici, Arzu; Carman, Kürşat Bora

2012-12-01

Neurofibromatosis-Noonan syndrome is a rare autosomal dominant disorder which combines neurofibromatosis type 1 (NF1) features with Noonan syndrome. NF1 gene mutations are reported in the majority of these patients. Sequence analysis of the established genes for Noonan syndrome revealed no mutation; a heterozygous NF1 point mutation c.7549C>T in exon 51, creating a premature stop codon (p.R2517X), had been demonstrated. Neurofibromatosis-Noonan syndrome recently has been considered a subtype of NF1 and caused by different NF1 mutations. We report the case of a 14-year-old boy with neurofibromatosis type 1 with Noonan-like features, who complained of headache with triventricular hydrocephaly and a heterozygous NF1 point mutation c.7549C>T in exon 51.

Mutation analysis of GM1 gangliosidosis in a Siamese cat from Japan in the 1960s.

PubMed

Uddin, Mohammad M; Tanimoto, Takeshi; Yabuki, Akira; Kotani, Takao; Kuwamura, Mitsuru; Chang, Hye-Sook; Yamato, Osamu

2012-12-01

GM1 gangliosidosis is a fatal, progressive neurodegenerative lysosomal storage disease caused by mutations of the β-galactosidase (GLB1) gene. In feline GM1 gangliosidosis, a pathogenic mutation (c.1448G>C) of the feline GLB1 gene was identified in Siamese and Korat cats previously diagnosed with the disease in the USA and Italy, respectively. The present study demonstrated the same mutation in a Siamese cat that had been diagnosed with GM1 gangliosidosis in Japan in the 1960s. The mutation was confirmed using DNA extracted from stored paraffin-embedded brain tissue by a direct sequencing method and a polymerase chain reaction-restriction fragment length polymorphism assay. This pathogenic mutation seems to have been distributed around the world.

Trafficking Defect and Proteasomal Degradation Contribute to the Phenotype of a Novel KCNH2 Long QT Syndrome Mutation

PubMed Central

Mihic, Anton; Chauhan, Vijay S.; Gao, Xiaodong; Oudit, Gavin Y.; Tsushima, Robert G.

2011-01-01

The Kv11.1 (hERG) K+ channel plays a fundamental role in cardiac repolarization. Missense mutations in KCNH2, the gene encoding Kv11.1, cause long QT syndrome (LQTS) and frequently cause channel trafficking-deficiencies. This study characterized the properties of a novel KCNH2 mutation discovered in a LQT2 patient resuscitated from a ventricular fibrillation arrest. Proband genotyping was performed by SSCP and DNA sequencing. The electrophysiological and biochemical properties of the mutant channel were investigated after expression in HEK293 cells. The proband manifested a QTc of 554 ms prior to electrolyte normalization. Mutation analysis revealed an autosomal dominant frameshift mutation at proline 1086 (P1086fs+32X; 3256InsG). Co-immunoprecipitation demonstrated that wild-type Kv11.1 and mutant channels coassemble. Western blot showed that the mutation did not produce mature complex-glycosylated Kv11.1 channels and coexpression resulted in reduced channel maturation. Electrophysiological recordings revealed mutant channel peak currents to be similar to untransfected cells. Co-expression of channels in a 1∶1 ratio demonstrated dominant negative suppression of peak Kv11.1 currents. Immunocytochemistry confirmed that mutant channels were not present at the plasma membrane. Mutant channel trafficking rescue was attempted by incubation at reduced temperature or with the pharmacological agents E-4031. These treatments did not significantly increase peak mutant currents or induce the formation of mature complex-glycosylated channels. The proteasomal inhibitor lactacystin increased the protein levels of the mutant channels demonstrating proteasomal degradation, but failed to induce mutant Kv11.1 protein trafficking. Our study demonstrates a novel dominant-negative Kv11.1 mutation, which results in degraded non-functional channels leading to a LQT2 phenotype. PMID:21483829

Significance of TP53 Mutation in Wilms Tumors with Diffuse Anaplasia: A Report from the Children's Oncology Group.

PubMed

Ooms, Ariadne H A G; Gadd, Samantha; Gerhard, Daniela S; Smith, Malcolm A; Guidry Auvil, Jaime M; Meerzaman, Daoud; Chen, Qing-Rong; Hsu, Chih Hao; Yan, Chunhua; Nguyen, Cu; Hu, Ying; Ma, Yussanne; Zong, Zusheng; Mungall, Andrew J; Moore, Richard A; Marra, Marco A; Huff, Vicki; Dome, Jeffrey S; Chi, Yueh-Yun; Tian, Jing; Geller, James I; Mullighan, Charles G; Ma, Jing; Wheeler, David A; Hampton, Oliver A; Walz, Amy L; van den Heuvel-Eibrink, Marry M; de Krijger, Ronald R; Ross, Nicole; Gastier-Foster, Julie M; Perlman, Elizabeth J

2016-11-15

To investigate the role and significance of TP53 mutation in diffusely anaplastic Wilms tumors (DAWTs). All DAWTs registered on National Wilms Tumor Study-5 (n = 118) with available samples were analyzed for TP53 mutations and copy loss. Integrative genomic analysis was performed on 39 selected DAWTs. Following analysis of a single random sample, 57 DAWTs (48%) demonstrated TP53 mutations, 13 (11%) copy loss without mutation, and 48 (41%) lacked both [defined as TP53-wild-type (wt)]. Patients with stage III/IV TP53-wt DAWTs (but not those with stage I/II disease) had significantly lower relapse and death rates than those with TP53 abnormalities. In-depth analysis of a subset of 39 DAWTs showed seven (18%) to be TP53-wt: These demonstrated gene expression evidence of an active p53 pathway. Retrospective pathology review of TP53-wt DAWT revealed no or very low volume of anaplasia in six of seven tumors. When samples from TP53-wt tumors known to contain anaplasia histologically were available, abnormal p53 protein accumulation was observed by immunohistochemistry. These data support the key role of TP53 loss in the development of anaplasia in WT, and support its significant clinical impact in patients with residual anaplastic tumor following surgery. These data also suggest that most DAWTs will show evidence of TP53 mutation when samples selected for the presence of anaplasia are analyzed. This suggests that modifications of the current criteria to also consider volume of anaplasia and documentation of TP53 aberrations may better reflect the risk of relapse and death and enable optimization of therapeutic stratification. Clin Cancer Res; 22(22); 5582-91. ©2016 AACR. ©2016 American Association for Cancer Research.

Significance of TP53 Mutation in Wilms Tumors with Diffuse Anaplasia: A Report from the Children’s Oncology Group

PubMed Central

Ooms, Ariadne H.A.G.; Gadd, Samantha; Gerhard, Daniela S.; Smith, Malcolm A.; Guidry Auvil, Jaime M.; Meerzaman, Daoud; Chen, Qing-Rong; Hsu, Chih Hao; Yan, Chunhua; Nguyen, Cu; Hu, Ying; Ma, Yussanne; Zong, Zusheng; Mungall, Andrew J.; Moore, Richard A.; Marra, Marco A.; Huff, Vicki; Dome, Jeffrey S.; Chi, Yueh-Yun; Tian, Jing; Geller, James I.; Mullighan, Charles G.; Ma, Jing; Wheeler, David A.; Hampton, Oliver A.; Walz, Amy L.; van den Heuvel-Eibrink, Marry M.; de Krijger, Ronald R.; Ross, Nicole; Gastier-Foster, Julie M.; Perlman, Elizabeth J.

2016-01-01

Purpose To investigate the role and significance of TP53 mutation in diffusely anaplastic Wilms tumor (DAWT). Experimental Design All DAWTs registered on National Wilms Tumor Study-5 (n=118) with available samples were analyzed for TP53 mutations and copy loss. Integrative genomic analysis was performed on 39 selected DAWTs. Results Following analysis of a single random sample, 57 DAWT (48%) demonstrated TP53 mutations, 13(11%) copy loss without mutation, and 48(41%) lacked both (defined as TP53-wildtype (wt)). Patients with Stage III/IV TP53-wt DAWTs (but not those with Stage I/II disease) had significantly lower relapse and death rates than those with TP53 abnormalities. In-depth analysis of a subset of 39 DAWT showed 7(18%) to be TP53-wt: these demonstrated gene expression evidence of an active p53 pathway. Retrospective pathology review of TP53-wt DAWT revealed no or very low volume of anaplasia in 6/7 tumors. When samples from TP53-wt tumors known to contain anaplasia histologically were available, abnormal p53 protein accumulation was observed by immunohistochemistry. Conclusion These data support the key role of TP53 loss in the development of anaplasia in WT, and support its significant clinical impact in patients with residual anaplastic tumor following surgery. These data also suggest that most DAWTs will show evidence of TP53 mutation when samples selected for the presence of anaplasia are analyzed. This suggests that modifications of the current criteria to also consider volume of anaplasia and documentation of TP53 aberrations may better reflect the risk of relapse and death and enable optimization of therapeutic stratification. PMID:27702824

A de novo novel missense mutation in AVPR2 with severe nephrogenic diabetes insipidus

PubMed Central

Kobayashi, Daisuke; Nagaraj, Shashi K.; Lin, Jen-Jar; Bichet, Daniel G.

2010-01-01

We describe a paediatric case of nephrogenic diabetes insipidus (NDI) with a novel mutation in the arginine vasopressin receptor 2 gene (AVPR2) in the absence of a family history of congenital polyuria. The patient, a 5-month-old Caucasian boy, had failure to thrive and hypernatraemia. On admission to hospital, he had a plasma sodium of 171 mEq/L with a concomittant urine osmolality of 131 mOsm/kg. Molecular genetic analysis demonstrated that the patient had an AVPR2 mutation (c.861C > G) resulting in a substitution of tryptophan for serine at amino acid position 167 (p.Ser167Trp). His mother was heterozygous for the same Ser167Trp mutation which was found to be de novo from the DNA analysis of the maternal grandparents. PMID:25949462

Congenital amegakaryocytic thrombocytopenia in three siblings: molecular analysis of atypical clinical presentation.

PubMed

Gandhi, Manish J; Pendergrass, Thomas W; Cummings, Carrie C; Ihara, Kenji; Blau, C Anthony; Drachman, Jonathan G

2005-10-01

An 11-year-old girl, presenting with fatigue and bruising, was found to be profoundly pancytopenic. Bone marrow exam and clinical evaluation were consistent with aplastic anemia. Family members were studied as potential stem cell donors, revealing that both younger siblings displayed significant thrombocytopenia, whereas both parents had normal blood counts. We evaluated this pedigree to understand the unusually late presentation of congenital amegakaryocytic thrombocytopenia (CAMT). The coding region and the intron/exon junctions of MPL were sequenced from each family member. Vectors representing each of the mutations were constructed and tested for the ability to support growth of Baf3/Mpl(mutant) cells. All three siblings had elevated thrombopoietin levels. Analysis of genomic DNA demonstrated that each parent had mutations/polymorphisms in a single MPL allele and that each child was a compound heterozygote, having inherited both abnormal alleles. The maternal allele encoded a mutation of the donor splice-junction at the exon-3/intron-3 boundary. A mini-gene construct encoding normal vs mutant versions of the intron-3 donor-site demonstrated that physiologic splicing was significantly reduced in the mutant construct. Mutations that incompletely eliminate Mpl expression/function may result in delayed diagnosis of CAMT and confusion with aplastic anemia.

Accumulation of Pol Mutations Selected by HLA-B*52:01-C*12:02 Protective Haplotype-Restricted Cytotoxic T Lymphocytes Causes Low Plasma Viral Load Due to Low Viral Fitness of Mutant Viruses

PubMed Central

Murakoshi, Hayato; Koyanagi, Madoka; Chikata, Takayuki; Rahman, Mohammad Arif; Kuse, Nozomi; Sakai, Keiko; Gatanaga, Hiroyuki; Oka, Shinichi

2016-01-01

ABSTRACT HLA-B*52:01-C*12:02, which is the most abundant haplotype in Japan, has a protective effect on disease progression in HIV-1-infected Japanese individuals, whereas HLA-B*57 and -B*27 protective alleles are very rare in Japan. A previous study on HLA-associated polymorphisms demonstrated that the number of HLA-B*52:01-associated mutations at four Pol positions was inversely correlated with plasma viral load (pVL) in HLA-B*52:01-negative individuals, suggesting that the transmission of HIV-1 with these mutations could modulate the pVL in the population. However, it remains unknown whether these mutations were selected by HLA-B*52:01-restricted CTLs and also reduced viral fitness. In this study, we identified two HLA-B*52:01-restricted and one HLA-C*12:02-restricted novel cytotoxic T-lymphocyte (CTL) epitopes in Pol. Analysis using CTLs specific for these three epitopes demonstrated that these CTLs failed to recognize mutant epitopes or more weakly recognized cells infected with mutant viruses than wild-type virus, supporting the idea that these mutations were selected by the HLA-B*52:01- or HLA-C*12:02-restricted T cells. We further showed that these mutations reduced viral fitness, although the effect of each mutation was weak. The present study demonstrated that the accumulation of these Pol mutations selected by HLA-B*52:01- or HLA-C*12:02-restricted CTLs impaired viral replication capacity and thus reduced the pVL. The fitness cost imposed by the mutations partially accounted for the effect of the HLA-B*52:01-C*12:02 haplotype on clinical outcome, together with the effect of HLA-B*52:01-restricted CTLs on viral replication, which had been previously demonstrated. IMPORTANCE Numerous population-based studies identified HLA-associated HIV-1 mutations to predict HIV-1 escape mutations from cytotoxic T lymphocytes (CTLs). However, the majority of these HLA-associated mutations have not been identified as CTL escape mutations. Our previous population-based study showed that five HLA-B*52:01-associated mutations at four Pol positions were inversely correlated with the plasma viral load in HLA-B*52:01-negative Japanese individuals. In the present study, we demonstrated that these mutations were indeed selected by CTLs specific for novel B*52:01- and C*12:02-restricted epitopes and that the accumulation of these mutations reduced the viral fitness in vitro. This study elucidated the mechanism by which the accumulation of these CTL escape mutations contributed to the protective effect of the HLA-B*52:01-HLA-C*12:02 haplotype on disease progression in HIV-1-infected Japanese individuals. PMID:27903797

Accumulation of Pol Mutations Selected by HLA-B*52:01-C*12:02 Protective Haplotype-Restricted Cytotoxic T Lymphocytes Causes Low Plasma Viral Load Due to Low Viral Fitness of Mutant Viruses.

PubMed

Murakoshi, Hayato; Koyanagi, Madoka; Chikata, Takayuki; Rahman, Mohammad Arif; Kuse, Nozomi; Sakai, Keiko; Gatanaga, Hiroyuki; Oka, Shinichi; Takiguchi, Masafumi

2017-02-15

HLA-B*52:01-C*12:02, which is the most abundant haplotype in Japan, has a protective effect on disease progression in HIV-1-infected Japanese individuals, whereas HLA-B*57 and -B*27 protective alleles are very rare in Japan. A previous study on HLA-associated polymorphisms demonstrated that the number of HLA-B*52:01-associated mutations at four Pol positions was inversely correlated with plasma viral load (pVL) in HLA-B*52:01-negative individuals, suggesting that the transmission of HIV-1 with these mutations could modulate the pVL in the population. However, it remains unknown whether these mutations were selected by HLA-B*52:01-restricted CTLs and also reduced viral fitness. In this study, we identified two HLA-B*52:01-restricted and one HLA-C*12:02-restricted novel cytotoxic T-lymphocyte (CTL) epitopes in Pol. Analysis using CTLs specific for these three epitopes demonstrated that these CTLs failed to recognize mutant epitopes or more weakly recognized cells infected with mutant viruses than wild-type virus, supporting the idea that these mutations were selected by the HLA-B*52:01- or HLA-C*12:02-restricted T cells. We further showed that these mutations reduced viral fitness, although the effect of each mutation was weak. The present study demonstrated that the accumulation of these Pol mutations selected by HLA-B*52:01- or HLA-C*12:02-restricted CTLs impaired viral replication capacity and thus reduced the pVL. The fitness cost imposed by the mutations partially accounted for the effect of the HLA-B*52:01-C*12:02 haplotype on clinical outcome, together with the effect of HLA-B*52:01-restricted CTLs on viral replication, which had been previously demonstrated. Numerous population-based studies identified HLA-associated HIV-1 mutations to predict HIV-1 escape mutations from cytotoxic T lymphocytes (CTLs). However, the majority of these HLA-associated mutations have not been identified as CTL escape mutations. Our previous population-based study showed that five HLA-B*52:01-associated mutations at four Pol positions were inversely correlated with the plasma viral load in HLA-B*52:01-negative Japanese individuals. In the present study, we demonstrated that these mutations were indeed selected by CTLs specific for novel B*52:01- and C*12:02-restricted epitopes and that the accumulation of these mutations reduced the viral fitness in vitro This study elucidated the mechanism by which the accumulation of these CTL escape mutations contributed to the protective effect of the HLA-B*52:01-HLA-C*12:02 haplotype on disease progression in HIV-1-infected Japanese individuals. Copyright © 2017 American Society for Microbiology.

Novel and recurrent NDP gene mutations in familial cases of Norrie disease and X-linked exudative vitreoretinopathy.

PubMed

Pelcastre, Erika L; Villanueva-Mendoza, Cristina; Zenteno, Juan C

2010-05-01

To present the results of molecular analysis of the NDP gene in Mexican families with Norrie disease (ND) and X-linked familial exudative vitreoretinopathy (XL-FEVR). Two unrelated families with ND and two with XL-FEVR were studied. Clinical diagnosis was suspected on the basis of a complete ophthalmologic examination. Molecular methods included DNA isolation from peripheral blood leucocytes, polymerase chain reaction amplification and direct nucleotide sequencing analysis of the complete coding region and exon-intron junctions of NDP. Haplotype analysis using NDP-linked microsatellites markers was performed in both ND families. A novel Norrin missense mutation, p.Arg41Thr, was identified in two apparently unrelated families with ND. Haplotype analysis demonstrated that affected males in these two families shared the same ND-linked haplotype, suggesting a common origin for this novel mutation. The previously reported p.Arg121Trp and p.Arg121Gln Norrin mutations were identified in the two families with XL-FEVR. Our results expand the mutational spectrum in ND. This is the first report of ND resulting from mutation at arginine position 41 of Norrin. Interestingly, mutations at the same residue but resulting in a different missense change were previously described in subjects with XL-FEVR (p.Arg41Lys) or persistent fetal vasculature syndrome (p.Arg41Ser), indicating that the novel p.Arg41Thr change causes a more severe retinal phenotype. Preliminary data suggest a founder effect for the ND p.Arg41Thr mutation in these two Mexican families.

In vivo evolution of resistant subpopulations of KPC-producing Klebsiella pneumoniae during ceftazidime/avibactam treatment.

PubMed

Gaibani, Paolo; Campoli, Caterina; Lewis, Russell E; Volpe, Silvia Lidia; Scaltriti, Erika; Giannella, Maddalena; Pongolini, Stefano; Berlingeri, Andrea; Cristini, Francesco; Bartoletti, Michele; Tedeschi, Sara; Ambretti, Simone

2018-06-01

KPC-producing Klebsiella pneumoniae (KPC-Kp) represent a serious problem worldwide. Herein, we describe the evolution of ceftazidime/avibactam resistance by sequencing longitudinal clinical isolates from a patient with KPC-Kp bloodstream infection undergoing ceftazidime/avibactam treatment. WGS was performed on one ceftazidime/avibactam-susceptible KPC-Kp (BOT-CA-S) and two phenotypically different ceftazidime/avibactam-resistant KPC-Kp with low (BOT-CA-R) and high (BOT-EMO) carbapenem MICs. The population diversity was assessed by the frequency of allele mutations and population analysis profiles (PAPs). Phylogenetic analysis demonstrated clonal relatedness of the KPC-Kp isolates, all belonging to the clone ST1519. The D179Y mutation in blaKPC-3 was detected in both of the ceftazidime/avibactam-resistant KPC-Kp, whereas it was absent in the ceftazidime/avibactam-susceptible isolate. The mutation emerged independently in the two ceftazidime/avibactam-resistant isolates and was associated with a significant reduction in carbapenem MICs in BOT-CA-R, but not in BOT-EMO. WGS analysis revealed that the frequency of the D179Y mutation was 96.32% and 51.05% in BOT-CA-R and BOT-EMO, respectively. PAP results demonstrated that carbapenem resistance in BOT-EMO was due to the coexistence of mixed subpopulations harbouring WT and mutated blaKPC-3. A bacterial subpopulation with high ceftazidime/avibactam resistance for BOT-EMO KPC-Kp showed low carbapenem MICs, whereas a subpopulation with high meropenem resistance had a low MIC of ceftazidime/avibactam. Our analysis indicates that mixed subpopulations of ceftazidime/avibactam-resistant KPC-Kp emerge after ceftazidime/avibactam treatment. The evolution of different subpopulations that are highly resistant to ceftazidime/avibactam likely contributes to treatment failure, thereby highlighting the need for combination treatment strategies to limit selection of ceftazidime/avibactam-resistant KPC-Kp subpopulations.

[Clinical features and COMP gene mutation in a family with a pseudoachondroplasia child].

PubMed

Lu, Chun-Ting; Guo, Li; Zahng, Zhan-Hui; Lin, Wei-Xia; Song, Yuan-Zong; Feng, Lie

2013-11-01

This study aimed to report the clinical characteristics and COMP gene mutation of a family with pseudoachondroplasia (PSACH), a relatively rare spinal and epiphyseal dysplasia that is inherited as an autosomal dominant trait. Clinical information on a 5-year-2-month-old PSACH child and his parents was collected and analyzed. Diagnosis was confirmed by PCR amplification and direct sequencing of all the 19 exons and their flanking sequences of COMP gene, and the mutation was further ascertained by cloning analysis of exon 10. The child presented with short and stubby fingers, bow leg, short limb dwarfism and metaphysic broadening in long bone as well as lumbar lordosis. A mutation c.1048_1116del (p.Asn350_Asp372del) in exon 10, inherited from his father who did not demonstrate any phenotypic feature of PSACH, was detected in the child. PSACH was diagnosed definitively by means of COMP mutation analysis, on the basis of the child's clinical and imaging features. The non-penetrance phenomenon of COMP mutation was described for the first time in PSACH.

A Japanese family with nonautoimmune hyperthyroidism caused by a novel heterozygous thyrotropin receptor gene mutation.

PubMed

Nakamura, Akie; Morikawa, Shuntaro; Aoyagi, Hayato; Ishizu, Katsura; Tajima, Toshihiro

2014-06-01

Hyperthyroidism caused by activating mutations of the thyrotropin receptor gene (TSHR) is rare in the pediatric population. We found a Japanese family with hyperthyroidism without autoantibody. DNA sequence analysis of TSHR was undertaken in this family. The functional consequences for the Gs-adenylyl cyclase and Gq/11-phospholipase C signaling pathways and cell surface expression of receptors were determined in vitro using transiently transfected human embryonic kidney 293 cells. We identified a heterozygous mutation (M453R) in exon 10 of TSHR. In this family, this mutation was found in all individuals who exhibited hyperthyroidism. The results showed that this mutation resulted in constitutive activation of the Gs-adenylyl cyclase system. However, this mutation also caused a reduction in the activation capacity of the Gq/11-phospholipase C pathway, compared with the wild type. We demonstrate that the M453R mutation is the cause of nonautoimmune hyperthyroidism.

Functional and splicing defect analysis of 23 ACVRL1 mutations in a cohort of patients affected by Hereditary Hemorrhagic Telangiectasia

PubMed Central

Alaa el Din, Ferdos; Patri, Sylvie; Thoreau, Vincent; Rodriguez-Ballesteros, Montserrat; Hamade, Eva; Bailly, Sabine; Gilbert-Dussardier, Brigitte; Abou Merhi, Raghida; Kitzis, Alain

2015-01-01

Hereditary Hemorrhagic Telangiectasia syndrome (HHT) or Rendu-Osler-Weber (ROW) syndrome is an autosomal dominant vascular disorder. Two most common forms of HHT, HHT1 and HHT2, have been linked to mutations in the endoglin (ENG) and activin receptor-like kinase 1 (ACVRL1or ALK1) genes respectively. This work was designed to examine the pathogenicity of 23 nucleotide variations in ACVRL1 gene detected in more than 400 patients. Among them, 14 missense mutations and one intronic variant were novels, and 8 missense mutations were previously identified with questionable implication in HHT2. The functionality of missense mutations was analyzed in response to BMP9 (specific ligand of ALK1), the maturation of the protein products and their localization were analyzed by western blot and fluorescence microscopy. The splicing impairment of the intronic and of two missense mutations was examined by minigene assay. Functional analysis showed that 18 out of 22 missense mutations were defective. Splicing analysis revealed that one missense mutation (c.733A>G, p.Ile245Val) affects the splicing of the harboring exon 6. Similarly, the intronic mutation outside the consensus splicing sites (c.1048+5G>A in intron 7) was seen pathogenic by splicing study. Both mutations induce a frame shift creating a premature stop codon likely resulting in mRNA degradation by NMD surveillance mechanism. Our results confirm the haploinsufficiency model proposed for HHT2. The affected allele of ACVRL1 induces mRNA degradation or the synthesis of a protein lacking the receptor activity. Furthermore, our data demonstrate that functional and splicing analyses together, represent two robust diagnostic tools to be used by geneticists confronted with novel or conflicted ACVRL1 mutations. PMID:26176610

Integrated Analysis of Mutation Data from Various Sources Identifies Key Genes and Signaling Pathways in Hepatocellular Carcinoma

PubMed Central

Wei, Lin; Tang, Ruqi; Lian, Baofeng; Zhao, Yingjun; He, Xianghuo; Xie, Lu

2014-01-01

Background Recently, a number of studies have performed genome or exome sequencing of hepatocellular carcinoma (HCC) and identified hundreds or even thousands of mutations in protein-coding genes. However, these studies have only focused on a limited number of candidate genes, and many important mutation resources remain to be explored. Principal Findings In this study, we integrated mutation data obtained from various sources and performed pathway and network analysis. We identified 113 pathways that were significantly mutated in HCC samples and found that the mutated genes included in these pathways contained high percentages of known cancer genes, and damaging genes and also demonstrated high conservation scores, indicating their important roles in liver tumorigenesis. Five classes of pathways that were mutated most frequently included (a) proliferation and apoptosis related pathways, (b) tumor microenvironment related pathways, (c) neural signaling related pathways, (d) metabolic related pathways, and (e) circadian related pathways. Network analysis further revealed that the mutated genes with the highest betweenness coefficients, such as the well-known cancer genes TP53, CTNNB1 and recently identified novel mutated genes GNAL and the ADCY family, may play key roles in these significantly mutated pathways. Finally, we highlight several key genes (e.g., RPS6KA3 and PCLO) and pathways (e.g., axon guidance) in which the mutations were associated with clinical features. Conclusions Our workflow illustrates the increased statistical power of integrating multiple studies of the same subject, which can provide biological insights that would otherwise be masked under individual sample sets. This type of bioinformatics approach is consistent with the necessity of making the best use of the ever increasing data provided in valuable databases, such as TCGA, to enhance the speed of deciphering human cancers. PMID:24988079

Integrated analysis of mutation data from various sources identifies key genes and signaling pathways in hepatocellular carcinoma.

PubMed

Zhang, Yuannv; Qiu, Zhaoping; Wei, Lin; Tang, Ruqi; Lian, Baofeng; Zhao, Yingjun; He, Xianghuo; Xie, Lu

2014-01-01

Recently, a number of studies have performed genome or exome sequencing of hepatocellular carcinoma (HCC) and identified hundreds or even thousands of mutations in protein-coding genes. However, these studies have only focused on a limited number of candidate genes, and many important mutation resources remain to be explored. In this study, we integrated mutation data obtained from various sources and performed pathway and network analysis. We identified 113 pathways that were significantly mutated in HCC samples and found that the mutated genes included in these pathways contained high percentages of known cancer genes, and damaging genes and also demonstrated high conservation scores, indicating their important roles in liver tumorigenesis. Five classes of pathways that were mutated most frequently included (a) proliferation and apoptosis related pathways, (b) tumor microenvironment related pathways, (c) neural signaling related pathways, (d) metabolic related pathways, and (e) circadian related pathways. Network analysis further revealed that the mutated genes with the highest betweenness coefficients, such as the well-known cancer genes TP53, CTNNB1 and recently identified novel mutated genes GNAL and the ADCY family, may play key roles in these significantly mutated pathways. Finally, we highlight several key genes (e.g., RPS6KA3 and PCLO) and pathways (e.g., axon guidance) in which the mutations were associated with clinical features. Our workflow illustrates the increased statistical power of integrating multiple studies of the same subject, which can provide biological insights that would otherwise be masked under individual sample sets. This type of bioinformatics approach is consistent with the necessity of making the best use of the ever increasing data provided in valuable databases, such as TCGA, to enhance the speed of deciphering human cancers.

Integrated single-cell genetic and transcriptional analysis suggests novel drivers of chronic lymphocytic leukemia

PubMed Central

Wang, Lili; Fan, Jean; Francis, Joshua M.; Georghiou, George; Hergert, Sarah; Li, Shuqiang; Gambe, Rutendo; Zhou, Chensheng W.; Yang, Chunxiao; Xiao, Sheng; Cin, Paola Dal; Bowden, Michaela; Kotliar, Dylan; Shukla, Sachet A.; Brown, Jennifer R.; Neuberg, Donna; Alessi, Dario R.; Zhang, Cheng-Zhong; Kharchenko, Peter V.; Livak, Kenneth J.; Wu, Catherine J.

2017-01-01

Intra-tumoral genetic heterogeneity has been characterized across cancers by genome sequencing of bulk tumors, including chronic lymphocytic leukemia (CLL). In order to more accurately identify subclones, define phylogenetic relationships, and probe genotype–phenotype relationships, we developed methods for targeted mutation detection in DNA and RNA isolated from thousands of single cells from five CLL samples. By clearly resolving phylogenic relationships, we uncovered mutated LCP1 and WNK1 as novel CLL drivers, supported by functional evidence demonstrating their impact on CLL pathways. Integrative analysis of somatic mutations with transcriptional states prompts the idea that convergent evolution generates phenotypically similar cells in distinct genetic branches, thus creating a cohesive expression profile in each CLL sample despite the presence of genetic heterogeneity. Our study highlights the potential for single-cell RNA-based targeted analysis to sensitively determine transcriptional and mutational profiles of individual cancer cells, leading to increased understanding of driving events in malignancy. PMID:28679620

Functional characterization of mutations in the myosin Vb gene associated with microvillus inclusion disease

PubMed Central

Szperl, Agata M.; Golachowska, Magdalena R.; Bruinenberg, Marcel; Prekeris, Rytis; Thunnissen, Andy-Mark W. H.; Karrenbeld, Arend; Dijkstra, Gerard; Hoekstra, Dick; Mercer, David; Ksiazyk, Janusz; Wijmenga, Cisca; Wapenaar, Martin C.; Rings, Edmond H. H. M.; van IJzendoorn, Sven C. D.

2010-01-01

Objectives Microvillus inclusion disease (MVID) is a rare autosomal recessive enteropathy characterized by intractable diarrhea and malabsorption. Recently, various MYO5B gene mutations have been identified in MVID patients. Interestingly, several MVID patients showed only a MYO5B mutation in one allele (heterozygous) or no mutations in the MYO5B gene, illustrating the need to further functionally characterize the cell biological effects of the MYO5B mutations. Methods The genomic DNA of nine patients diagnosed with microvillus inclusion disease was screened for MYO5B mutations, and qPCR and immunohistochemistry on the material of two patients was performed to investigate resultant cellular consequences. Results We demonstrate for the first time that MYO5B mutations can be correlated with altered myosin Vb mRNA expression and with an aberrant subcellular distribution of the myosin Vb protein. Moreover, we demonstrate that the typical and myosin Vb–controlled accumulation of rab11a-and FIP5-positive recycling endosomes in the apical cytoplasm of the cells is abolished in MVID enterocytes, which is indicative for altered myosin Vb function. Also, we report 8 novel MYO5B mutations in 9 MVID patients of various etnic backgrounds, including compound heterozygous mutations. Conclusions Our functional analysis indicate that MYO5B mutations can be correlated with an aberrant subcellular distribution of the myosin Vb protein and apical recycling endosomes which, together with the additional compound heterozygous mutations, significantly strengthen the link between MYO5B and MVID. PMID:21206382

An improved method on stimulated T-lymphocytes to functionally characterize novel and known LDLR mutations[S

PubMed Central

Romano, Maria; Di Taranto, Maria Donata; Mirabelli, Peppino; D'Agostino, Maria Nicoletta; Iannuzzi, Arcangelo; Marotta, Gennaro; Gentile, Marco; Raia, Maddalena; Di Noto, Rosa; Del Vecchio, Luigi; Rubba, Paolo; Fortunato, Giuliana

2011-01-01

The main causes of familial hypercholesterolemia (FH) are mutations in LDL receptor (LDLR) gene. Functional studies are necessary to demonstrate the LDLR function impairment caused by mutations and would be useful as a diagnostic tool if they allow discrimination between FH patients and controls. In order to identify the best method to detect LDLR activity, we compared continuous Epstein-Barr virus (EBV)-transformed B-lymphocytes and mitogen stimulated T-lymphocytes. In addition, we characterized both novel and known mutations in the LDLR gene. T-lymphocytes and EBV-transformed B-lymphocytes were obtained from peripheral blood of 24 FH patients and 24 control subjects. Functional assays were performed by incubation with fluorescent LDL followed by flow cytometry analysis. Residual LDLR activity was calculated normalizing fluorescence for the mean fluorescence of controls. With stimulated T-lymphocytes we obtained a better discrimination capacity between controls and FH patients compared with EBV-transformed B-lymphocytes as demonstrated by receiver operating characteristic (ROC) curve analysis (the areas under the curve are 1.000 and 0.984 respectively; P < 0.0001 both). The characterization of LDLR activity through T-lymphocytes is more simple and faster than the use of EBV-transformed B-lymphocytes and allows a complete discrimination between controls and FH patients. Therefore the evaluation of residual LDLR activity could be helpful not only for mutation characterization but also for diagnostic purposes. PMID:21865347

Genetic heterogeneity in type 1 Gaucher disease: Multiple genotypes in Ashkenazic and non-Ashkenazic individuals

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

Tsuji, Shoji; Martin, B.M.; Stubblefield, B.K.

1988-04-01

Nucleotide sequence analysis of a genomic clone from an Ashkenazic Jewish patient with type 1 Gaucher disease revealed a single-base mutation (adenosine to guanosine transition) in exon 9 of the glucocerebrosidase gene. This change results in the amino acid substitution of serine for asparagine. Transient expression studies following oligonucleotide-directed mutagenesis of the normal cDNA confirmed that the mutation results in loss of glucocerebrosidase activity. Allele-specific hybridization with oligonucleotide probes demonstrated that this mutation was found exclusively in type 1 phenotype. None of the 6 type 2 patients, 11 type 3 patients, or 12 normal controls had this allele. In contrast,more » 15 of 24 type 1 patients had one allele with this mutation, and 3 others were homozygous for the mutation. Furthermore, some of the Ashkenazic Jewish type 1 patients had only one allele with this mutation, suggesting that even in this population there is allelic heterozygosity. These findings indicate that there are multiple allelic mutations responsible for type 1 Gaucher disease in both the Jewish and non-Jewish populations. Allelic-specific hybridization demonstrating this mutation in exon 9, used in conjunction with the Nci I restriction fragment length polymorphism described as a marker for neuronopathic Gaucher disease, provides a tool for diagnosis and genetic counseling that is {approx}80% informative in all Gaucher patients studied.« less

Statistical Coupling Analysis-Guided Library Design for the Discovery of Mutant Luciferases.

PubMed

Liu, Mira D; Warner, Elliot A; Morrissey, Charlotte E; Fick, Caitlyn W; Wu, Taia S; Ornelas, Marya Y; Ochoa, Gabriela V; Zhang, Brendan S; Rathbun, Colin M; Porterfield, William B; Prescher, Jennifer A; Leconte, Aaron M

2018-02-06

Directed evolution has proven to be an invaluable tool for protein engineering; however, there is still a need for developing new approaches to continue to improve the efficiency and efficacy of these methods. Here, we demonstrate a new method for library design that applies a previously developed bioinformatic method, Statistical Coupling Analysis (SCA). SCA uses homologous enzymes to identify amino acid positions that are mutable and functionally important and engage in synergistic interactions between amino acids. We use SCA to guide a library of the protein luciferase and demonstrate that, in a single round of selection, we can identify luciferase mutants with several valuable properties. Specifically, we identify luciferase mutants that possess both red-shifted emission spectra and improved stability relative to those of the wild-type enzyme. We also identify luciferase mutants that possess a >50-fold change in specificity for modified luciferins. To understand the mutational origin of these improved mutants, we demonstrate the role of mutations at N229, S239, and G246 in altered function. These studies show that SCA can be used to guide library design and rapidly identify synergistic amino acid mutations from a small library.

Involvement of ER Stress in Dysmyelination of Pelizaeus-Merzbacher Disease with PLP1 Missense Mutations Shown by iPSC-Derived Oligodendrocytes

PubMed Central

Numasawa-Kuroiwa, Yuko; Okada, Yohei; Shibata, Shinsuke; Kishi, Noriyuki; Akamatsu, Wado; Shoji, Masanobu; Nakanishi, Atsushi; Oyama, Manabu; Osaka, Hitoshi; Inoue, Ken; Takahashi, Kazutoshi; Yamanaka, Shinya; Kosaki, Kenjiro; Takahashi, Takao; Okano, Hideyuki

2014-01-01

Summary Pelizaeus-Merzbacher disease (PMD) is a form of X-linked leukodystrophy caused by mutations in the proteolipid protein 1 (PLP1) gene. Although PLP1 proteins with missense mutations have been shown to accumulate in the rough endoplasmic reticulum (ER) in disease model animals and cell lines transfected with mutant PLP1 genes, the exact pathogenetic mechanism of PMD has not previously been clarified. In this study, we established induced pluripotent stem cells (iPSCs) from two PMD patients carrying missense mutation and differentiated them into oligodendrocytes in vitro. In the PMD iPSC-derived oligodendrocytes, mislocalization of mutant PLP1 proteins to the ER and an association between increased susceptibility to ER stress and increased numbers of apoptotic oligodendrocytes were observed. Moreover, electron microscopic analysis demonstrated drastically reduced myelin formation accompanied by abnormal ER morphology. Thus, this study demonstrates the involvement of ER stress in pathogenic dysmyelination in the oligodendrocytes of PMD patients with the PLP1 missense mutation. PMID:24936452

Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

PubMed Central

Uchibori, Ken; Inase, Naohiko; Araki, Mitsugu; Kamada, Mayumi; Sato, Shigeo; Okuno, Yasushi; Fujita, Naoya; Katayama, Ryohei

2017-01-01

Osimertinib has been demonstrated to overcome the epidermal growth factor receptor (EGFR)-T790M, the most relevant acquired resistance to first-generation EGFR–tyrosine kinase inhibitors (EGFR–TKIs). However, the C797S mutation, which impairs the covalent binding between the cysteine residue at position 797 of EGFR and osimertinib, induces resistance to osimertinib. Currently, there are no effective therapeutic strategies to overcome the C797S/T790M/activating-mutation (triple-mutation)-mediated EGFR–TKI resistance. In the present study, we identify brigatinib to be effective against triple-mutation-harbouring cells in vitro and in vivo. Our original computational simulation demonstrates that brigatinib fits into the ATP-binding pocket of triple-mutant EGFR. The structure–activity relationship analysis reveals the key component in brigatinib to inhibit the triple-mutant EGFR. The efficacy of brigatinib is enhanced markedly by combination with anti-EGFR antibody because of the decrease of surface and total EGFR expression. Thus, the combination therapy of brigatinib with anti-EGFR antibody is a powerful candidate to overcome triple-mutant EGFR. PMID:28287083

Brigatinib combined with anti-EGFR antibody overcomes osimertinib resistance in EGFR-mutated non-small-cell lung cancer

NASA Astrophysics Data System (ADS)

Uchibori, Ken; Inase, Naohiko; Araki, Mitsugu; Kamada, Mayumi; Sato, Shigeo; Okuno, Yasushi; Fujita, Naoya; Katayama, Ryohei

2017-03-01

Osimertinib has been demonstrated to overcome the epidermal growth factor receptor (EGFR)-T790M, the most relevant acquired resistance to first-generation EGFR-tyrosine kinase inhibitors (EGFR-TKIs). However, the C797S mutation, which impairs the covalent binding between the cysteine residue at position 797 of EGFR and osimertinib, induces resistance to osimertinib. Currently, there are no effective therapeutic strategies to overcome the C797S/T790M/activating-mutation (triple-mutation)-mediated EGFR-TKI resistance. In the present study, we identify brigatinib to be effective against triple-mutation-harbouring cells in vitro and in vivo. Our original computational simulation demonstrates that brigatinib fits into the ATP-binding pocket of triple-mutant EGFR. The structure-activity relationship analysis reveals the key component in brigatinib to inhibit the triple-mutant EGFR. The efficacy of brigatinib is enhanced markedly by combination with anti-EGFR antibody because of the decrease of surface and total EGFR expression. Thus, the combination therapy of brigatinib with anti-EGFR antibody is a powerful candidate to overcome triple-mutant EGFR.

Mutational analysis of multiple lung cancers: Discrimination between primary and metastatic lung cancers by genomic profile.

PubMed

Goto, Taichiro; Hirotsu, Yosuke; Mochizuki, Hitoshi; Nakagomi, Takahiro; Shikata, Daichi; Yokoyama, Yujiro; Oyama, Toshio; Amemiya, Kenji; Okimoto, Kenichiro; Omata, Masao

2017-05-09

In cases of multiple lung cancers, individual tumors may represent either a primary lung cancer or both primary and metastatic lung cancers. Treatment selection varies depending on such features, and this discrimination is critically important in predicting prognosis. The present study was undertaken to determine the efficacy and validity of mutation analysis as a means of determining whether multiple lung cancers are primary or metastatic in nature. The study involved 12 patients who underwent surgery in our department for multiple lung cancers between July 2014 and March 2016. Tumor cells were collected from formalin-fixed paraffin-embedded tissues of the primary lesions by using laser capture microdissection, and targeted sequencing of 53 lung cancer-related genes was performed. In surgically treated patients with multiple lung cancers, the driver mutation profile differed among the individual tumors. Meanwhile, in a case of a solitary lung tumor that appeared after surgery for double primary lung cancers, gene mutation analysis using a bronchoscopic biopsy sample revealed a gene mutation profile consistent with the surgically resected specimen, thus demonstrating that the tumor in this case was metastatic. In cases of multiple lung cancers, the comparison of driver mutation profiles clarifies the clonal origin of the tumors and enables discrimination between primary and metastatic tumors.

Late-onset nonketotic hyperglycinemia with a heterozygous novel point mutation of the GLDC gene.

PubMed

Brenton, J Nicholas; Rust, Robert S

2014-05-01

Atypical nonketotic hyperglycinemia is characterized by heterogeneous phenotypes that often include nonspecific behavioral problems, cognitive deficits, and developmental delays. We describe a girl with late-onset nonketotic hyperglycinemia presenting at 5 years of age with hypotonia, chorea, ataxia, and alterations in consciousness in the setting of febrile illness. Serum amino acid analysis was mildly elevated; however, urine amino acid analysis was instrumental in demonstrating marked hyperglycinuria. Mutation testing showed a heterozygous novel sequence change/point mutation in the glycine decarboxylase gene. This patient illustrates the importance of obtaining urine amino acids in individuals whose clinical manifestations are suspicious for any form of nonketotic hyperglycinemia, because this testing may provide more prominent evidence of elevations in glycine. She also illustrates the potential for a heterozygous mutation to result in manifestations of an atypical form of nonketotic hyperglycinemia. Copyright © 2014 Elsevier Inc. All rights reserved.

Prognostic Implications of Multiplex Detection of KRAS Mutations in Cell-Free DNA from Patients with Pancreatic Ductal Adenocarcinoma.

PubMed

Kim, Min Kyeong; Woo, Sang Myung; Park, Boram; Yoon, Kyong-Ah; Kim, Yun-Hee; Joo, Jungnam; Lee, Woo Jin; Han, Sung-Sik; Park, Sang-Jae; Kong, Sun-Young

2018-04-01

Cell-free DNA (cfDNA) is known to provide potential biomarkers for predicting clinical outcome, but its value in pancreatic ductal adenocarcinoma (PDAC) has not been fully evaluated. The aim of this study was to evaluate the clinical applicability of quantitative analysis of multiplex KRAS mutations in cell-free DNA from patients with PDAC. A total of 106 patients with PDAC were enrolled in this prospective study. The concentration and fraction of KRAS mutations were determined through multiplex detection of KRAS mutations in plasma samples by use of a droplet digital PCR kit (Bio-Rad). KRAS mutations were detected in 96.1% of tissue samples. Eighty patients (80.5%) harbored KRAS mutations in cfDNA, with a median KRAS mutation concentration of 0.165 copies/μL and a median fractional abundance of 0.415%. Multivariable analyses demonstrated that the KRAS mutation concentration [hazard ratio (HR), 2.08; 95% CI, 1.20-3.63] and KRAS fraction (HR, 1.73; 95% CI, 1.02-2.95) were significant factors for progression-free survival. KRAS mutation concentration (HR, 1.97; 95% CI, 1.05-3.67) also had prognostic implications for overall survival. Subgroup analyses showed that KRAS mutation concentration and fractional abundance significantly affected progression-free survival in resectable PDAC ( P = 0.016). Moreover, when combined with the cancer biomarker CA19-9, the KRAS mutation concentration in cfDNA showed additive benefits for the prediction of overall survival. This study demonstrates that multiplex detection of KRAS mutations in plasma cfDNA is clinically relevant, providing a potential candidate biomarker for prognosis of PDAC. © 2018 American Association for Clinical Chemistry.

Role of GnRH receptor mutations in patients with a wide spectrum of pubertal delay

PubMed Central

Beneduzzi, Daiane; Trarbach, Ericka B.; Min, Le; Jorge, Alexander A. L.; Garmes, Heraldo M.; Renk, Alessandra Covallero; Fichna, Marta; Fichna, Piotr; Arantes, Karina A.; Costa, Elaine M. F.; Zhang, Anna; Adeola, Oluwaseun; Wen, Junping; Carroll, Rona S.; Mendonça, Berenice B.; Kaiser, Ursula B.; Latronico, Ana Claudia; Silveira, Letícia F. G.

2014-01-01

Objective To analyze the GNRHR in patients with normosmic isolated hypogonadotropic hypogonadism (IHH) and constitutional delay of growth and puberty (CDGP). Design Molecular analysis and in vitro experiments correlated with phenotype. Setting Academic medical center. Patient(s) 110 individuals with normosmic IHH (74 males) and 50 with CGDP. Intervention(s) GNRHR coding region was amplified and sequenced. Main Outcome Measure(s) Novel variants were submitted to in vitro analysis. Frequency of mutations and genotype-phenotype correlation were analyzed. Microsatellite markers flanking GNRHR were examined in patients carrying the same mutation to investigate a possible founder effect. Result(s) Eleven IHH patients (10%) carried biallelic GNRHR mutations. In vitro analysis of novel variants (p.Y283H and p.V134G) demonstrated complete inactivation. The founder effect study revealed that Brazilian patients carrying the p.R139H mutation shared the same haplotype. Phenotypic spectrum in patients with GNRHR mutations varied from complete GnRH deficiency to partial and reversible IHH, with a relatively good genotype-phenotype correlation. One boy with CDGP was heterozygous for the p.Q106R variant, which was not considered pathogenic. Conclusion(s) GNRHR mutations are a frequent cause of congenital normosmic IHH and should be the first candidate gene for genetic screening in this condition, especially in autosomal recessive familial cases. The founder effect study suggested that the p.R139H mutation arises from a common ancestor in the Brazilian population. Finally, mutations in GNRHR do not appear to be involved in the pathogenesis of CDGP. PMID:25016926

Drug Resistance Missense Mutations in Cancer Are Subject to Evolutionary Constraints

PubMed Central

Friedman, Ran

2013-01-01

Several tumour types are sensitive to deactivation of just one or very few genes that are constantly active in the cancer cells, a phenomenon that is termed ‘oncogene addiction’. Drugs that target the products of those oncogenes can yield a temporary relief, and even complete remission. Unfortunately, many patients receiving oncogene-targeted therapies relapse on treatment. This often happens due to somatic mutations in the oncogene (‘resistance mutations’). ‘Compound mutations’, which in the context of cancer drug resistance are defined as two or more mutations of the drug target in the same clone may lead to enhanced resistance against the most selective inhibitors. Here, it is shown that the vast majority of the resistance mutations occurring in cancer patients treated with tyrosin kinase inhibitors aimed at three different proteins follow an evolutionary pathway. Using bioinformatic analysis tools, it is found that the drug-resistance mutations in the tyrosine kinase domains of Abl1, ALK and exons 20 and 21 of EGFR favour transformations to residues that can be identified in similar positions in evolutionary related proteins. The results demonstrate that evolutionary pressure shapes the mutational landscape in the case of drug-resistance somatic mutations. The constraints on the mutational landscape suggest that it may be possible to counter single drug-resistance point mutations. The observation of relatively many resistance mutations in Abl1, but not in the other genes, is explained by the fact that mutations in Abl1 tend to be biochemically conservative, whereas mutations in EGFR and ALK tend to be radical. Analysis of Abl1 compound mutations suggests that such mutations are more prevalent than hitherto reported and may be more difficult to counter. This supports the notion that such mutations may provide an escape route for targeted cancer drug resistance. PMID:24376513

Using information content and base frequencies to distinguish mutations from genetic polymorphisms in splice junction recognition sites.

PubMed

Rogan, P K; Schneider, T D

1995-01-01

Predicting the effects of nucleotide substitutions in human splice sites has been based on analysis of consensus sequences. We used a graphic representation of sequence conservation and base frequency, the sequence logo, to demonstrate that a change in a splice acceptor of hMSH2 (a gene associated with familial nonpolyposis colon cancer) probably does not reduce splicing efficiency. This confirms a population genetic study that suggested that this substitution is a genetic polymorphism. The information theory-based sequence logo is quantitative and more sensitive than the corresponding splice acceptor consensus sequence for detection of true mutations. Information analysis may potentially be used to distinguish polymorphisms from mutations in other types of transcriptional, translational, or protein-coding motifs.

Activating HER2 mutations in HER2 gene amplification negative breast cancer

PubMed Central

Bose, Ron; Kavuri, Shyam M.; Searleman, Adam C.; Shen, Wei; Shen, Dong; Koboldt, Daniel C.; Monsey, John; Goel, Nicholas; Aronson, Adam B.; Li, Shunqiang; Ma, Cynthia X.; Ding, Li; Mardis, Elaine R.; Ellis, Matthew J.

2012-01-01

Data from eight breast cancer genome sequencing projects identified 25 patients with HER2 somatic mutations in cancers lacking HER2 gene amplification. To determine the phenotype of these mutations, we functionally characterized thirteen HER2 mutations using in vitro kinase assays, protein structure analysis, cell culture and xenograft experiments. Seven of these mutations are activating mutations, including G309A, D769H, D769Y, V777L, P780ins, V842I, and R896C. HER2 in-frame deletion 755-759, which is homologous to EGFR exon 19 in-frame deletions, had a neomorphic phenotype with increased phosphorylation of EGFR or HER3. L755S produced lapatinib resistance, but was not an activating mutation in our experimental systems. All of these mutations were sensitive to the irreversible kinase inhibitor, neratinib. These findings demonstrate that HER2 somatic mutation is an alternative mechanism to activate HER2 in breast cancer and they validate HER2 somatic mutations as drug targets for breast cancer treatment. PMID:23220880

In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics

PubMed Central

Grundberg, Ida; Kiflemariam, Sara; Mignardi, Marco; Imgenberg-Kreuz, Juliana; Edlund, Karolina; Micke, Patrick; Sundström, Magnus; Sjöblom, Tobias

2013-01-01

Current assays for somatic mutation analysis are based on extracts from tissue sections that often contain morphologically heterogeneous neoplastic regions with variable contents of genetically normal stromal and inflammatory cells, obscuring the results of the assays. We have developed an RNA-based in situ mutation assay that targets oncogenic mutations in a multiplex fashion that resolves the heterogeneity of the tissue sample. Activating oncogenic mutations are targets for a new generation of cancer drugs. For anti-EGFR therapy prediction, we demonstrate reliable in situ detection of KRAS mutations in codon 12 and 13 in colon and lung cancers in three different types of routinely processed tissue materials. High-throughput screening of KRAS mutation status was successfully performed on a tissue microarray. Moreover, we show how the patterns of expressed mutated and wild-type alleles can be studied in situ in tumors with complex combinations of mutated EGFR, KRAS and TP53. This in situ method holds great promise as a tool to investigate the role of somatic mutations during tumor progression and for prediction of response to targeted therapy. PMID:24280411

The Prevalence of Gap Junction Protein Beta 2 (GJB2) Mutations in Non Syndromic Sensorineural Hearing Loss in Çukurova Region.

PubMed

Bozdoğan, Sevcan Tuğ; Kuran, Gökhan; Yüregir, Özge Özalp; Aslan, Hüseyin; Haytoğlu, Süheyl; Ayaz, Akif; Arıkan, Osman Kürşat

2015-08-01

To date, studies in all populations showed that mutations in the gene of Gap junction protein beta 2 (GJB2) play an important role in non-syndromic autosomal recessive congenital hearing loss. The aim of this study was to evaluate GJB2 gene of patients with hearing loss in our region using deoxyribonucleic acid (DNA) sequencing method and to demonstrate region-specific mutation and polymorphism distribution. Patients who had bilateral severe sensorineural non-syndromic hearing loss identified by audiologic evaluation were included. Peripheral blood samples were collected and the GJB2 gene exon1 and exon 2 regions were amplified by polymerase chain reaction (PCR). Obtained PCR products were sequenced by the DNA sequence analysis method (SeqFinder Sequencing System; ABI 3130; Foster City, CA, USA) and analyzed using the SeqScape software. Of the 77 patients, 16 had homozygous or heterozygous mutation. The mutation of 35delG, which is known as the most frequent mutation of GJB2 gene, was also the most frequently seen mutation at a ratio of 5.5% in patients with hearing loss in our region; this was followed by the V27I mutation. As this is the first study conducted by sequence analysis in our region, it was worth to be presented in terms of showing the distribution of mutation.

SDHB-related pheochromocytoma and paraganglioma penetrance and genotype-phenotype correlations.

PubMed

Jochmanova, Ivana; Wolf, Katherine I; King, Kathryn S; Nambuba, Joan; Wesley, Robert; Martucci, Victoria; Raygada, Margarita; Adams, Karen T; Prodanov, Tamara; Fojo, Antonio Tito; Lazurova, Ivica; Pacak, Karel

2017-08-01

Succinate dehydrogenase subunit B (SDHB) gene mutations are associated with an aggressive clinical disease course of pheochromocytoma/paraganglioma (PHEO/PGL). Limited information is available concerning PHEO/PGL penetrance among SDHB mutation carriers with regards to primary tumor location, specific mutation type, and gender. We assessed PHEO/PGL penetrance in SDHB mutation carriers and described the clinical presentation and disease course. Asymptomatic relatives (N = 611) of 103 index patients were tested for SDHB mutations. Mutation carriers (N = 328) were offered PHEO/PGL screening, of which 241 participated and were included in penetrance analysis. For additional disease outcome analysis, the 103 index patients and 40 screened individuals who developed PHEO/PGL were included. Clinical data were collected between October 2004 and June 2016. Forty (16.60%) of the 241 screened individuals developed PHEO/PGL during the study. The penetrance estimate in this population was 49.80% (95% CI 29-74.9) at 85 years. A significantly higher age-related penetrance of disease was observed in males compared to females, with 50% penetrance achieved at age 74 vs. not reached. Age-related penetrance analysis demonstrated 4 mutations (Ile127Ser, IVS1+1G>T, Exon 1 deletion, Arg90X) presenting with a slower rate of disease development (50% penetrance ages, respectively: not achieved, 70, 63, 61 years) compared to Arg46X and Val140Phe mutations (50% penetrance at 38 years). Here, we found a higher estimated penetrance compared to several other studies, and a striking difference in age-related penetrance between male and female SDHB mutation carriers with no association between mutation and gender or tumor location.

BRAFV600E mutation and its association with clinicopathological features of colorectal cancer: a systematic review and meta-analysis.

PubMed

Chen, Dong; Huang, Jun-Fu; Liu, Kai; Zhang, Li-Qun; Yang, Zhao; Chuai, Zheng-Ran; Wang, Yun-Xia; Shi, Da-Chuan; Huang, Qing; Fu, Wei-Ling

2014-01-01

Colorectal cancer (CRC) is a heterogeneous disease with multiple underlying causative genetic mutations. The B-type Raf proto-oncogene (BRAF) plays an important role in the mitogen-activated protein kinase (MAPK) signaling cascade during CRC. The presence of BRAFV600E mutation can determine the response of a tumor to chemotherapy. However, the association between the BRAFV600E mutation and the clinicopathological features of CRC remains controversial. We performed a systematic review and meta-analysis to estimate the effect of BRAFV600E mutation on the clinicopathological characteristics of CRC. We identified studies that examined the effect of BRAFV600E mutation on CRC within the PubMed, ISI Science Citation Index, and Embase databases. The effect of BRAFV600E on outcome parameters was estimated by odds ratios (ORs) with 95% confidence intervals (CIs) for each study using a fixed effects or random effects model. 25 studies with a total of 11,955 CRC patients met inclusion criteria. The rate of BRAFV600 was 10.8% (1288/11955). The BRAFV600E mutation in CRC was associated with advanced TNM stage, poor differentiation, mucinous histology, microsatellite instability (MSI), CpG island methylator phenotype (CIMP). This mutation was also associated with female gender, older age, proximal colon, and mutL homolog 1 (MLH1) methylation. This meta-analysis demonstrated that BRAFV600E mutation was significantly correlated with adverse pathological features of CRC and distinct clinical characteristics. These data suggest that BRAFV600E mutation could be used to supplement standard clinical and pathological staging for the better management of individual CRC patients, and could be considered as a poor prognostic marker for CRC.

Clinicopathological comparison of colorectal and endometrial carcinomas in patients with Lynch-like syndrome versus patients with Lynch syndrome.

PubMed

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

2015-11-01

Screening for DNA mismatch repair (MMR) deficiency in colorectal and endometrial carcinomas identifies patients at risk for Lynch syndrome. Some patients with MMR-deficient tumors have no evidence of a germline mutation and have been described as having Lynch-like syndrome. We compared the clinicopathological features of colorectal and endometrial carcinomas in patients with Lynch-like syndrome and Lynch syndrome. Universal screening identified 356 (10.6%) of 3352 patients with colorectal carcinoma and 72 (33%) of 215 patients with endometrial carcinoma with deficient DNA MMR. Sixty-six patients underwent germline mutation analysis with 45 patients (68%) having evidence of a germline MMR gene mutation confirming Lynch syndrome and 21 patients (32%) having Lynch-like syndrome with no evidence of a germline mutation. Most patients with Lynch-like syndrome had carcinoma involving the right colon compared to patients with Lynch syndrome (93% versus 45%; P < .002). All patients with colorectal carcinomas demonstrating isolated loss of MSH6 expression had Lynch syndrome confirmed by germline mutation analysis. Synchronous or metachronous Lynch syndrome-associated carcinoma was more frequently identified in patients with Lynch syndrome compared to Lynch-like syndrome (38% versus 7%; P = .04). There were no significant differences in clinicopathological variables between patients with Lynch-like syndrome and Lynch syndrome with endometrial carcinoma. In summary, 32% of patients with MMR deficiency concerning Lynch syndrome will have Lynch-like syndrome. Our results demonstrate that patients with Lynch-like syndrome are more likely to have right-sided colorectal carcinoma, less likely to have synchronous or metachronous Lynch syndrome-associated carcinoma, and less likely to demonstrate isolated loss of MSH6 expression within their tumor. Copyright © 2015 Elsevier Inc. All rights reserved.

A new SETX mutation producing AOA2 in two siblings.

PubMed

Datta, Neil; Hohler, Anna

2013-09-01

In this paper, we document two cases of a new SETX mutation (820:A>G) combined with an established recessive SETX mutation (5927:T>G) causing ataxia with oculomotor apraxia type 2 (AOA2). The patients had a detailed neurological history and examination performed. Radiological imaging was obtained and genetic analysis was obtained. Both siblings demonstrated healthy and normal growth until adolescence. At that time, slowed speech, hypophonia, dysarthria, extraocular muscle dysfunction and some mild choreiform movements began to appear. Family history included some movement disorder difficulties in second degree relatives. The diagnosis of AOA2 was confirmed by genetic testing. We describe a new SETX gene mutation, which when combined with a recognized SETX mutation results in AOA2. The clinical, radiographic and ancillary testing are described.

Activation of Antibiotic Production in Bacillus spp. by Cumulative Drug Resistance Mutations

PubMed Central

Tojo, Shigeo; Tanaka, Yukinori

2015-01-01

Bacillus subtilis strains produce a wide range of antibiotics, including ribosomal and nonribosomal peptide antibiotics, as well as bacilysocin and neotrehalosadiamine. Mutations in B. subtilis strain 168 that conferred resistance to drugs such as streptomycin and rifampin resulted in overproduction of the dipeptide antibiotic bacilysin. Cumulative drug resistance mutations, such as mutations in the mthA and rpsL genes, which confer low- and high-level resistance, respectively, to streptomycin, and mutations in rpoB, which confer resistance to rifampin, resulted in cells that overproduced bacilysin. Transcriptional analysis demonstrated that the enhanced transcription of biosynthesis genes was responsible for the overproduction of bacilysin. This approach was effective also in activating the cryptic genes of Bacillus amyloliquefaciens, leading to actual production of antibiotic(s). PMID:26369962

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

Epidermolysis bullosa with congenital pyloric atresia: novel mutations in the beta 4 integrin gene (ITGB4) and genotype/phenotype correlations.

PubMed

Nakano, A; Pulkkinen, L; Murrell, D; Rico, J; Lucky, A W; Garzon, M; Stevens, C A; Robertson, S; Pfendner, E; Uitto, J

2001-05-01

Epidermolysis bullosa with pyloric atresia (EB-PA: OMIM 226730), also known as Carmi syndrome, is a rare autosomal recessive genodermatosis that manifests with neonatal mucocutaneous fragility associated with congenital pyloric atresia. The disease is frequently lethal within the first year, but nonlethal cases have been reported. Mutations in the genes encoding subunit polypeptides of the alpha 6 beta 4 integrin (ITGA6 and ITGB4) have been demonstrated in EB-PA patients. To extend the repertoire of mutations and to identify genotype-phenotype correlations, we examined seven new EB-PA families, four with lethal and three with nonlethal disease variants. DNA from patients was screened for mutations using heteroduplex analysis followed by nucleotide sequencing of PCR products spanning all beta 4 integrin-coding sequences. Mutation analysis disclosed 12 distinct mutations, 11 of them novel. Four mutations predicted a premature termination codon as a result of nonsense mutations or small out-of-frame insertions or deletions, whereas seven were missense mutations. This brings the total number of distinct ITGB4 mutations to 33. The mutation database indicates that premature termination codons are associated predominantly with the lethal EB-PA variants, whereas missense mutations are more prevalent in nonlethal forms. However, the consequences of the missense mutations are position dependent, and substitutions of highly conserved amino acids may have lethal consequences. In general, indirect immunofluorescence studies of affected skin revealed negative staining for beta 4 integrin in lethal cases and positive, but attenuated, staining in nonlethal cases and correlated with clinical phenotype. The data on specific mutations in EB-PA patients allows prenatal testing and preimplantation genetic diagnosis in families at risk.

CHEK2 mutation and risk of prostate cancer: a systematic review and meta-analysis

PubMed Central

Wang, Yue; Dai, Bo; Ye, Dingwei

2015-01-01

Background: CHEK2 encodes for a G2 checkpoint kinase which plays a critical role in DNA repair. Its mutation confers an increased risk of breast cancer. It has also been suggested to increase risks of prostate cancer, but its involvement with this type of cancer has not been confirmed. Methods: We performed a systematic review and meta-analysis to clarify the association between CHEK2 1100delC, IVS2+1G>A, I157T mutation and risk of Prostate Cancer. A comprehensive, computerized literature search of PubMed until December 27, 2014 was carried out. Eligible studies were included according to specific inclusion criteria. Pooled hazard ratio was estimated using the fixed effects model or random effects model according to heterogeneity between studies. Results: Eight eligible studies were included in the analysis, all were retrospective studies. The overall meta-analysis demonstrated that the CHEK2 1100delC mutation (OR 3.29; 95% confidence interval: 1.85-5.85; P = 0.00) and I157T missense mutation (OR 1.80; 95% confidence interval: 1.51-2.14; P = 0.00) was associated with higher risk of Prostate Cancer, and CHEK2 1100delC mutation is irrelevant to familial aggregation phenomenon of prostate cancer (OR 1.59; 95% confidence interval: 0.79-3.20; P = 0.20). The IVS2+1G>A mutation is also irrelevant to Prostate Cancer (OR = 1.59, 95% CI = 0.93-2.71, P = 0.09). None of the single studies materially altered the original results and no evidence of publication bias was found. Conclusion: CHEK2 1100delC mutation and I157T missense mutation in males indicates higher risk of Prostate Cancer, but there’s no evidence to prove the CHEK2 1100delC mutation was associated with Familial prostate cancer. PMID:26629066

CHEK2 mutation and risk of prostate cancer: a systematic review and meta-analysis.

PubMed

Wang, Yue; Dai, Bo; Ye, Dingwei

2015-01-01

CHEK2 encodes for a G2 checkpoint kinase which plays a critical role in DNA repair. Its mutation confers an increased risk of breast cancer. It has also been suggested to increase risks of prostate cancer, but its involvement with this type of cancer has not been confirmed. We performed a systematic review and meta-analysis to clarify the association between CHEK2 1100delC, IVS2+1G>A, I157T mutation and risk of Prostate Cancer. A comprehensive, computerized literature search of PubMed until December 27, 2014 was carried out. Eligible studies were included according to specific inclusion criteria. Pooled hazard ratio was estimated using the fixed effects model or random effects model according to heterogeneity between studies. Eight eligible studies were included in the analysis, all were retrospective studies. The overall meta-analysis demonstrated that the CHEK2 1100delC mutation (OR 3.29; 95% confidence interval: 1.85-5.85; P = 0.00) and I157T missense mutation (OR 1.80; 95% confidence interval: 1.51-2.14; P = 0.00) was associated with higher risk of Prostate Cancer, and CHEK2 1100delC mutation is irrelevant to familial aggregation phenomenon of prostate cancer (OR 1.59; 95% confidence interval: 0.79-3.20; P = 0.20). The IVS2+1G>A mutation is also irrelevant to Prostate Cancer (OR = 1.59, 95% CI = 0.93-2.71, P = 0.09). None of the single studies materially altered the original results and no evidence of publication bias was found. CHEK2 1100delC mutation and I157T missense mutation in males indicates higher risk of Prostate Cancer, but there's no evidence to prove the CHEK2 1100delC mutation was associated with Familial prostate cancer.

Assessment of molecular markers demonstrates concordance between samples acquired via stereotactic biopsy and open craniotomy in both anaplastic astrocytomas and glioblastomas.

PubMed

Gessler, Florian; Baumgarten, Peter; Bernstock, Joshua D; Harter, Patrick; Lescher, Stephanie; Senft, Christian; Seifert, Volker; Marquardt, Gerhard; Weise, Lutz

2017-06-01

The classification, treatment and prognosis of high-grade gliomas has been shown to correlate with the expression of molecular markers (e.g. MGMT promotor methylation and IDH1 mutations). Acquisition of tumor samples may be obtained via stereotactic biopsy or open craniotomy. Between the years 2009 and 2013, 22 patients initially diagnosed with HGGs via stereotactic biopsy, that ultimately underwent open craniotomy for resection of their tumor were prospectively included in an institutional glioma database. MGMT promotor analysis was performed using methylation-specific (MS)-PCR and IDH1R132H mutation analysis was performed using immunohistochemistry. Three patients (13.7%) exhibited IDH1R132H mutations in samples obtained via stereotactic biopsy. Tissue derived from stereotaxic biopsy was demonstrated to have MGMT promotor methylation in ten patients (45.5%), while a non-methylated MGMT promotor was demonstrated in ten patients (45.5%); inconclusive results were obtained for the remaining two patients (9%) within our cohort. The initial histologic grading, IDH1R132H mutation and MGMT promotor methylation results were confirmed using samples obtained during open craniotomy in all but one patient; here inconclusive MGMT promotor analysis was obtained in contrast to that which was obtained via stereotactic biopsy. Tumor samples acquired via stereotactic biopsy provide accurate information with regard to clinically relevant molecular markers that have been shown to impact patient care decisions. The profile of markers analyzed in our cohort was nearly concordant between those samples obtained via stereotactic biopsy or open craniotomy thereby suggesting that clinical decisions may be based on the molecular profile of the tumor samples obtained via stereotactic biopsy.

Congenital nephrogenic diabetes insipidus with a novel mutation in the aquaporin 2 gene.

PubMed

Park, Youn Jong; Baik, Haing Woon; Cheong, Hae Il; Kang, Ju Hyung

2014-07-01

Congenital nephrogenic diabetes insipidus (CNDI) is a rare disorder caused by mutations of the arginine vasopressin (AVP) V2 receptor or aquaporin 2 ( AQP2 ) genes. The current study presented the case of CNDI in a 1-month-old male with a novel mutation in the AQP2 gene. The patient was referred due to the occurrence of hypernatremia and mild-intermittent fever since birth. An AVP stimulation test was compatible with CNDI as there was no significant response to desmopressin. Molecular genetic analysis demonstrated two mutations in exon 1 of the AQP2 gene: C to T transition, which resulted in a missense mutation of 108 Thr (ACG) to Met (ATG); and a 127, 128 delCA, which resulted in a deletion mutation of glutamine in position 43 at codon CAG as the first affected amino acid, with the new reading frame endign in a termination codon at position 62. The molecular genetic analysis of the parents showed that the missense mutation was inherited maternally and the deletion mutation was inherited paternally. The parents showed no signs or symptoms of CNDI, indicating autosomal recessive inheritance. The 108 Thr (ACG) to Met (ATG) mutation was confirmed as a novel mutation. Therefore, the molecular identification of the AQP2 gene has clinical significance, as early recognition of CNDI in infants that show only non-specific symptoms, can be facilitated. Thus, repeated episodes of dehydration, which may cause physical and mental retardation can be avoided.

Congenital nephrogenic diabetes insipidus with a novel mutation in the aquaporin 2 gene

PubMed Central

PARK, YOUN JONG; BAIK, HAING WOON; CHEONG, HAE IL; KANG, JU HYUNG

2014-01-01

Congenital nephrogenic diabetes insipidus (CNDI) is a rare disorder caused by mutations of the arginine vasopressin (AVP) V2 receptor or aquaporin 2 (AQP2) genes. The current study presented the case of CNDI in a 1-month-old male with a novel mutation in the AQP2 gene. The patient was referred due to the occurrence of hypernatremia and mild-intermittent fever since birth. An AVP stimulation test was compatible with CNDI as there was no significant response to desmopressin. Molecular genetic analysis demonstrated two mutations in exon 1 of the AQP2 gene: C to T transition, which resulted in a missense mutation of 108Thr (ACG) to Met (ATG); and a 127, 128 delCA, which resulted in a deletion mutation of glutamine in position 43 at codon CAG as the first affected amino acid, with the new reading frame endign in a termination codon at position 62. The molecular genetic analysis of the parents showed that the missense mutation was inherited maternally and the deletion mutation was inherited paternally. The parents showed no signs or symptoms of CNDI, indicating autosomal recessive inheritance. The 108Thr (ACG) to Met (ATG) mutation was confirmed as a novel mutation. Therefore, the molecular identification of the AQP2 gene has clinical significance, as early recognition of CNDI in infants that show only non-specific symptoms, can be facilitated. Thus, repeated episodes of dehydration, which may cause physical and mental retardation can be avoided. PMID:24944815

Leigh syndrome associated with a novel mutation in the COX15 gene.

PubMed

Miryounesi, Mohammad; Fardaei, Majid; Tabei, Seyed Mohammadbagher; Ghafouri-Fard, Soudeh

2016-06-01

Leigh syndrome (LS) is a subacute necrotizing encephalomyelopathy with a diverse range of symptoms, such as psychomotor delay or regression, weakness, hypotonia, truncal ataxia, intention tremor as well as lactic acidosis in the blood, cerebrospinal fluid or urine. Both nuclear gene defects and mutations of the mitochondrial genome have been detected in these patients. Here we report a 7-year-old girl with hypotonia, tremor, developmental delay and psychomotor regression. However, serum lactate level as well as brain magnetic resonance imaging were normal. Mutational analysis has revealed a novel mutation in exon 4 of COX15 gene (c.415C>G) which results in p.Leu139Val. Previous studies have demonstrated that COX15 mutations are associated with typical LS as well as fatal infantile hypertrophic cardiomyopathy. Consequently, clinical manifestations of COX15 mutations may be significantly different in patients. Such information is of practical importance in genetic counseling.

Simultaneous Profiling of DNA Mutation and Methylation by Melting Analysis Using Magnetoresistive Biosensor Array.

PubMed

Rizzi, Giovanni; Lee, Jung-Rok; Dahl, Christina; Guldberg, Per; Dufva, Martin; Wang, Shan X; Hansen, Mikkel F

2017-09-26

Epigenetic modifications, in particular DNA methylation, are gaining increasing interest as complementary information to DNA mutations for cancer diagnostics and prognostics. We introduce a method to simultaneously profile DNA mutation and methylation events for an array of sites with single site specificity. Genomic (mutation) or bisulphite-treated (methylation) DNA is amplified using nondiscriminatory primers, and the amplicons are then hybridized to a giant magnetoresistive (GMR) biosensor array followed by melting curve measurements. The GMR biosensor platform offers scalable multiplexed detection of DNA hybridization, which is insensitive to temperature variation. The melting curve approach further enhances the assay specificity and tolerance to variations in probe length. We demonstrate the utility of this method by simultaneously profiling five mutation and four methylation sites in human melanoma cell lines. The method correctly identified all mutation and methylation events and further provided quantitative assessment of methylation density validated by bisulphite pyrosequencing.

Schwannomatosis associated with multiple meningiomas due to a familial SMARCB1 mutation.

PubMed

Bacci, Costanza; Sestini, Roberta; Provenzano, Aldesia; Paganini, Irene; Mancini, Irene; Porfirio, Berardino; Vivarelli, Rossella; Genuardi, Maurizio; Papi, Laura

2010-02-01

Schwannomatosis (MIM 162091) is a condition predisposing to the development of central and peripheral schwannomas; most cases are sporadic without a clear family history but a few families with a clear autosomal dominant pattern of transmission have been described. Germline mutations in SMARCB1 are associated with schwannomatosis. We report a family with multiple schwannomas and meningiomas. A SMARCB1 germline mutation in exon 1 was identified. The mutation, c.92A>T (p.Glu31Val), occurs in a highly conserved amino acid in the SMARCB1 protein. In addition, in silico analysis demonstrated that the mutation disrupts the donor consensus sequence of exon 1. RNA studies verified the absence of mRNA transcribed by the mutant allele. This is the first report of a SMARCB1 germline mutation in a family with schwannomatosis characterized by the development of multiple meningiomas.

Distinct molecular profile of diffuse cerebellar gliomas.

PubMed

Nomura, Masashi; Mukasa, Akitake; Nagae, Genta; Yamamoto, Shogo; Tatsuno, Kenji; Ueda, Hiroki; Fukuda, Shiro; Umeda, Takayoshi; Suzuki, Tomonari; Otani, Ryohei; Kobayashi, Keiichi; Maruyama, Takashi; Tanaka, Shota; Takayanagi, Shunsaku; Nejo, Takahide; Takahashi, Satoshi; Ichimura, Koichi; Nakamura, Taishi; Muragaki, Yoshihiro; Narita, Yoshitaka; Nagane, Motoo; Ueki, Keisuke; Nishikawa, Ryo; Shibahara, Junji; Aburatani, Hiroyuki; Saito, Nobuhito

2017-12-01

Recent studies have demonstrated that tumor-driving alterations are often different among gliomas that originated from different brain regions and have underscored the importance of analyzing molecular characteristics of gliomas stratified by brain region. Therefore, to elucidate molecular characteristics of diffuse cerebellar gliomas (DCGs), 27 adult, mostly glioblastoma cases were analyzed. Comprehensive analysis using whole-exome sequencing, RNA sequencing, and Infinium methylation array (n = 17) demonstrated their distinct molecular profile compared to gliomas in other brain regions. Frequent mutations in chromatin-modifier genes were identified including, noticeably, a truncating mutation in SETD2 (n = 4), which resulted in loss of H3K36 trimethylation and was mutually exclusive with H3F3A K27M mutation (n = 3), suggesting that epigenetic dysregulation may lead to DCG tumorigenesis. Alterations that cause loss of p53 function including TP53 mutation (n = 9), PPM1D mutation (n = 2), and a novel type of PPM1D fusion (n = 1), were also frequent. On the other hand, mutations and copy number changes commonly observed in cerebral gliomas were infrequent. DNA methylation profile analysis demonstrated that all DCGs except for those with H3F3A mutations were categorized in the "RTK I (PDGFRA)" group, and those DCGs had a gene expression signature that was highly associated with PDGFRA. Furthermore, compared with the data of 315 gliomas derived from different brain regions, promoter methylation of transcription factors genes associated with glial development showed a characteristic pattern presumably reflecting their tumor origin. Notably, SOX10, a key transcription factor associated with oligodendroglial differentiation and PDGFRA regulation, was up-regulated in both DCG and H3 K27M-mutant diffuse midline glioma, suggesting their developmental and biological commonality. In contrast, SOX10 was silenced by promoter methylation in most cerebral gliomas. These findings may suggest potential tailored targeted therapy for gliomas according to their brain region, in addition to providing molecular clues to identify the region-related cellular origin of DCGs.

Detecting recurrent gene mutation in interaction network context using multi-scale graph diffusion.

PubMed

Babaei, Sepideh; Hulsman, Marc; Reinders, Marcel; de Ridder, Jeroen

2013-01-23

Delineating the molecular drivers of cancer, i.e. determining cancer genes and the pathways which they deregulate, is an important challenge in cancer research. In this study, we aim to identify pathways of frequently mutated genes by exploiting their network neighborhood encoded in the protein-protein interaction network. To this end, we introduce a multi-scale diffusion kernel and apply it to a large collection of murine retroviral insertional mutagenesis data. The diffusion strength plays the role of scale parameter, determining the size of the network neighborhood that is taken into account. As a result, in addition to detecting genes with frequent mutations in their genomic vicinity, we find genes that harbor frequent mutations in their interaction network context. We identify densely connected components of known and putatively novel cancer genes and demonstrate that they are strongly enriched for cancer related pathways across the diffusion scales. Moreover, the mutations in the clusters exhibit a significant pattern of mutual exclusion, supporting the conjecture that such genes are functionally linked. Using multi-scale diffusion kernel, various infrequently mutated genes are found to harbor significant numbers of mutations in their interaction network neighborhood. Many of them are well-known cancer genes. The results demonstrate the importance of defining recurrent mutations while taking into account the interaction network context. Importantly, the putative cancer genes and networks detected in this study are found to be significant at different diffusion scales, confirming the necessity of a multi-scale analysis.

Truncation- and motif-based pan-cancer analysis reveals tumor-suppressing kinases.

PubMed

Hudson, Andrew M; Stephenson, Natalie L; Li, Cynthia; Trotter, Eleanor; Fletcher, Adam J; Katona, Gitta; Bieniasz-Krzywiec, Patrycja; Howell, Matthew; Wirth, Chris; Furney, Simon; Miller, Crispin J; Brognard, John

2018-04-17

A major challenge in cancer genomics is identifying "driver" mutations from the many neutral "passenger" mutations within a given tumor. To identify driver mutations that would otherwise be lost within mutational noise, we filtered genomic data by motifs that are critical for kinase activity. In the first step of our screen, we used data from the Cancer Cell Line Encyclopedia and The Cancer Genome Atlas to identify kinases with truncation mutations occurring within or before the kinase domain. The top 30 tumor-suppressing kinases were aligned, and hotspots for loss-of-function (LOF) mutations were identified on the basis of amino acid conservation and mutational frequency. The functional consequences of new LOF mutations were biochemically validated, and the top 15 hotspot LOF residues were used in a pan-cancer analysis to define the tumor-suppressing kinome. A ranked list revealed MAP2K7, an essential mediator of the c-Jun N-terminal kinase (JNK) pathway, as a candidate tumor suppressor in gastric cancer, despite its mutational frequency falling within the mutational noise for this cancer type. The majority of mutations in MAP2K7 abolished its catalytic activity, and reactivation of the JNK pathway in gastric cancer cells harboring LOF mutations in MAP2K7 or the downstream kinase JNK suppressed clonogenicity and growth in soft agar, demonstrating the functional relevance of inactivating the JNK pathway in gastric cancer. Together, our data highlight a broadly applicable strategy to identify functional cancer driver mutations and define the JNK pathway as tumor-suppressive in gastric cancer. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

MELAS syndrome in a patient with a point mutation in MTTS1.

PubMed

Lindberg, C; Moslemi, A-R; Oldfors, A

2008-02-01

BACKGROUND, OBJECTIVE AND METHODS: We describe a female patient with a mitochondrial encephalopathy, lactic acidosis and stroke-like episodes syndrome. As a child, she developed epilepsy and stroke-like episodes giving cognitive impairment and ataxia but no hearing impairment. At the age of 44 years, she suffered a cerebral sinus thrombosis which was warfarin treated. One month later, she developed an episode of severe acidosis associated with encephalopathy and myelopathy. She was found to harbour a 7512T>C mutation in the mitochondrial encoded tRNA(Ser(UCN)) gene (MTTS1). The mutation load was 91% in muscle and 24% in blood. Enzyme histochemical analysis of the muscle tissue showed numerous cytochrome c oxidase (COX)-negative fibres. Restriction fragment length polymorphism (RFLP) analysis of single muscle fibres showed significantly higher level (median 97%, range: 94-99%) of the mutation in the COX-negative fibres compared with COX-positive fibres (median 36%, range: 12-91%), demonstrating the pathogenic effect of the mutation. Different levels of heteroplasmy (range 34-61%) were detected in hair shafts analysed by RFLP. This case adds to the spectrum of clinical presentations, i.e. sinus thrombosis, in patients having MTTS1 mutations.

Glucocerebrosidase mutations and neuropsychiatric phenotypes in Parkinson's disease and Lewy body dementias: Review and meta-analyses.

PubMed

Creese, Byron; Bell, Emily; Johar, Iskandar; Francis, Paul; Ballard, Clive; Aarsland, Dag

2018-03-01

Heterozygous mutations in glucocerebrosidase gene (GBA) are a major genetic risk factor for Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Recently, there has been a considerable focus on the relationship between GBA mutations and emergence of cognitive impairment and neuropsychiatric symptoms in these diseases. Here, we review the literature in this area, with a particular focus, including meta-analysis, on the key neuropsychiatric symptoms of cognitive impairment, psychosis, and depression in Parkinson's disease. Our meta-analysis demonstrated that GBA mutations are associated with a 2.4-fold increased risk of cognitive impairment. In addition, our novel meta-analyses of psychosis and depression showed a 1.8- and 2.2-fold increased risk respectively associated with GBA mutations, although due to possible bias and heterogeneity the depression findings should be interpreted with caution. While the precise mechanisms which increase susceptibility to neurodegeneration in GBA carriers are not known, evidence of greater cortical Lewy body pathology, reduced patterns of cortical activation, and hippocampal pathology in animal models are all consistent with a direct effect of GBA mutations on these symptoms. Extension of this work in DLB and individuals without neurodegeneration will be important in further characterizing how GBA mutations increase risk for PD and DLB and influence disease course. © 2017 Wiley Periodicals, Inc.

VPS53 mutations cause progressive cerebello-cerebral atrophy type 2 (PCCA2).

PubMed

Feinstein, Miora; Flusser, Hagit; Lerman-Sagie, Tally; Ben-Zeev, Bruria; Lev, Dorit; Agamy, Orly; Cohen, Idan; Kadir, Rotem; Sivan, Sara; Leshinsky-Silver, Esther; Markus, Barak; Birk, Ohad S

2014-05-01

Progressive cerebello-cerebral atrophy (PCCA) leading to profound mental retardation, progressive microcephaly, spasticity and early onset epilepsy, was diagnosed in four non-consanguineous apparently unrelated families of Jewish Moroccan ancestry. Common founder mutation(s) were assumed. Genome-wide linkage analysis and whole exome sequencing were done, followed by realtime PCR and immunofluorescent microscopy. Genome-wide linkage analysis mapped the disease-associated gene to 0.5 Mb on chromosome 17p13.3. Whole exome sequencing identified only two mutations within this locus, which were common to the affected individuals: compound heterozygous mutations in VPS53, segregating as expected for autosomal recessive heredity within all four families, and common in Moroccan Jews (∼1:37 carrier rate). The Golgi-associated retrograde protein (GARP) complex is involved in the retrograde pathway recycling endocytic vesicles to Golgi; c.2084A>G and c.1556+5G>A VPS53 founder mutations are predicted to affect the C-terminal domain of VPS53, known to be critical to its role as part of this complex. Immunofluorescent microscopy demonstrated swollen and abnormally numerous CD63 positive vesicular bodies, likely intermediate recycling/late endosomes, in fibroblasts of affected individuals. Autosomal recessive PCCA type 2 is caused by VPS53 mutations.

A novel mutation of α-galactosidase A gene causes Fabry disease mimicking primary erythromelalgia in a Chinese family.

PubMed

Ge, Wei; Wei, Bin; Zhu, Hao; Miao, Zhigang; Zhang, Weimin; Leng, Cuihua; Li, Jizhen; Zhang, Dan; Sun, Miao; Xu, Xingshun

2017-05-01

Fabry disease is an X-linked genetic disorder caused by the mutations of α-galactosidase A (GLA, MIM 300644) gene presenting with various clinical symptoms including small-fiber peripheral neuropathy and limb burning pain. Here, we reported a Chinese pedigree with the initial diagnosis of primary erythromelalgia in an autosomal dominant (AD)-inherited pattern. Mutation analysis of SCN9A and GLA genes by direct sequencing and functional analysis of a novel mutation of GLA in cells were performed. Our data did not show any pathological mutations in SCN9A gene; however, a novel missense mutation c.139T>C (p.W47R) of GLA was identified in a male proband as well as two female carriers in this family. Enzyme assay of α-galactosidase A activity showed deficient enzyme activity in male patients and female carriers, further confirming the diagnosis of Fabry disease. Finally, a functional analysis indicated that the replacement of the 47th amino acid tryptophan (W47) with arginine (W47R) or glycine (W47G) led to reduced activity of α-galactosidase A in 293T cells. Therefore, these findings demonstrated that the novel mutation p.W47R of GLA is the cause of Fabry disease. Because Fabry disease and primary erythromelalgia share similar symptoms, it is a good strategy for clinical physicians to perform genetic mutation screenings on both SCN9A and GLA genes in those patients with limb burning pain but without a clear inheritant pattern.

Stearoyl-Acyl Carrier Protein Desaturase Mutations Uncover an Impact of Stearic Acid in Leaf and Nodule Structure.

PubMed

Lakhssassi, Naoufal; Colantonio, Vincent; Flowers, Nicholas D; Zhou, Zhou; Henry, Jason; Liu, Shiming; Meksem, Khalid

2017-07-01

Stearoyl-acyl carrier protein desaturase (SACPD-C) has been reported to control the accumulation of seed stearic acid; however, no study has previously reported its involvement in leaf stearic acid content and impact on leaf structure and morphology. A subset of an ethyl methanesulfonate mutagenized population of soybean ( Glycine max ) 'Forrest' was screened to identify mutants within the GmSACPD-C gene. Using a forward genetics approach, one nonsense and four missense Gmsacpd-c mutants were identified to have high levels of seed, nodule, and leaf stearic acid content. Homology modeling and in silico analysis of the GmSACPD-C enzyme revealed that most of these mutations were localized near or at conserved residues essential for diiron ion coordination. Soybeans carrying Gmsacpd-c mutations at conserved residues showed the highest stearic acid content, and these mutations were found to have deleterious effects on nodule development and function. Interestingly, mutations at nonconserved residues show an increase in stearic acid content yet retain healthy nodules. Thus, random mutagenesis and mutational analysis allows for the achievement of high seed stearic acid content with no associated negative agronomic characteristics. Additionally, expression analysis demonstrates that nodule leghemoglobin transcripts were significantly more abundant in soybeans with deleterious mutations at conserved residues of GmSACPD-C. Finally, we report that Gmsacpd-c mutations cause an increase in leaf stearic acid content and an alteration of leaf structure and morphology in addition to differences in nitrogen-fixing nodule structure. © 2017 American Society of Plant Biologists. All Rights Reserved.

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

Golbus, Jessica R.; Puckelwartz, Megan J.; Dellefave-Castillo, Lisa

Background—Cardiomyopathy is highly heritable but genetically diverse. At present, genetic testing for cardiomyopathy uses targeted sequencing to simultaneously assess the coding regions of more than 50 genes. New genes are routinely added to panels to improve the diagnostic yield. With the anticipated $1000 genome, it is expected that genetic testing will shift towards comprehensive genome sequencing accompanied by targeted gene analysis. Therefore, we assessed the reliability of whole genome sequencing and targeted analysis to identify cardiomyopathy variants in 11 subjects with cardiomyopathy. Methods and Results—Whole genome sequencing with an average of 37× coverage was combined with targeted analysis focused onmore » 204 genes linked to cardiomyopathy. Genetic variants were scored using multiple prediction algorithms combined with frequency data from public databases. This pipeline yielded 1-14 potentially pathogenic variants per individual. Variants were further analyzed using clinical criteria and/or segregation analysis. Three of three previously identified primary mutations were detected by this analysis. In six subjects for whom the primary mutation was previously unknown, we identified mutations that segregated with disease, had clinical correlates, and/or had additional pathological correlation to provide evidence for causality. For two subjects with previously known primary mutations, we identified additional variants that may act as modifiers of disease severity. In total, we identified the likely pathological mutation in 9 of 11 (82%) subjects. We conclude that these pilot data demonstrate that ~30-40× coverage whole genome sequencing combined with targeted analysis is feasible and sensitive to identify rare variants in cardiomyopathy-associated genes.« less

Fulminant neurological deterioration in a neonate with Leigh syndrome due to a maternally transmitted missense mutation in the mitochondrial ND3 gene.

PubMed

Leshinsky-Silver, E; Lev, D; Tzofi-Berman, Z; Cohen, S; Saada, A; Yanoov-Sharav, M; Gilad, E; Lerman-Sagie, T

2005-08-26

Leigh syndrome can result from both nuclear and mitochondrial DNA defects. Mutations in complex V genes of the respiratory chain were considered until recently as the most frequent cause for mitochondrial inherited Leigh syndrome, while gene defects in complex I were related to recessive Leigh syndrome. Recently few reports of mutations in the mitochondrial-encoded complex I subunit genes causing Leigh syndrome have been reported. We describe a 1-month-old baby who acutely deteriorated, with abrupt onset of brainstem dysfunction, due to basal ganglia lesions extending to the brainstem. A muscle biopsy demonstrated complex I deficiency. Subsequent analysis of the mitochondrial genome revealed a homoplastic T10191C mutation in the ND3 gene (in blood and muscle), resulting in a substitution of serine to proline. Hair root analysis revealed a 50% mutant load, reflecting heteroplasmy in early embryonic stages. The mutation was also detected in his mother (5%). Western blot analysis revealed a decrease of the 20 kDa subunit (likely ND6) and of the 30 kDa subunit (NDUFA9), which is probably due to instability attributed to the inability to form subcomplexes with ND3. This is the first description of infantile Leigh syndrome due to a maternally transmitted T10191C substitution in ND3 and not due to a de novo mutation. This mutation is age and tissue dependent and therefore may not be amenable to prenatal testing.

Multi-center analysis of glucocerebrosidase mutations in Parkinson disease

PubMed Central

Sidransky, Ellen; Nalls, Michael A.; Aasly, Jan O.; Aharon-Peretz, Judith; Annesi, Grazia; Barbosa, Egberto Reis; Bar-Shira, Anat; Berg, Daniela; Bras, Jose; Brice, Alexis; Chen, Chiung-Mei; Clark, Lorraine N.; Condroyer, Christel; De Marco, Elvira Valeria; Dürr, Alexandra; Eblan, Michael J.; Fahn, Stanley; Farrer, Matthew; Fung, Hon-Chung; Gan-Or, Ziv; Gasser, Thomas; Gershoni-Baruch, Ruth; Giladi, Nir; Griffith, Alida; Gurevich, Tanya; Januario, Cristina; Kropp, Peter; Lang, Anthony E.; Lee-Chen, Guey-Jen; Lesage, Suzanne; Marder, Karen; Mata, Ignacio F.; Mirelman, Anat; Mitsui, Jun; Mizuta, Ikuko; Nicoletti, Giuseppe; Oliveira, Catarina; Ottman, Ruth; Orr-Urtreger, Avi; Pereira, Lygia V.; Quattrone, Aldo; Rogaeva, Ekaterina; Rolfs, Arndt; Rosenbaum, Hanna; Rozenberg, Roberto; Samii, Ali; Samaddar, Ted; Schulte, Claudia; Sharma, Manu; Singleton, Andrew; Spitz, Mariana; Tan, Eng-King; Tayebi, Nahid; Toda, Tatsushi; Troiano, André; Tsuji, Shoji; Wittstock, Matthias; Wolfsberg, Tyra G.; Wu, Yih-Ru; Zabetian, Cyrus P.; Zhao, Yi; Ziegler, Shira G.

2010-01-01

Background Recent studies indicate an increased frequency of mutations in the gene for Gaucher disease, glucocerebrosidase (GBA), among patients with Parkinson disease. An international collaborative study was conducted to ascertain the frequency of GBA mutations in ethnically diverse patients with Parkinson disease. Methods Sixteen centers participated, including five from the Americas, six from Europe, two from Israel and three from Asia. Each received a standard DNA panel to compare genotyping results. Genotypes and phenotypic data from patients and controls were analyzed using multivariate logistic regression models and the Mantel Haenszel procedure to estimate odds ratios (ORs) across studies. The sample included 5691 patients (780 Ashkenazi Jews) and 4898 controls (387 Ashkenazi Jews). Results All 16 centers could detect GBA mutations, L444P and N370S, and the two were found in 15.3% of Ashkenazi patients with Parkinson disease (ORs = 4.95 for L444P and 5.62 for N370S), and in 3.2% of non-Ashkenazi patients (ORs = 9.68 for L444P and 3.30 for N370S). GBA was sequenced in 1642 non-Ashkenazi subjects, yielding a frequency of 6.9% for all mutations, demonstrate that limited mutation screens miss half the mutant alleles. The presence of any GBA mutation was associated with an OR of 5.43 across studies. Clinically, although phenotypes varied, subjects with a GBA mutation presented earlier, and were more likely to have affected relatives and atypical manifestations. Conclusion Data collected from sixteen centers demonstrate that there is a strong association between GBA mutations and Parkinson disease. PMID:19846850

A case study of an integrative genomic and experimental therapeutic approach for rare tumors: identification of vulnerabilities in a pediatric poorly differentiated carcinoma.

PubMed

Dela Cruz, Filemon S; Diolaiti, Daniel; Turk, Andrew T; Rainey, Allison R; Ambesi-Impiombato, Alberto; Andrews, Stuart J; Mansukhani, Mahesh M; Nagy, Peter L; Alvarez, Mariano J; Califano, Andrea; Forouhar, Farhad; Modzelewski, Beata; Mitchell, Chelsey M; Yamashiro, Darrell J; Marks, Lianna J; Glade Bender, Julia L; Kung, Andrew L

2016-10-31

Precision medicine approaches are ideally suited for rare tumors where comprehensive characterization may have diagnostic, prognostic, and therapeutic value. We describe the clinical case and molecular characterization of an adolescent with metastatic poorly differentiated carcinoma (PDC). Given the rarity and poor prognosis associated with PDC in children, we utilized genomic analysis and preclinical models to validate oncogenic drivers and identify molecular vulnerabilities. We utilized whole exome sequencing (WES) and transcriptome analysis to identify germline and somatic alterations in the patient's tumor. In silico and in vitro studies were used to determine the functional consequences of genomic alterations. Primary tumor was used to generate a patient-derived xenograft (PDX) model, which was used for in vivo assessment of predicted therapeutic options. WES revealed a novel germline frameshift variant (p.E1554fs) in APC, establishing a diagnosis of Gardner syndrome, along with a somatic nonsense (p.R790*) APC mutation in the tumor. Somatic mutations in TP53, MAX, BRAF, ROS1, and RPTOR were also identified and transcriptome and immunohistochemical analyses suggested hyperactivation of the Wnt/ß-catenin and AKT/mTOR pathways. In silico and biochemical assays demonstrated that the MAX p.R60Q and BRAF p.K483E mutations were activating mutations, whereas the ROS1 and RPTOR mutations were of lower utility for therapeutic targeting. Utilizing a patient-specific PDX model, we demonstrated in vivo activity of mTOR inhibition with temsirolimus and partial response to inhibition of MEK. This clinical case illustrates the depth of investigation necessary to fully characterize the functional significance of the breadth of alterations identified through genomic analysis.

Clear cell papillary renal cell carcinoma: a chromosomal microarray analysis of two cases using a novel Molecular Inversion Probe (MIP) technology.

PubMed

Alexiev, Borislav A; Zou, Ying S

2014-12-01

Chromosomal microarray analysis using novel Molecular Inversion Probe (MIP) technology demonstrated 2,570 kb copy neutral LOH of 10q11.22 in two clear cell papillary renal cell carcinomas. In addition, one of the tumors had a big 29,784 kb deletion of 13q11-q14.2. There were two variants of unknown significance, a 2,509 kb gain of Xp22.33 and a 257 kb homozygous deletion of 8p11.22. The somatic mutation panel containing 74 mutations in nine genes did not reveal any mutations. Besides identification of submicroscopic duplications or deletions, SNP microarrays can reveal abnormal allelic imbalances including LOH and copy neutral LOH, which cannot be recognized by chromosome, FISH, and non-SNP microarray arrays. To the best of our knowledge, this is the first study demonstrating copy neutral LOH of 10q11.22 in clear cell papillary renal cell carcinomas using the new MIP SNP OncoScan FFPE Assay Kit on formalin-fixed paraffin-embedded tumor samples. Copyright © 2014 Elsevier GmbH. All rights reserved.

Duplication within the SEPT9 gene associated with a founder effect in North American families with hereditary neuralgic amyotrophy

PubMed Central

Landsverk, Megan L.; Ruzzo, Elizabeth K.; Mefford, Heather C.; Buysse, Karen; Buchan, Jillian G.; Eichler, Evan E.; Petty, Elizabeth M.; Peterson, Esther A.; Knutzen, Dana M.; Barnett, Karen; Farlow, Martin R.; Caress, Judy; Parry, Gareth J.; Quan, Dianna; Gardner, Kathy L.; Hong, Ming; Simmons, Zachary; Bird, Thomas D.; Chance, Phillip F.; Hannibal, Mark C.

2009-01-01

Hereditary neuralgic amyotrophy (HNA) is an autosomal dominant disorder associated with recurrent episodes of focal neuropathy primarily affecting the brachial plexus. Point mutations in the SEPT9 gene have been previously identified as the molecular basis of HNA in some pedigrees. However in many families, including those from North America demonstrating a genetic founder haplotype, no sequence mutations have been detected. We report an intragenic 38 Kb SEPT9 duplication that is linked to HNA in 12 North American families that share the common founder haplotype. Analysis of the breakpoints showed that the duplication is identical in all pedigrees, and molecular analysis revealed that the duplication includes the 645 bp exon in which previous HNA mutations were found. The SEPT9 transcript variants that span this duplication contain two in-frame repeats of this exon, and immunoblotting demonstrates larger molecular weight SEPT9 protein isoforms. This exon also encodes for a majority of the SEPT9 N-terminal proline rich region suggesting that this region plays a role in the pathogenesis of HNA. PMID:19139049

Duplication within the SEPT9 gene associated with a founder effect in North American families with hereditary neuralgic amyotrophy.

PubMed

Landsverk, Megan L; Ruzzo, Elizabeth K; Mefford, Heather C; Buysse, Karen; Buchan, Jillian G; Eichler, Evan E; Petty, Elizabeth M; Peterson, Esther A; Knutzen, Dana M; Barnett, Karen; Farlow, Martin R; Caress, Judy; Parry, Gareth J; Quan, Dianna; Gardner, Kathy L; Hong, Ming; Simmons, Zachary; Bird, Thomas D; Chance, Phillip F; Hannibal, Mark C

2009-04-01

Hereditary neuralgic amyotrophy (HNA) is an autosomal dominant disorder associated with recurrent episodes of focal neuropathy primarily affecting the brachial plexus. Point mutations in the SEPT9 gene have been previously identified as the molecular basis of HNA in some pedigrees. However in many families, including those from North America demonstrating a genetic founder haplotype, no sequence mutations have been detected. We report an intragenic 38 Kb SEPT9 duplication that is linked to HNA in 12 North American families that share the common founder haplotype. Analysis of the breakpoints showed that the duplication is identical in all pedigrees, and molecular analysis revealed that the duplication includes the 645 bp exon in which previous HNA mutations were found. The SEPT9 transcript variants that span this duplication contain two in-frame repeats of this exon, and immunoblotting demonstrates larger molecular weight SEPT9 protein isoforms. This exon also encodes for a majority of the SEPT9 N-terminal proline rich region suggesting that this region plays a role in the pathogenesis of HNA.

The SHOX region and its mutations.

PubMed

Capone, L; Iughetti, L; Sabatini, S; Bacciaglia, A; Forabosco, A

2010-06-01

The short stature homeobox-containing (SHOX) gene lies in the pseudoautosomal region 1 (PAR1) that comprises 2.6 Mb of the short-arm tips of both the X and Y chromosomes. It is known that its heterozygous mutations cause Leri-Weill dyschondrosteosis (LWD) (OMIM #127300), while its homozygous mutations cause a severe form of dwarfism known as Langer mesomelic dysplasia (LMD) (OMIM #249700). The analysis of 238 LWD patients between 1998 and 2007 by multiple authors shows a prevalence of deletions (46.4%) compared to point mutations (21.2%). On the whole, deletions and point mutations account for about 67% of LWD patients. SHOX is located within a 1000 kb desert region without genes. The comparative genomic analysis of this region between genomes of different vertebrates has led to the identification of evolutionarily conserved non-coding DNA elements (CNE). Further functional studies have shown that one of these CNE downstream of the SHOX gene is necessary for the expression of SHOX; this is considered to be typical "enhancer" activity. Including the enhancer, the overall mutation of the SHOX region in LWD patients does not hold in 100% of cases. Various authors have demonstrated the existence of other CNE both downstream and upstream of SHOX regions. The resulting conclusion is that it is necessary to reanalyze all LWD/LMD patients without SHOX mutations for the presence of mutations in the 5'- and 3'-flanking SHOX regions.

Whole-Genome and Epigenomic Landscapes of Etiologically Distinct Subtypes of Cholangiocarcinoma

PubMed Central

Jusakul, Apinya; Cutcutache, Ioana; Yong, Chern Han; Lim, Jing Quan; Huang, Mi Ni; Padmanabhan, Nisha; Nellore, Vishwa; Kongpetch, Sarinya; Ng, Alvin Wei Tian; Ng, Ley Moy; Choo, Su Pin; Myint, Swe Swe; Thanan, Raynoo; Nagarajan, Sanjanaa; Lim, Weng Khong; Ng, Cedric Chuan Young; Boot, Arnoud; Liu, Mo; Ong, Choon Kiat; Rajasegaran, Vikneswari; Lie, Stefanus; Lim, Alvin Soon Tiong; Lim, Tse Hui; Tan, Jing; Loh, Jia Liang; McPherson, John R.; Khuntikeo, Narong; Bhudhisawasdi, Vajaraphongsa; Yongvanit, Puangrat; Wongkham, Sopit; Totoki, Yasushi; Nakamura, Hiromi; Arai, Yasuhito; Yamasaki, Satoshi; Chow, Pierce Kah-Hoe; Chung, Alexander Yaw Fui; Ooi, London Lucien Peng Jin; Lim, Kiat Hon; Dima, Simona; Duda, Dan G.; Popescu, Irinel; Broet, Philippe; Hsieh, Sen-Yung; Yu, Ming-Chin; Scarpa, Aldo; Lai, Jiaming; Luo, Di-Xian; Carvalho, André Lopes; Vettore, André Luiz; Rhee, Hyungjin; Park, Young Nyun; Alexandrov, Ludmil B.; Gordân, Raluca; Rozen, Steven G.; Shibata, Tatsuhiro; Pairojkul, Chawalit; Teh, Bin Tean; Tan, Patrick

2017-01-01

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy exhibiting high incidence in countries with endemic liver-fluke infection. We analysed 489 CCAs from 10 countries, combining whole-genome (71 cases), targeted/exome, copy-number, gene expression, and DNA methylation information. Integrative clustering defined four CCA clusters – Fluke-Positive CCAs (Clusters 1/2) are enriched in ERBB2 amplifications and TP53 mutations, conversely Fluke-Negative CCAs (Clusters 3/4) exhibit high copy-number alterations and PD-1/PD-L2 expression, or epigenetic mutations (IDH1/2, BAP1) and FGFR/PRKA-related gene rearrangements. Whole-genome analysis highlighted FGFR2 3′UTR deletion as a mechanism of FGFR2 upregulation. Integration of non-coding promoter mutations with protein-DNA binding profiles demonstrates pervasive modulation of H3K27me3-associated sites in CCA. Clusters 1 and 4 exhibit distinct DNA hypermethylation patterns targeting either CpG islands or shores – mutation signature and subclonality analysis suggests that these reflect different mutational pathways. Our results exemplify how genetics, epigenetics and environmental carcinogens can interplay across different geographies to generate distinct molecular subtypes of cancer. PMID:28667006

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

Jusakul, Apinya; Cutcutache, Ioana; Yong, Chern Han

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy exhibiting high incidence in countries with endemic liver-fluke infection. We analysed 489 CCAs from 10 countries, combining whole-genome (71 cases), targeted/exome, copy-number, gene expression, and DNA methylation information. Integrative clustering defined four CCA clusters - Fluke- Positive CCAs (Clusters 1/2) are enriched in ERBB2 amplifications and TP53 mutations, conversely Fluke-Negative CCAs (Clusters 3/4) exhibit high copy-number alterations and PD-1/PD-L2 expression, or epigenetic mutations (IDH1/2, BAP1) and FGFR/PRKA-related gene rearrangements. Whole-genome analysis highlighted FGFR2 3’UTR deletion as a mechanism of FGFR2 upregulation. Integration of non-coding promoter mutations with protein-DNA binding profiles demonstrates pervasive modulation ofmore » H3K27me3-associated sites in CCA. Clusters 1 and 4 exhibit distinct DNA hypermethylation patterns targeting either CpG islands or shores - mutation signature and subclonality analysis suggests that these reflect different mutational pathways. Lastly, our results exemplify how genetics, epigenetics and environmental carcinogens can interplay across different geographies to generate distinct molecular subtypes of cancer.« less

Scalable Open Science Approach for Mutation Calling of Tumor Exomes Using Multiple Genomic Pipelines.

PubMed

Ellrott, Kyle; Bailey, Matthew H; Saksena, Gordon; Covington, Kyle R; Kandoth, Cyriac; Stewart, Chip; Hess, Julian; Ma, Singer; Chiotti, Kami E; McLellan, Michael; Sofia, Heidi J; Hutter, Carolyn; Getz, Gad; Wheeler, David; Ding, Li

2018-03-28

The Cancer Genome Atlas (TCGA) cancer genomics dataset includes over 10,000 tumor-normal exome pairs across 33 different cancer types, in total >400 TB of raw data files requiring analysis. Here we describe the Multi-Center Mutation Calling in Multiple Cancers project, our effort to generate a comprehensive encyclopedia of somatic mutation calls for the TCGA data to enable robust cross-tumor-type analyses. Our approach accounts for variance and batch effects introduced by the rapid advancement of DNA extraction, hybridization-capture, sequencing, and analysis methods over time. We present best practices for applying an ensemble of seven mutation-calling algorithms with scoring and artifact filtering. The dataset created by this analysis includes 3.5 million somatic variants and forms the basis for PanCan Atlas papers. The results have been made available to the research community along with the methods used to generate them. This project is the result of collaboration from a number of institutes and demonstrates how team science drives extremely large genomics projects. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

Identification of somatic mutations in cancer through Bayesian-based analysis of sequenced genome pairs

PubMed Central

2013-01-01

Background The field of cancer genomics has rapidly adopted next-generation sequencing (NGS) in order to study and characterize malignant tumors with unprecedented resolution. In particular for cancer, one is often trying to identify somatic mutations – changes specific to a tumor and not within an individual’s germline. However, false positive and false negative detections often result from lack of sufficient variant evidence, contamination of the biopsy by stromal tissue, sequencing errors, and the erroneous classification of germline variation as tumor-specific. Results We have developed a generalized Bayesian analysis framework for matched tumor/normal samples with the purpose of identifying tumor-specific alterations such as single nucleotide mutations, small insertions/deletions, and structural variation. We describe our methodology, and discuss its application to other types of paired-tissue analysis such as the detection of loss of heterozygosity as well as allelic imbalance. We also demonstrate the high level of sensitivity and specificity in discovering simulated somatic mutations, for various combinations of a) genomic coverage and b) emulated heterogeneity. Conclusion We present a Java-based implementation of our methods named Seurat, which is made available for free academic use. We have demonstrated and reported on the discovery of different types of somatic change by applying Seurat to an experimentally-derived cancer dataset using our methods; and have discussed considerations and practices regarding the accurate detection of somatic events in cancer genomes. Seurat is available at https://sites.google.com/site/seuratsomatic. PMID:23642077

Identification of somatic mutations in cancer through Bayesian-based analysis of sequenced genome pairs.

PubMed

Christoforides, Alexis; Carpten, John D; Weiss, Glen J; Demeure, Michael J; Von Hoff, Daniel D; Craig, David W

2013-05-04

The field of cancer genomics has rapidly adopted next-generation sequencing (NGS) in order to study and characterize malignant tumors with unprecedented resolution. In particular for cancer, one is often trying to identify somatic mutations--changes specific to a tumor and not within an individual's germline. However, false positive and false negative detections often result from lack of sufficient variant evidence, contamination of the biopsy by stromal tissue, sequencing errors, and the erroneous classification of germline variation as tumor-specific. We have developed a generalized Bayesian analysis framework for matched tumor/normal samples with the purpose of identifying tumor-specific alterations such as single nucleotide mutations, small insertions/deletions, and structural variation. We describe our methodology, and discuss its application to other types of paired-tissue analysis such as the detection of loss of heterozygosity as well as allelic imbalance. We also demonstrate the high level of sensitivity and specificity in discovering simulated somatic mutations, for various combinations of a) genomic coverage and b) emulated heterogeneity. We present a Java-based implementation of our methods named Seurat, which is made available for free academic use. We have demonstrated and reported on the discovery of different types of somatic change by applying Seurat to an experimentally-derived cancer dataset using our methods; and have discussed considerations and practices regarding the accurate detection of somatic events in cancer genomes. Seurat is available at https://sites.google.com/site/seuratsomatic.

Lynch syndrome and Lynch syndrome mimics: The growing complex landscape of hereditary colon cancer

PubMed Central

Carethers, John M; Stoffel, Elena M

2015-01-01

Hereditary non-polyposis colorectal cancer (HNPCC) was previously synonymous with Lynch syndrome; however, identification of the role of germline mutations in the DNA mismatch repair (MMR) genes has made it possible to differentiate Lynch syndrome from other conditions associated with familial colorectal cancer (CRC). Broadly, HNPCC may be dichotomized into conditions that demonstrate defective DNA MMR and microsatellite instability (MSI) vs those conditions that demonstrate intact DNA MMR. Conditions characterized by MMR deficient CRCs include Lynch syndrome (germline MMR mutation), Lynch-like syndrome (biallelic somatic MMR mutations), constitutional MMR deficiency syndrome (biallelic germline MMR mutations), and sporadic MSI CRC (somatic biallelic methylation of MLH1). HNPCC conditions with intact DNA MMR associated with familial CRC include polymerase proofreading associated polyposis and familial colorectal cancer type X. Although next generation sequencing technologies have elucidated the genetic cause for some HNPCC conditions, others remain genetically undefined. Differentiating between Lynch syndrome and the other HNPCC disorders has profound implications for cancer risk assessment and surveillance of affected patients and their at-risk relatives. Clinical suspicion coupled with molecular tumor analysis and testing for germline mutations can help differentiate the clinical mimicry within HNPCC and facilitate diagnosis and management. PMID:26309352

Lynch syndrome and Lynch syndrome mimics: The growing complex landscape of hereditary colon cancer.

PubMed

Carethers, John M; Stoffel, Elena M

2015-08-21

Hereditary non-polyposis colorectal cancer (HNPCC) was previously synonymous with Lynch syndrome; however, identification of the role of germline mutations in the DNA mismatch repair (MMR) genes has made it possible to differentiate Lynch syndrome from other conditions associated with familial colorectal cancer (CRC). Broadly, HNPCC may be dichotomized into conditions that demonstrate defective DNA MMR and microsatellite instability (MSI) vs those conditions that demonstrate intact DNA MMR. Conditions characterized by MMR deficient CRCs include Lynch syndrome (germline MMR mutation), Lynch-like syndrome (biallelic somatic MMR mutations), constitutional MMR deficiency syndrome (biallelic germline MMR mutations), and sporadic MSI CRC (somatic biallelic methylation of MLH1). HNPCC conditions with intact DNA MMR associated with familial CRC include polymerase proofreading associated polyposis and familial colorectal cancer type X. Although next generation sequencing technologies have elucidated the genetic cause for some HNPCC conditions, others remain genetically undefined. Differentiating between Lynch syndrome and the other HNPCC disorders has profound implications for cancer risk assessment and surveillance of affected patients and their at-risk relatives. Clinical suspicion coupled with molecular tumor analysis and testing for germline mutations can help differentiate the clinical mimicry within HNPCC and facilitate diagnosis and management.

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

Leshinsky-Silver, E.; Mitochondrial Disease Center, Wolfson Medical Center, Holon; E-mail: leshinsky@wolfson.health.gov.il

Leigh syndrome can result from both nuclear and mitochondrial DNA defects. Mutations in complex V genes of the respiratory chain were considered until recently as the most frequent cause for mitochondrial inherited Leigh syndrome, while gene defects in complex I were related to recessive Leigh syndrome. Recently few reports of mutations in the mitochondrial-encoded complex I subunit genes causing Leigh syndrome have been reported. We describe a 1-month-old baby who acutely deteriorated, with abrupt onset of brainstem dysfunction, due to basal ganglia lesions extending to the brainstem. A muscle biopsy demonstrated complex I deficiency. Subsequent analysis of the mitochondrial genomemore » revealed a homoplastic T10191C mutation in the ND3 gene (in blood and muscle), resulting in a substitution of serine to proline. Hair root analysis revealed a 50% mutant load, reflecting heteroplasmy in early embryonic stages. The mutation was also detected in his mother (5%). Western blot analysis revealed a decrease of the 20 kDa subunit (likely ND6) and of the 30 kDa subunit (NDUFA9), which is probably due to instability attributed to the inability to form subcomplexes with ND3. This is the first description of infantile Leigh syndrome due to a maternally transmitted T10191C substitution in ND3 and not due to a de novo mutation. This mutation is age and tissue dependent and therefore may not be amenable to prenatal testing.« less

Application of tissue mesodissection to molecular cancer diagnostics.

PubMed

Krizman, David; Adey, Nils; Parry, Robert

2015-02-01

To demonstrate clinical application of a mesodissection platform that was developed to combine advantages of laser-based instrumentation with the speed/ease of manual dissection for automated dissection of tissue off standard glass slides. Genomic analysis for KRAS gene mutation was performed on formalin fixed paraffin embedded (FFPE) cancer patient tissue that was dissected using the mesodissection platform. Selected reaction monitoring proteomic analysis for quantitative Her2 protein expression was performed on FFPE patient tumour tissue dissected by a laser-based instrument and the MilliSect instrument. Genomic analysis demonstrates highly confident detection of KRAS mutation specifically in lung cancer cells and not the surrounding benign, non-tumour tissue. Proteomic analysis demonstrates Her2 quantitative protein expression in breast cancer cells dissected manually, by laser-based instrumentation and by MilliSect instrumentation (mesodissection). Slide-mounted tissue dissection is commonly performed using laser-based instruments or manually scraping tissue by scalpel. Here we demonstrate that the mesodissection platform as performed by the MilliSect instrument for tissue dissection is cost-effective; it functions comparably to laser-based dissection and which can be adopted into a clinical diagnostic workflow. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Activation of Antibiotic Production in Bacillus spp. by Cumulative Drug Resistance Mutations.

PubMed

Tojo, Shigeo; Tanaka, Yukinori; Ochi, Kozo

2015-12-01

Bacillus subtilis strains produce a wide range of antibiotics, including ribosomal and nonribosomal peptide antibiotics, as well as bacilysocin and neotrehalosadiamine. Mutations in B. subtilis strain 168 that conferred resistance to drugs such as streptomycin and rifampin resulted in overproduction of the dipeptide antibiotic bacilysin. Cumulative drug resistance mutations, such as mutations in the mthA and rpsL genes, which confer low- and high-level resistance, respectively, to streptomycin, and mutations in rpoB, which confer resistance to rifampin, resulted in cells that overproduced bacilysin. Transcriptional analysis demonstrated that the enhanced transcription of biosynthesis genes was responsible for the overproduction of bacilysin. This approach was effective also in activating the cryptic genes of Bacillus amyloliquefaciens, leading to actual production of antibiotic(s). Copyright © 2015, American Society for Microbiology. All Rights Reserved.

High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes.

PubMed

Lochlainn, Seosamh Ó; Amoah, Stephen; Graham, Neil S; Alamer, Khalid; Rios, Juan J; Kurup, Smita; Stoute, Andrew; Hammond, John P; Østergaard, Lars; King, Graham J; White, Phillip J; Broadley, Martin R

2011-12-08

Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes. We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner™ system we were able to detect mutations in heterozygous and homozygous states for both genes. Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M3 seed of Brassica rapa from the RevGenUK TILLING service.

High Resolution Melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes

PubMed Central

2011-01-01

Background Targeted Induced Loci Lesions IN Genomes (TILLING) is increasingly being used to generate and identify mutations in target genes of crop genomes. TILLING populations of several thousand lines have been generated in a number of crop species including Brassica rapa. Genetic analysis of mutants identified by TILLING requires an efficient, high-throughput and cost effective genotyping method to track the mutations through numerous generations. High resolution melt (HRM) analysis has been used in a number of systems to identify single nucleotide polymorphisms (SNPs) and insertion/deletions (IN/DELs) enabling the genotyping of different types of samples. HRM is ideally suited to high-throughput genotyping of multiple TILLING mutants in complex crop genomes. To date it has been used to identify mutants and genotype single mutations. The aim of this study was to determine if HRM can facilitate downstream analysis of multiple mutant lines identified by TILLING in order to characterise allelic series of EMS induced mutations in target genes across a number of generations in complex crop genomes. Results We demonstrate that HRM can be used to genotype allelic series of mutations in two genes, BraA.CAX1a and BraA.MET1.a in Brassica rapa. We analysed 12 mutations in BraA.CAX1.a and five in BraA.MET1.a over two generations including a back-cross to the wild-type. Using a commercially available HRM kit and the Lightscanner™ system we were able to detect mutations in heterozygous and homozygous states for both genes. Conclusions Using HRM genotyping on TILLING derived mutants, it is possible to generate an allelic series of mutations within multiple target genes rapidly. Lines suitable for phenotypic analysis can be isolated approximately 8-9 months (3 generations) from receiving M3 seed of Brassica rapa from the RevGenUK TILLING service. PMID:22152063

EG-08IDH MUTATIONS IN GLIOMAS ASSOCIATED WITH ENCHONDROMATOSIS

PubMed Central

Nicholas, M. Kelly; Joseph, Loren; Venneti, Sriram; Daher, Ahmad; Pytel, Peter

2014-01-01

The enchondromatoses, Ollier's disease and Maffucci syndrome, are non-heritable developmental disorders characterized by multiple enchondromas (Olllier's) in association with hemangiomas (Maffucci). Glial neoplasms are reported in both disorders but a pathogenic mechanism underlying this association has not been identified. We report a case of anaplastic astrocytoma in a 23 year old man with Maffucci syndrome whose tumor carried a substitution mutation of arginine for cysteine at position 132 (R132C) of the isocitrate dehydrogenase 1 (IDH1) protein. This mutation, commonly found in Maffucci-associated enchondromas and hemangiomas, was not detected on routine immunohistochemical (IHC) analysis of the astrocytoma using the R132H mutation-specific antibody, commonly applied in clinical laboratories. The R132C mutation was detected by polymerase chain reaction (PCR) and subsequently confirmed using a SNaPshot assay. Because somatic mosaic IDH mutations are associated with enchondromas and hemangiomas in Maffucci syndrome, we looked for the R132C mutation in a hemangioma, peripheral blood mononuclear cells (PBMNC) and histologically normal brain surrounding the tumor from this patient. The mutation was present in the hemangioma, absent in PBMNC, and present in 2% of alleles in ‘normal’ brain. The low level in surrounding brain tissue is consistent with tumor cell infiltration, not mosaicism, as a S173T p53 mutation in the tumor showed similar results. Using IHC, we further demonstrated that the mutant IDH1 protein in this glioma functions as an oncometabolite. Two repressive histone trimethylation marks were strongly positive in the tumor, supporting a role for 2-hydroxyglutarate in the inhibition of histone demethylation. Together, these data demonstrate that an IDH1 mutation common in enchodromatoses underlies the association of glial tumors reported in both Ollier's disease and Maffucci syndrome.

Genetic heterogeneity in patients with Bartter syndrome type 1

PubMed Central

Sun, Mingran; Ning, Jing; Xu, Weihong; Zhang, Han; Zhao, Kaishu; Li, Wenfu; Li, Guiying; Li, Shibo

2017-01-01

Bartter syndrome (BS) type 1 is an autosomal recessive kidney disorder caused by loss-of-function mutations in the solute carrier family 12 member 1 (SLC12A1) gene. To date, 72 BS type 1 patients harboring SLC12A1 mutations have been documented. Of these 144 alleles studied, 68 different disease-causing mutations have been detected in 129 alleles, and no mutation was detected in the remaining 15 alleles. The mutation types included missense/nonsense mutations, splicing mutations and small insertions and deletions ranging from 1 to 4 nucleotides. A large deletion encompassing a whole exon in the SLC12A1 gene has not yet been reported. The current study initially identified an undocumented homozygous frameshift mutation (c.1833delT) by Sanger sequencing analysis of a single infant with BS type 1. However, in a subsequent analysis, the mutation was detected only in the father's DNA. Upon further investigation using a next-generation sequencing approach, a deletion in exons 14 and 15 in both the patient and patient's mother was detected. The deletion was subsequently confirmed by use of a long-range polymerase chain reaction and was determined to be 3.16 kb in size based on sequencing of the junction fragment. The results of the present study demonstrated that pathogenic variants of SLC12A1 are heterogeneous. Large deletions appear to serve an etiological role in BS type 1, and may be more prevalent than previously thought. PMID:28000888

Genetic heterogeneity in patients with Bartter syndrome type 1.

PubMed

Sun, Mingran; Ning, Jing; Xu, Weihong; Zhang, Han; Zhao, Kaishu; Li, Wenfu; Li, Guiying; Li, Shibo

2017-02-01

Bartter syndrome (BS) type 1 is an autosomal recessive kidney disorder caused by loss‑of‑function mutations in the solute carrier family 12 member 1 (SLC12A1) gene. To date, 72 BS type 1 patients harboring SLC12A1 mutations have been documented. Of these 144 alleles studied, 68 different disease‑causing mutations have been detected in 129 alleles, and no mutation was detected in the remaining 15 alleles. The mutation types included missense/nonsense mutations, splicing mutations and small insertions and deletions ranging from 1 to 4 nucleotides. A large deletion encompassing a whole exon in the SLC12A1 gene has not yet been reported. The current study initially identified an undocumented homozygous frameshift mutation (c.1833delT) by Sanger sequencing analysis of a single infant with BS type 1. However, in a subsequent analysis, the mutation was detected only in the father's DNA. Upon further investigation using a next‑generation sequencing approach, a deletion in exons 14 and 15 in both the patient and patient's mother was detected. The deletion was subsequently confirmed by use of a long‑range polymerase chain reaction and was determined to be 3.16 kb in size based on sequencing of the junction fragment. The results of the present study demonstrated that pathogenic variants of SLC12A1 are heterogeneous. Large deletions appear to serve an etiological role in BS type 1, and may be more prevalent than previously thought.

Analysis of the surface expression of c-kit and occurrence of the c-kit Asp816Val activating mutation in T cells, B cells, and myelomonocytic cells in patients with mastocytosis.

PubMed

Akin, C; Kirshenbaum, A S; Semere, T; Worobec, A S; Scott, L M; Metcalfe, D D

2000-02-01

The Asp816Val c-kit activating mutation is detectable in the peripheral blood cells of some patients with mastocytosis and in lesional skin biopsies obtained from adult patients with urticaria pigmentosa. These observations led to the conclusion that this mutation is present in mast cells and mast cell precursors that express c-kit. However, the distribution of the Asp816Val mutation among hematopoietic lineages is unknown. To determine the distribution of the Asp816Val mutation among hematopoietic lineages and to explore its relationship to clinical disease, we examined cells bearing differentiation markers for myelomonocytic cells as well as T and B lymphocytes, in both peripheral blood and bone marrow obtained from patients with mastocytosis. The presence of Asp816Val c-kit mutation in cells magnetically sorted from peripheral blood or bone marrow according to surface differentiation markers was studied by reverse transcriptase polymerase chain reaction (RT-PCR) restriction fragment length polymorphism (RFLP) analysis. The surface expression of c-kit was determined by flow cytometry. The mutation was detectable by RT-PCR in at least one cell lineage in the bone marrow in 7 of 7 patients examined and in the peripheral blood of 11 of 11 adult patients with urticaria pigmentosa and indolent disease. The mutation was identified most frequently in B cells and myeloid cells. Flow cytometric analysis demonstrated that the differentiated cells expressing mutated c-kit were negative for surface KIT. These results are consistent with the conclusion that the c-kit Asp816Val mutation occurs in an early progenitor cell and is carried by myelomonocytic cells, T cells, and B cells in addition to mast cells. However, unlike mast cells, these myelomonocytic cells, T cells, and B cells do not concomitantly express surface c-kit and thus may be less susceptible to the effects of this mutation.

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.

Analysis of ESR1 mutation in circulating tumor DNA demonstrates evolution during therapy for metastatic breast cancer

PubMed Central

Schiavon, Gaia; Hrebien, Sarah; Garcia-Murillas, Isaac; Cutts, Rosalind J; Pearson, Alex; Tarazona, Noelia; Fenwick, Kerry; Kozarewa, Iwanka; Lopez-Knowles, Elena; Ribas, Ricardo; Nerurkar, Ashutosh; Osin, Peter; Chandarlapaty, Sarat; Martin, Lesley-Ann; Dowsett, Mitch; Smith, Ian E; Turner, Nicholas C.

2016-01-01

Acquired ESR1 mutations are a major mechanism of resistance to aromatase inhibitors (AI). We developed ultra-high sensitivity multiplexed digital PCR assays for ESR1 mutations in circulating tumor DNA (ctDNA) and used these to investigate the clinical relevance and origin of ESR1 mutations in a cohort of 171 women with advanced breast cancer. ESR1 mutation status in ctDNA showed high concordance with contemporaneous tumor biopsies, and could be assessed in samples shipped at room temperature in preservative tubes without loss of accuracy. ESR1 mutations were found exclusively in patients with estrogen receptor positive breast cancer previously exposed to AI. Patients with ESR1 mutations had a substantially shorter progression-free survival on subsequent AI-based therapy (HR 3.1, 95%CI 1.9-23.1, log rank p=0.0041). ESR1 mutation prevalence differed markedly between patients that were first exposed to AI during the adjuvant and metastatic settings (5.8% (3/52) vs 36.4% (16/44) respectively, p=0.0002). In an independent cohort, ESR1 mutations were identified in 0% (0/32, 95%CI 0-10.9%) tumor biopsies taken after progression on adjuvant AI. In a patient with serial samples taken during metastatic treatment, ESR1 mutation was selected during metastatic AI therapy, to become the dominant clone in the cancer. ESR1 mutations can be robustly identified with ctDNA analysis and predict for resistance to subsequent AI therapy. ESR1 mutations are rarely acquired during adjuvant AI therapy, but are commonly selected by therapy for metastatic disease, providing evidence that the mechanisms of resistance to targeted therapy may be substantially different between the treatment of micro-metastatic and overt metastatic cancer. PMID:26560360

Analysis of ESR1 mutation in circulating tumor DNA demonstrates evolution during therapy for metastatic breast cancer.

PubMed

Schiavon, Gaia; Hrebien, Sarah; Garcia-Murillas, Isaac; Cutts, Rosalind J; Pearson, Alex; Tarazona, Noelia; Fenwick, Kerry; Kozarewa, Iwanka; Lopez-Knowles, Elena; Ribas, Ricardo; Nerurkar, Ashutosh; Osin, Peter; Chandarlapaty, Sarat; Martin, Lesley-Ann; Dowsett, Mitch; Smith, Ian E; Turner, Nicholas C

2015-11-11

Acquired ESR1 mutations are a major mechanism of resistance to aromatase inhibitors (AIs). We developed ultra high-sensitivity multiplex digital polymerase chain reaction assays for ESR1 mutations in circulating tumor DNA (ctDNA) and investigated the clinical relevance and origin of ESR1 mutations in 171 women with advanced breast cancer. ESR1 mutation status in ctDNA showed high concordance with contemporaneous tumor biopsies and was accurately assessed in samples shipped at room temperature in preservative tubes. ESR1 mutations were found exclusively in estrogen receptor-positive breast cancer patients previously exposed to AI. Patients with ESR1 mutations had a substantially shorter progression-free survival on subsequent AI-based therapy [hazard ratio, 3.1; 95% confidence interval (CI), 1.9 to 23.1; P = 0.0041]. ESR1 mutation prevalence differed markedly between patients who were first exposed to AI during the adjuvant and metastatic settings [5.8% (3 of 52) versus 36.4% (16 of 44), respectively; P = 0.0002]. In an independent cohort, ESR1 mutations were identified in 0% (0 of 32; 95% CI, 0 to 10.9) tumor biopsies taken after progression on adjuvant AI. In a patient with serial sampling, ESR1 mutation was selected during metastatic AI therapy to become the dominant clone in the cancer. ESR1 mutations can be robustly identified with ctDNA analysis and predict for resistance to subsequent AI therapy. ESR1 mutations are rarely acquired during adjuvant AI but are commonly selected by therapy for metastatic disease, providing evidence that mechanisms of resistance to targeted therapy may be substantially different between the treatment of micrometastatic and overt metastatic cancer. Copyright © 2015, American Association for the Advancement of Science.

Identification and Functional Analysis of ZIC3 Mutations in Heterotaxy and Related Congenital Heart Defects

PubMed Central

Ware, Stephanie M.; Peng, Jianlan; Zhu, Lirong; Fernbach, Susan; Colicos, Suzanne; Casey, Brett; Towbin, Jeffrey; Belmont, John W.

2004-01-01

Mutations in the zinc finger transcription factor ZIC3 cause X-linked heterotaxy and have also been identified in patients with isolated congenital heart disease (CHD). To determine the relative contribution of ZIC3 mutations to both heterotaxy and isolated CHD, we screened the coding region of ZIC3 in 194 unrelated patients, including 61 patients with classic heterotaxy, 93 patients with heart defects characteristic of heterotaxy, and 11 patients with situs inversus totalis. Five novel ZIC3 mutations in three classic heterotaxy kindreds and two sporadic CHD cases were identified. None of these alleles was found in 97 ethnically matched control samples. On the basis of these analyses, we conclude that the phenotypic spectrum of ZIC3 mutations should be expanded to include affected females and CHD not typical for heterotaxy. This screening of a cohort of patients with sporadic heterotaxy indicates that ZIC3 mutations account for ∼1% of affected individuals. Missense and nonsense mutations were found in the highly conserved zinc finger–binding domain and in the N-terminal protein domain. Functional analysis of all currently known ZIC3 point mutations indicates that mutations in the putative zinc finger DNA binding domain and in the N-terminal domain result in loss of reporter gene transactivation. It is surprising that transfection studies demonstrate aberrant cytoplasmic localization resulting from mutations between amino acids 253–323 of the ZIC3 protein, indicating that the pathogenesis of a subset of ZIC3 mutations results at least in part from failure of appropriate nuclear localization. These results further expand the phenotypic and genotypic spectrum of ZIC3 mutations and provide initial mechanistic insight into their functional consequences. PMID:14681828

Germ-line mutations of the p53 tumor suppressor gene in patients with high risk for cancer inactivate the p53 protein.

PubMed Central

Frebourg, T; Kassel, J; Lam, K T; Gryka, M A; Barbier, N; Andersen, T I; Børresen, A L; Friend, S H

1992-01-01

Germ-line mutations in the p53 tumor suppressor gene have been observed in patients with Li-Fraumeni syndrome, brain tumors, second malignancies, and breast cancers. It is unclear whether all of these mutations have inactivated p53 and thereby provide an increased risk for cancer. Therefore, it is necessary to establish the biological significance of these germ-line mutations by the functional and structural analysis of the resulting mutant p53 proteins. We analyzed the ability of seven germ-line mutant proteins observed in patients with Li-Fraumeni syndrome, second primary neoplasms, or familial breast cancer to block the growth of malignant cells and compared the structural properties of the mutant proteins to that of the wild-type protein. Six of seven missense mutations disrupted the growth inhibitory properties and structure of the wild-type protein. One germ-line mutation retained the features of the wild-type p53. Genetic analysis of the breast cancer family in which this mutation was observed indicated that this germ-line mutation was not associated with the development of cancer. These results demonstrate that germ-line p53 mutations observed in patients with Li-Fraumeni syndrome and with second malignancies have inactivated the p53 tumor suppressor gene. The inability of the germ-line p53 mutants to block the growth of malignant cells can explain why patients with these germ-line mutations have an increased risk for cancer. The observation of a functionally silent germ-line mutation indicates that, before associating a germ-line tumor suppressor gene mutation with cancer risk, it is prudent to consider its functional significance. Images PMID:1631137

Germ-line mutations of the p53 tumor suppressor gene in patients with high risk for cancer inactivate the p53 protein.

PubMed

Frebourg, T; Kassel, J; Lam, K T; Gryka, M A; Barbier, N; Andersen, T I; Børresen, A L; Friend, S H

1992-07-15

Germ-line mutations in the p53 tumor suppressor gene have been observed in patients with Li-Fraumeni syndrome, brain tumors, second malignancies, and breast cancers. It is unclear whether all of these mutations have inactivated p53 and thereby provide an increased risk for cancer. Therefore, it is necessary to establish the biological significance of these germ-line mutations by the functional and structural analysis of the resulting mutant p53 proteins. We analyzed the ability of seven germ-line mutant proteins observed in patients with Li-Fraumeni syndrome, second primary neoplasms, or familial breast cancer to block the growth of malignant cells and compared the structural properties of the mutant proteins to that of the wild-type protein. Six of seven missense mutations disrupted the growth inhibitory properties and structure of the wild-type protein. One germ-line mutation retained the features of the wild-type p53. Genetic analysis of the breast cancer family in which this mutation was observed indicated that this germ-line mutation was not associated with the development of cancer. These results demonstrate that germ-line p53 mutations observed in patients with Li-Fraumeni syndrome and with second malignancies have inactivated the p53 tumor suppressor gene. The inability of the germ-line p53 mutants to block the growth of malignant cells can explain why patients with these germ-line mutations have an increased risk for cancer. The observation of a functionally silent germ-line mutation indicates that, before associating a germ-line tumor suppressor gene mutation with cancer risk, it is prudent to consider its functional significance.

Functional analysis of mutations in SLC7A9, and genotype-phenotype correlation in non-Type I cystinuria.

PubMed

Font, M A; Feliubadaló, L; Estivill, X; Nunes, V; Golomb, E; Kreiss, Y; Pras, E; Bisceglia, L; d'Adamo, A P; Zelante, L; Gasparini, P; Bassi, M T; George , A L; Manzoni, M; Riboni, M; Ballabio, A; Borsani, G; Reig, N; Fernández, E; Zorzano, A; Bertran, J; Palacín, M

2001-02-15

Cystinuria (OMIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids that results in nephrolithiasis of cystine. Mutations in SLC3A1, which encodes rBAT, cause Type I cystinuria, and mutations in SLC7A9, which encodes a putative subunit of rBAT (b(o,+)AT), cause non-Type I cystinuria. Here we describe the genomic structure of SLC7A9 (13 exons) and 28 new mutations in this gene that, together with the seven previously reported, explain 79% of the alleles in 61 non-Type I cystinuria patients. These data demonstrate that SLC7A9 is the main non-Type I cystinuria gene. Mutations G105R, V170M, A182T and R333W are the most frequent SLC7A9 missense mutations found. Among heterozygotes carrying these mutations, A182T heterozygotes showed the lowest urinary excretion values of cystine and dibasic amino acids. Functional analysis of mutation A182T after co-expression with rBAT in HeLa cells revealed significant residual transport activity. In contrast, mutations G105R, V170M and R333W are associated to a complete or almost complete loss of transport activity, leading to a more severe urinary phenotype in heterozygotes. SLC7A9 mutations located in the putative transmembrane domains of b(o,+)AT and affecting conserved amino acid residues with a small side chain generate a severe phenotype, while mutations in non-conserved residues give rise to a mild phenotype. These data provide the first genotype-phenotype correlation in non-Type I cystinuria, and show that a mild urinary phenotype in heterozygotes may associate with mutations with significant residual transport activity.

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

PubMed

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

2016-11-01

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

Targeted Analysis of Whole Genome Sequence Data to Diagnose Genetic Cardiomyopathy

DOE PAGES

Golbus, Jessica R.; Puckelwartz, Megan J.; Dellefave-Castillo, Lisa; ...

2014-09-01

Background—Cardiomyopathy is highly heritable but genetically diverse. At present, genetic testing for cardiomyopathy uses targeted sequencing to simultaneously assess the coding regions of more than 50 genes. New genes are routinely added to panels to improve the diagnostic yield. With the anticipated $1000 genome, it is expected that genetic testing will shift towards comprehensive genome sequencing accompanied by targeted gene analysis. Therefore, we assessed the reliability of whole genome sequencing and targeted analysis to identify cardiomyopathy variants in 11 subjects with cardiomyopathy. Methods and Results—Whole genome sequencing with an average of 37× coverage was combined with targeted analysis focused onmore » 204 genes linked to cardiomyopathy. Genetic variants were scored using multiple prediction algorithms combined with frequency data from public databases. This pipeline yielded 1-14 potentially pathogenic variants per individual. Variants were further analyzed using clinical criteria and/or segregation analysis. Three of three previously identified primary mutations were detected by this analysis. In six subjects for whom the primary mutation was previously unknown, we identified mutations that segregated with disease, had clinical correlates, and/or had additional pathological correlation to provide evidence for causality. For two subjects with previously known primary mutations, we identified additional variants that may act as modifiers of disease severity. In total, we identified the likely pathological mutation in 9 of 11 (82%) subjects. We conclude that these pilot data demonstrate that ~30-40× coverage whole genome sequencing combined with targeted analysis is feasible and sensitive to identify rare variants in cardiomyopathy-associated genes.« less

A distinct X-linked syndrome involving joint contractures, keloids, large optic cup-to-disc ratio, and renal stones results from a filamin A (FLNA) mutation.

PubMed

Lah, Melissa; Niranjan, Tejasvi; Srikanth, Sujata; Holloway, Lynda; Schwartz, Charles E; Wang, Tao; Weaver, David D

2016-04-01

We further evaluated a previously reported family with an apparently undescribed X-linked syndrome involving joint contractures, keloids, an increased optic cup-to-disc ratio, and renal stones to elucidate the genetic cause. To do this, we obtained medical histories and performed physical examination on 14 individuals in the family, five of whom are affected males and three are obligate carrier females. Linkage analysis was performed on all but one individual and chromosome X-exome sequencing was done on two affected males. The analysis localized the putative gene to Xq27-qter and chromosome X-exome sequencing revealed a mutation in exon 28 (c.4726G>A) of the filamin A (FLNA) gene, predicting that a conserved glycine had been replaced by arginine at amino acid 1576 (p.G1576R). Segregation analysis demonstrated that all known carrier females tested were heterozygous (G/A), all affected males were hemizygous for the mutation (A allele) and all normal males were hemizygous for the normal G allele. The data and the bioinformatic analysis indicate that the G1576R mutation in the FLNA gene is very likely pathogenic in this family. The syndrome affecting the family shares phenotypic overlap with other syndromes caused by FLNA mutations, but appears to be a distinct phenotype, likely representing a unique genetic syndrome. © 2016 Wiley Periodicals, Inc.

A BAC transgenic mouse model reveals neuron subtype-specific effects of a Generalized Epilepsy with Febrile Seizures Plus (GEFS+) mutation

PubMed Central

Tang, Bin; Dutt, Karoni; Papale, Ligia; Rusconi, Raffaella; Shankar, Anupama; Hunter, Jessica; Tufik, Sergio; Yu, Frank H.; Catterall, William A.; Mantegazza, Massimo; Goldin, Alan L.; Escayg, Andrew

2009-01-01

Mutations in the voltage-gated sodium channel SCN1A are responsible for a number of seizure disorders including Generalized Epilepsy with Febrile Seizures Plus (GEFS+) and Severe Myoclonic Epilepsy of Infancy (SMEI). To determine the effects of SCN1A mutations on channel function in vivo, we generated a bacterial artificial chromosome (BAC) transgenic mouse model that expresses the human SCN1A GEFS+ mutation, R1648H. Mice with the R1648H mutation exhibit a more severe response to the proconvulsant kainic acid compared with mice expressing a control Scn1a transgene. Electrophysiological analysis of dissociated neurons from mice with the R1648H mutation reveal delayed recovery from inactivation and increased use-dependent inactivation only in inhibitory bipolar neurons, as well as a hyperpolarizing shift in the voltage dependence of inactivation only in excitatory pyramidal neurons. These results demonstrate that the effects of SCN1A mutations are cell type-dependent and that the R1648H mutation specifically leads to a reduction in interneuron excitability. PMID:19409490

RNA-based mutation analysis identifies an unusual MSH6 splicing defect and circumvents PMS2 pseudogene interference.

PubMed

Etzler, J; Peyrl, A; Zatkova, A; Schildhaus, H-U; Ficek, A; Merkelbach-Bruse, S; Kratz, C P; Attarbaschi, A; Hainfellner, J A; Yao, S; Messiaen, L; Slavc, I; Wimmer, K

2008-02-01

Heterozygous germline mutations in one of the mismatch repair (MMR) genes MLH1, MSH2, MSH6, and PMS2 cause hereditary nonpolyposis colorectal cancer (HNPCC) or Lynch syndrome, a dominantly inherited cancer susceptibility syndrome. Recent reports provide evidence for a novel recessively inherited cancer syndrome with constitutive MMR deficiency due to biallelic germline mutations in one of the MMR genes. MMR-deficiency (MMR-D) syndrome is characterized by childhood brain tumors, hematological and/or gastrointestinal malignancies, and signs of neurofibromatosis type 1 (NF1). We established an RNA-based mutation detection assay for the four MMR genes, since 1) a number of splicing defects may escape detection by the analysis of genomic DNA, and 2) DNA-based mutation detection in the PMS2 gene is severely hampered by the presence of multiple highly similar pseudogenes, including PMS2CL. Using this assay, which is based on direct cDNA sequencing of RT-PCR products, we investigated two families with children suspected to suffer from MMR-D syndrome. We identified a homozygous complex MSH6 splicing alteration in the index patients of the first family and a novel homozygous PMS2 mutation (c.182delA) in the index patient of the second family. Furthermore, we demonstrate, by the analysis of a PMS2/PMS2CL "hybrid" allele carrier, that RNA-based PMS2 testing effectively avoids the caveats of genomic DNA amplification approaches; i.e., pseudogene coamplification as well as allelic dropout, and will, thus, allow more sensitive mutation analysis in MMR deficiency and in HNPCC patients with PMS2 defects. (c) 2007 Wiley-Liss, Inc.

Clinical phenotype and infertility treatment in a male with hypogonadotropic hypogonadism due to mutations Ala129Asp/Arg262Gln of the gonadotropin-releasing hormone receptor.

PubMed

Layman, Lawrence C; Cohen, David P; Xie, Jun; Smith, Gary D

2002-12-01

To characterize the genotype and phenotype of a man with idiopathic hypogonadism with infertility. Molecular analysis and clinical description. Medical school laboratory and reproductive endocrine clinic.A 40-year-old male with idiopathic hypogonadotropic hypogonadism. Denaturing gradient gel electrophoresis analysis and DNA sequencing of the gonadotropin-releasing hormone receptor (GNRHR) gene were performed. The patient was treated with hCG and FSH. GNRHR mutation detection, genotype/phenotype correlation, and testicular response to exogenous gonadotropin therapy. The proband demonstrated compound heterozygosity for Ala129Asp/Arg262Gln GNRHR mutations. He had a complete form of idiopathic hypogonadotropic hypogonadism, with descended testes and severe oligospermia but little response to exogenous gonadotropins. The phenotype of this patient differs from the one other family described with the same mutations. Exogenous gonadotropin therapy may not be as beneficial for increasing sperm concentration in older men with idiopathic hypogonadotropic hypogonadism.

[Myoclonus epilepsy with ragged-red fibers: a case report and literature review].

PubMed

Zhao, Man-man; Zhang, Yao; Bao, Xin-hua

2015-12-18

To demonstrate the clinical manifestation, diagnosis and treatment of myoclonus epilepsy with ragged-red-fibers (MERRF), a case of MERRF was presented with review of the literature. A 4-year-7-month-old girl was diagnosed with MERRF. She had tremor, fatigue and developmental delay for more than 2 years. Laboratory tests showed that the serum and urine lactic acid and pyruvic acid increased significantly. Electroencephalogram showed diffuse and focal spike slow wave and slow wave in right central and parietal regions. Electromyogram showed neurological damage. Gene mutational analysis showed mtDNA 8344 A>G mutation. The mutational rate was 78%. Mitochondrial disease MERRF syndrome was diagnosed. Cocktails therapy with vitamins B1, B6, B12, L-carnitine, and coenzyme Q10 was administrated to the patient. MERRF is a rare disease. The diagnosis can be made by gene mutational analysis. Cocktail therapy may slow down the deterioration of the disease. Gene therapy is still experimental.

RNA Recombination Enhances Adaptability and Is Required for Virus Spread and Virulence.

PubMed

Xiao, Yinghong; Rouzine, Igor M; Bianco, Simone; Acevedo, Ashley; Goldstein, Elizabeth Faul; Farkov, Mikhail; Brodsky, Leonid; Andino, Raul

2016-04-13

Mutation and recombination are central processes driving microbial evolution. A high mutation rate fuels adaptation but also generates deleterious mutations. Recombination between two different genomes may resolve this paradox, alleviating effects of clonal interference and purging deleterious mutations. Here we demonstrate that recombination significantly accelerates adaptation and evolution during acute virus infection. We identified a poliovirus recombination determinant within the virus polymerase, mutation of which reduces recombination rates without altering replication fidelity. By generating a panel of variants with distinct mutation rates and recombination ability, we demonstrate that recombination is essential to enrich the population in beneficial mutations and purge it from deleterious mutations. The concerted activities of mutation and recombination are key to virus spread and virulence in infected animals. These findings inform a mathematical model to demonstrate that poliovirus adapts most rapidly at an optimal mutation rate determined by the trade-off between selection and accumulation of detrimental mutations. Copyright © 2016 Elsevier Inc. All rights reserved.

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

PubMed

Thierry, Alain R

2016-01-01

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

Morphometric analysis and neuroanatomical mapping of the zebrafish brain.

PubMed

Gupta, Tripti; Marquart, Gregory D; Horstick, Eric J; Tabor, Kathryn M; Pajevic, Sinisa; Burgess, Harold A

2018-06-21

Large-scale genomic studies have recently identified genetic variants causative for major neurodevelopmental disorders, such as intellectual disability and autism. However, determining how underlying developmental processes are affected by these mutations remains a significant challenge in the field. Zebrafish is an established model system in developmental neurogenetics that may be useful in uncovering the mechanisms of these mutations. Here we describe the use of voxel-intensity, deformation field, and volume-based morphometric techniques for the systematic and unbiased analysis of gene knock-down and environmental exposure-induced phenotypes in zebrafish. We first present a computational method for brain segmentation based on transgene expression patterns to create a comprehensive neuroanatomical map. This map allowed us to disclose statistically significant changes in brain microstructure and composition in neurodevelopmental models. We demonstrate the effectiveness of morphometric techniques in measuring changes in the relative size of neuroanatomical subdivisions in atoh7 morphant larvae and in identifying phenotypes in larvae treated with valproic acid, a chemical demonstrated to increase the risk of autism in humans. These tools enable rigorous evaluation of the effects of gene mutations and environmental exposures on neural development, providing an entry point for cellular and molecular analysis of basic developmental processes as well as neurodevelopmental and neurodegenerative disorders. Published by Elsevier Inc.

Spinal Neurofibromatosis without Café-au-Lait Macules in Two Families with Null Mutations of the NF1 Gene

PubMed Central

Kaufmann, Dieter; Müller, Ralf; Bartelt, Britta; Wolf, Michael; Kunzi-Rapp, Karin; Hanemann, Clemens Oliver; Fahsold, Raimund; Hein, Christian; Vogel, Walther; Assum, Günter

2001-01-01

Spinal neurofibromatosis (SNF) is considered to be an alternative form of neurofibromatosis, showing multiple spinal tumors and café-au-lait macules. Involvement of the neurofibromatosis type 1 (NF1) locus has been demonstrated, by linkage analysis, for three families with SNF. In one of them, a cosegregating frameshift mutation in exon 46 of the NF1 gene was identified. In the present study, we report four individuals from two families who carry NF1 null mutations that would be expected to cause NF1. Three patients have multiple spinal tumors and no café-au-lait macules, and the fourth has no clinical signs of NF1. In the first family, a missense mutation (Leu2067Pro) in NF1 exon 33 was found, and, in the second, a splice-site mutation (IVS31-5A→G) enlarging exon 32 by 4 bp at the 5′ end was found. The latter mutation has also been observed in an unrelated patient with classical NF1. Both NF1 mutations cause a reduction in neurofibromin of ∼50%, with no truncated protein present in the cells. This demonstrates that typical NF1 null mutations can result in a phenotype that is distinct from classical NF1, showing only a small spectrum of the NF1 symptoms, such as multiple spinal tumors, but not completely fitting the current clinical criteria for SNF. We speculate that this phenotype is caused by an unknown modifying gene that compensates for some, but not all, of the effects caused by neurofibromin deficiency. PMID:11704931

Trade-offs with stability modulate innate and mutationally acquired drug-resistance in bacterial dihydrofolate reductase enzymes.

PubMed

Matange, Nishad; Bodkhe, Swapnil; Patel, Maitri; Shah, Pooja

2018-06-05

Structural stability is a major constraint on the evolution of protein sequences. However, under strong directional selection, mutations that confer novel phenotypes but compromise structural stability of proteins may be permissible. During the evolution of antibiotic resistance, mutations that confer drug resistance often have pleiotropic effects on the structure and function of antibiotic-target proteins, usually essential metabolic enzymes. In this study, we show that trimethoprim-resistant alleles of dihydrofolate reductase from Escherichia coli (EcDHFR) harbouring the Trp30Gly, Trp30Arg or Trp30Cys mutations are significantly less stable than the wild type making them prone to aggregation and proteolysis. This destabilization is associated with lower expression level resulting in a fitness cost and negative epistasis with other TMP-resistant mutations in EcDHFR. Using structure-based mutational analysis we show that perturbation of critical stabilizing hydrophobic interactions in wild type EcDHFR enzyme explains the phenotypes of Trp30 mutants. Surprisingly, though crucial for the stability of EcDHFR, significant sequence variation is found at this site among bacterial DHFRs. Mutational and computational analyses in EcDHFR as well as in DHFR enzymes from Staphylococcus aureus and Mycobacterium tuberculosis demonstrate that natural variation at this site and its interacting hydrophobic residues, modulates TMP-resistance in other bacterial DHFRs as well, and may explain the different susceptibilities of bacterial pathogens to trimethoprim. Our study demonstrates that trade-offs between structural stability and function can influence innate drug resistance as well as the potential for mutationally acquired drug resistance of an enzyme. ©2018 The Author(s).

CREBBP mutations in relapsed acute lymphoblastic leukaemia

PubMed Central

Mullighan, Charles G.; Zhang, Jinghui; Kasper, Lawryn H.; Lerach, Stephanie; Payne-Turner, Debbie; Phillips, Letha A.; Heatley, Sue L.; Holmfeldt, Linda; Collins-Underwood, J. Racquel; Ma, Jing; Buetow, Kenneth H.; Pui, Ching-Hon; Baker, Sharyn D.; Brindle, Paul K.; Downing, James R.

2010-01-01

Relapsed acute lymphoblastic leukaemia (ALL) is a leading cause of death due to disease in young people, but the biologic determinants of treatment failure remain poorly understood. Recent genome-wide profiling of structural DNA alterations in ALL have identified multiple submicroscopic somatic mutations targeting key cellular pathways1,2, and have demonstrated substantial evolution in genetic alterations from diagnosis to relapse3. However, detailed analysis of sequence mutations in ALL has not been performed. To identify novel mutations in relapsed ALL, we resequenced 300 genes in matched diagnosis and relapse samples from 23 patients with ALL. This identified 52 somatic non-synonymous mutations in 32 genes, many of which were novel, including the transcriptional coactivators CREBBP and NCOR1, the transcription factors ERG, SPI1, TCF4 and TCF7L2, components of the Ras signalling pathway, histone genes, genes involved in histone modification (CREBBP and CTCF), and genes previously shown to be targets of recurring DNA copy number alteration in ALL. Analysis of an extended cohort of 71 diagnosis-relapse cases and 270 acute leukaemia cases that did not relapse found that 18.3% of relapse cases had sequence or deletion mutations of CREBBP, which encodes the transcriptional coactivator and histone acetyltransferase (HAT) CREB-binding protein (CBP)4. The mutations were either present at diagnosis or acquired at relapse, and resulted in truncated alleles or deleterious substitutions in conserved residues of the HAT domain. Functionally, the mutations impaired histone acetylation and transcriptional regulation of CREBBP targets, including glucocorticoid responsive genes. Several mutations acquired at relapse were detected in subclones at diagnosis, suggesting that the mutations may confer resistance to therapy. These results extend the landscape of genetic alterations in leukaemia, and identify mutations targeting transcriptional and epigenetic regulation as a mechanism of resistance in ALL. PMID:21390130

Detection of Rare Mutations in EGFR-ARMS-PCR-Negative Lung Adenocarcinoma by Sanger Sequencing.

PubMed

Liang, Chaoyue; Wu, Zhuolin; Gan, Xiaohong; Liu, Yuanbin; You, You; Liu, Chenxian; Zhou, Chengzhi; Liang, Ying; Mo, Haiyun; Chen, Allen M; Zhang, Jiexia

2018-01-01

This study aimed to identify potential epidermal growth factor receptor (EGFR) gene mutations in non-small cell lung cancer that went undetected by amplification refractory mutation system-Scorpion real-time PCR (ARMS-PCR). A total of 200 specimens were obtained from the First Affiliated Hospital of Guangzhou Medical University from August 2014 to August 2015. In total, 100 ARMS-negative and 100 ARMS-positive specimens were evaluated for EGFR gene mutations by Sanger sequencing. The methodology and sensitivity of each method and the outcomes of EGFR-tyrosine kinase inhibitor (TKI) therapy were analyzed. Among the 100 ARMS-PCR-positive samples, 90 were positive by Sanger sequencing, while 10 cases were considered negative, because the mutation abundance was less than 10%. Among the 100 negative cases, three were positive for a rare EGFR mutation by Sanger sequencing. In the curative effect analysis of EGFR-TKIs, the progression-free survival (PFS) analysis based on ARMS and Sanger sequencing results showed no difference. However, the PFS of patients with a high abundance of EGFR mutation was 12.4 months [95% confidence interval (CI), 11.6-12.4 months], which was significantly higher than that of patients with a low abundance of mutations detected by Sanger sequencing (95% CI, 10.7-11.3 months) (p<0.001). The ARMS method demonstrated higher sensitivity than Sanger sequencing, but was prone to missing mutations due to primer design. Sanger sequencing was able to detect rare EGFR mutations and deemed applicable for confirming EGFR status. A clinical trial evaluating the efficacy of EGFR-TKIs in patients with rare EGFR mutations is needed. © Copyright: Yonsei University College of Medicine 2018

Fast maximum likelihood estimation of mutation rates using a birth-death process.

PubMed

Wu, Xiaowei; Zhu, Hongxiao

2015-02-07

Since fluctuation analysis was first introduced by Luria and Delbrück in 1943, it has been widely used to make inference about spontaneous mutation rates in cultured cells. Under certain model assumptions, the probability distribution of the number of mutants that appear in a fluctuation experiment can be derived explicitly, which provides the basis of mutation rate estimation. It has been shown that, among various existing estimators, the maximum likelihood estimator usually demonstrates some desirable properties such as consistency and lower mean squared error. However, its application in real experimental data is often hindered by slow computation of likelihood due to the recursive form of the mutant-count distribution. We propose a fast maximum likelihood estimator of mutation rates, MLE-BD, based on a birth-death process model with non-differential growth assumption. Simulation studies demonstrate that, compared with the conventional maximum likelihood estimator derived from the Luria-Delbrück distribution, MLE-BD achieves substantial improvement on computational speed and is applicable to arbitrarily large number of mutants. In addition, it still retains good accuracy on point estimation. Published by Elsevier Ltd.

A human-specific mutation leads to the origin of a novel splice form of neuropsin (KLK8), a gene involved in learning and memory.

PubMed

Lu, Zhi-xiang; Peng, Jia; Su, Bing

2007-10-01

Neuropsin (kallikrein 8, KLK8) is a secreted-type serine protease preferentially expressed in the central nervous system and involved in learning and memory. Its splicing pattern is different in human and mouse, with the longer form (type II) only expressed in human. Sequence analysis suggested a recent origin of type II during primate evolution. Here we demonstrate that the type II form is absent in nonhuman primates, and is thus a human-specific splice form. With the use of an in vitro splicing assay, we show that a human-specific T to A mutation (c.71-127T>A) triggers the change of splicing pattern, leading to the origin of a novel splice form in the human brain. Using mutation assay, we prove that this mutation is not only necessary but also sufficient for type II expression. Our results demonstrate a molecular mechanism for the creation of novel proteins through alternative splicing in the central nervous system during human evolution. Copyright 2007 Wiley-Liss, Inc.

Effect of a mutation at arginine 301 on the stability, crystal quality and the preliminary crystallographic analysis of recombinant canavalin from Canavalia ensiformis

NASA Astrophysics Data System (ADS)

Elizabeth Green, M.; Kirkland, Natalie; Ng, Joseph D.

2001-11-01

The technique of site-directed mutagenesis was used to implement rational amino acid changes in the plant storage protein canavalin, the major seed storage protein of the jack bean ( Canavali ensiformis). The mutations were targeted to amino acids previously demonstrated to be involved in either the intra- or intermolecular salt bridges, thought to be responsible for maintaining the three-dimensional structure of the trimer. The amino acid changes were designed to disrupt the salt bridge interactions by substituting a neutral alanine for a negatively charged aspartate or glutamate, or by substituting a negatively charged glutamate for a positively charged arginine. The resulting recombinant mutants were subsequently expressed, purified, and crystallized. The crystals of the mutant versions of canavalin were compared to those of the wild-type canavalin by visual inspection and X-ray analysis. Of the crystals obtained for the mutants, those for the Arg301Glu mutation appeared to be more stable with fewer surface defects than any of the other mutants or the wild-type protein. The I/ σ ratio of reflections versus the resolution for the Arg301Glu mutation was approximately 30% greater over the entire resolution range than that obtained for any of the other mutations or for the wild-type. Additionally, the crystals of Arg301Glu mutations displayed lower mosaicity. Finally, the Arg301Glu mutation displayed a striking increase in the transition temperature when subjected to thermal denaturation. This paper describes the rationale and techniques behind the mutation of canavalin and suggests possible explanations for the observed and measured differences between the Arg301Glu mutant and the wild-type protein. We show the initial crystallographic structure analysis of this mutant and its preliminary implications.

Novel mutations of MYO7A and USH1G in Israeli Arab families with Usher syndrome type 1.

PubMed

Rizel, Leah; Safieh, Christine; Shalev, Stavit A; Mezer, Eedy; Jabaly-Habib, Haneen; Ben-Neriah, Ziva; Chervinsky, Elena; Briscoe, Daniel; Ben-Yosef, Tamar

2011-01-01

This study investigated the genetic basis for Usher syndrome type 1 (USH1) in four consanguineous Israeli Arab families. Haplotype analysis for all known USH1 loci was performed in each family. In families for which haplotype analysis was inconclusive, we performed genome-wide homozygosity mapping using a single nucleotide polymorphism (SNP) array. For mutation analysis, specific primers were used to PCR amplify the coding exons of the MYO7A, USH1C, and USH1G genes including intron-exon boundaries. Mutation screening was performed with direct sequencing. A combination of haplotype analysis and genome-wide homozygosity mapping indicated linkage to the USH1B locus in two families, USH1C in one family and USH1G in another family. Sequence analysis of the relevant genes (MYO7A, USH1C, and USH1G) led to the identification of pathogenic mutations in all families. Two of the identified mutations are novel (c.1135-1147dup in MYO7A and c.206-207insC in USH1G). USH1 is a genetically heterogenous condition. Of the five USH1 genes identified to date, USH1C and USH1G are the rarest contributors to USH1 etiology worldwide. It is therefore interesting that two of the four Israeli Arab families reported here have mutations in these two genes. This finding further demonstrates the unique genetic structure of the Israeli population in general, and the Israeli Arab population in particular, which due to high rates of consanguinity segregates many rare autosomal recessive genetic conditions.

Kin-Driver: a database of driver mutations in protein kinases.

PubMed

Simonetti, Franco L; Tornador, Cristian; Nabau-Moretó, Nuria; Molina-Vila, Miguel A; Marino-Buslje, Cristina

2014-01-01

Somatic mutations in protein kinases (PKs) are frequent driver events in many human tumors, while germ-line mutations are associated with hereditary diseases. Here we present Kin-driver, the first database that compiles driver mutations in PKs with experimental evidence demonstrating their functional role. Kin-driver is a manual expert-curated database that pays special attention to activating mutations (AMs) and can serve as a validation set to develop new generation tools focused on the prediction of gain-of-function driver mutations. It also offers an easy and intuitive environment to facilitate the visualization and analysis of mutations in PKs. Because all mutations are mapped onto a multiple sequence alignment, analogue positions between kinases can be identified and tentative new mutations can be proposed for studying by transferring annotation. Finally, our database can also be of use to clinical and translational laboratories, helping them to identify uncommon AMs that can correlate with response to new antitumor drugs. The website was developed using PHP and JavaScript, which are supported by all major browsers; the database was built using MySQL server. Kin-driver is available at: http://kin-driver.leloir.org.ar/ © The Author(s) 2014. Published by Oxford University Press.

Activation and products of the cryptic secondary metabolite biosynthetic gene clusters by rifampin resistance (rpoB) mutations in actinomycetes.

PubMed

Tanaka, Yukinori; Kasahara, Ken; Hirose, Yutaka; Murakami, Kiriko; Kugimiya, Rie; Ochi, Kozo

2013-07-01

A subset of rifampin resistance (rpoB) mutations result in the overproduction of antibiotics in various actinomycetes, including Streptomyces, Saccharopolyspora, and Amycolatopsis, with H437Y and H437R rpoB mutations effective most frequently. Moreover, the rpoB mutations markedly activate (up to 70-fold at the transcriptional level) the cryptic/silent secondary metabolite biosynthetic gene clusters of these actinomycetes, which are not activated under general stressful conditions, with the exception of treatment with rare earth elements. Analysis of the metabolite profile demonstrated that the rpoB mutants produced many metabolites, which were not detected in the wild-type strains. This approach utilizing rifampin resistance mutations is characterized by its feasibility and potential scalability to high-throughput studies and would be useful to activate and to enhance the yields of metabolites for discovery and biochemical characterization.

NDUFS4 mutations cause Leigh syndrome with predominant brainstem involvement.

PubMed

Leshinsky-Silver, E; Lebre, Anne-Sophie; Minai, Limor; Saada, Ann; Steffann, Julie; Cohen, Sarit; Rötig, Agnes; Munnich, Arnold; Lev, Dorit; Lerman-Sagie, Tally

2009-07-01

Complex I deficiency is a frequent cause of Leigh syndrome. We describe a non-consanguineous Ashkenazi-Sephardic Jewish patient with Leigh syndrome due to complex I deficiency. The clinical and neuroradiological presentation showed predominant brainstem involvement. Blue native polyacrylamide gel electrophoresis analysis revealed an impaired assembly of complex I. The patient was found to be compound heterozygous of two mutations in the NDUFS4 gene: p.Asp119His (a novel mutation) and p.Lys154fs (recently described in an Ashkenazi Jewish family). These findings support the suggestion that the p.Lys154fs mutation in NDUFS4 should be evaluated in Ashkenazi Jewish patients presenting with early onset Leigh syndrome even before enzymatic studies. Our results further demonstrated that NDUFS4 presents a hotspot of mutations in the genetic apparatus of oxidative phosphorylation and the correct assembly of the subunit it encodes is essential for completion of the assembly of complex I.

Severe Bleeding In a Woman Heterozygous for the Fibrinogen γR275C Mutation

PubMed Central

Rein, Chantelle M.; Anderson, Brian L; Ballard, Morgan M.; Domes, Christopher M.; Johnston, Joshua M.; Madsen, R. Jared; Wolper, Kathryn K. M.; Terker, Andrew S.; Strother, John M.; Deloughery, Thomas G.; Farrell, David H.

2010-01-01

The dysfibrinogen γR275C can be a clinically silent mutation, with only two out of seventeen cases in the literature reporting a hemorrhagic presentation, and four cases reporting a thrombotic presentation. We describe here a particularly severe presentation in 54-year-old female patient who required a hysterectomy at 47 years of age due to heavy menstrual bleeding. Coagulation studies revealed a prolonged prothrombin time and thrombin time, a normal fibrinogen antigen level, and a low fibrinogen activity level. Molecular analysis of the patient’s DNA revealed a γ chain gene mutation resulting in an amino acid substitution at residue 275 (γR275C). Protein sequencing of the fibrinogen γ chain confirmed this mutation, which was named Fibrinogen Portland I. This case demonstrates that the γR275C mutation can lead to a severe hemorrhagic phenotype. PMID:20386430

Novel splice mutation in microthalmia-associated transcription factor in Waardenburg Syndrome.

PubMed

Brenner, Laura; Burke, Kelly; Leduc, Charles A; Guha, Saurav; Guo, Jiancheng; Chung, Wendy K

2011-01-01

Waardenburg Syndrome (WS) is a syndromic form of hearing loss associated with mutations in six different genes. We identified a large family with WS that had previously undergone clinical testing, with no reported pathogenic mutation. Using linkage analysis, a region on 3p14.1 with an LOD score of 6.6 was identified. Microthalmia-Associated Transcription Factor, a gene known to cause WS, is located within this region of linkage. Sequencing of Microthalmia-Associated Transcription Factor demonstrated a c.1212 G>A synonymous variant that segregated with the WS in the family and was predicted to cause a novel splicing site that was confirmed with expression analysis of the mRNA. This case illustrates the need to computationally analyze novel synonymous sequence variants for possible effects on splicing to maximize the clinical sensitivity of sequence-based genetic testing.

Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy

NASA Astrophysics Data System (ADS)

Kühnemund, Malte; Wei, Qingshan; Darai, Evangelia; Wang, Yingjie; Hernández-Neuta, Iván; Yang, Zhao; Tseng, Derek; Ahlford, Annika; Mathot, Lucy; Sjöblom, Tobias; Ozcan, Aydogan; Nilsson, Mats

2017-01-01

Molecular diagnostics is typically outsourced to well-equipped centralized laboratories, often far from the patient. We developed molecular assays and portable optical imaging designs that permit on-site diagnostics with a cost-effective mobile-phone-based multimodal microscope. We demonstrate that targeted next-generation DNA sequencing reactions and in situ point mutation detection assays in preserved tumour samples can be imaged and analysed using mobile phone microscopy, achieving a new milestone for tele-medicine technologies.

Compound heterozygosity for two GHR missense mutations in a patient affected by Laron Syndrome: a case report.

PubMed

Moia, Stefania; Tessaris, Daniele; Einaudi, Silvia; de Sanctis, Luisa; Bona, Gianni; Bellone, Simonetta; Prodam, Flavia

2017-10-12

Mutations localized in the Growth Hormone Receptor (GHR) gene are often associated with the pathogenesis of Laron Syndrome, an autosomal recessive hereditary disorder characterized by severe growth retardation. Biochemically, patients present normal to high circulating GH levels, in presence of very low or undetectable IGF-I levels, which do not rise after rhGH treatment. We describe the case of a 3.8 years old girl with symmetrical short stature (-3.76 SDS), low IGF-1 and IGFBP-3, in presence of normal GH levels. Parents were not relatives and there was no family history of short stature. During the second day of birth, she developed severe hypoglycaemia that required glucose infusion. She presented frontal bossing and depressed nasal bridge. IGF-1 generation test showed no response, suggesting a GH resistance evidence. In the hypothesis of Laron Syndrome, we decided to perform a molecular analysis of Growth Hormone Receptor (GHR) gene. This analysis demonstrated that the patient was compound heterozygote for two missense mutations. GHR gene mutations are a well demonstrated cause of GH insensitivity. In heterozygous patients, probably the normal stature may be achieved by a compensatory mechanism of GH secretion or signalling. On the contrary, in homozygous or compound heterozygous patients these compensatory mechanisms are inadequate, and short stature may be the consequence.

Comprehensive analysis of oculocutaneous albinism among non-Hispanic caucasians shows that OCA1 is the most prevalent OCA type.

PubMed

Hutton, Saunie M; Spritz, Richard A

2008-10-01

Oculocutaneous albinism (OCA) is a genetically heterogeneous group of disorders characterized by absent or reduced pigmentation of the skin, hair, and eyes. In humans, four genes have been associated with "classical" OCA and another 12 genes with syndromic forms of OCA. To assess the prevalence of different forms of OCA and different gene mutations among non-Hispanic Caucasian patients, we performed DNA sequence analysis of the four genes associated with "classical" OCA (TYR, OCA2, TYRP1, SLC45A2), the two principal genes associated with syndromic OCA (HPS1, HPS4), and a candidate OCA gene (SILV), in 121 unrelated, unselected non-Hispanic/Latino Caucasian patients carrying the clinical diagnosis of OCA. We identified apparent pathologic TYR gene mutations in 69% of patients, OCA2 mutations in 18%, SLC45A2 mutations in 6%, and no apparent pathological mutations in 7% of patients. We found no mutations of TYRP1, HPS1, HPS4, or SILV in any patients. Although we observed a diversity of mutations for each gene, a relatively small number of different mutant alleles account for a majority of the total. This study demonstrates that, contrary to long-held clinical lore, OCA1, not OCA2, is by far the most frequent cause of OCA among Caucasian patients.

Arrestin gene mutations in autosomal recessive retinitis pigmentosa.

PubMed

Nakazawa, M; Wada, Y; Tamai, M

1998-04-01

To assess the clinical and molecular genetic studies of patients with autosomal recessive retinitis pigmentosa associated with a mutation in the arrestin gene. Results of molecular genetic screening and case reports with DNA analysis and clinical features. University medical center. One hundred twenty anamnestically unrelated patients with autosomal recessive retinitis pigmentosa. DNA analysis was performed by single strand conformation polymorphism followed by nucleotide sequencing to search for a mutation in exon 11 of the arrestin gene. Clinical features were characterized by visual acuity slitlamp biomicroscopy, fundus examinations, fluorescein angiography, kinetic visual field testing, and electroretinography. We identified 3 unrelated patients with retinitis pigmentosa associated with a homozygous 1-base-pair deletion mutation in codon 309 of the arrestin gene designated as 1147delA. All 3 patients showed pigmentary retinal degeneration in the midperipheral area with or without macular involvement. Patient 1 had a sibling with Oguchi disease associated with the same mutation. Patient 2 demonstrated pigmentary retinal degeneration associated with a golden-yellow reflex in the peripheral fundus. Patients 1 and 3 showed features of retinitis pigmentosa without the golden-yellow fundus reflex. Although the arrestin 1147delA has been known as a frequent cause of Oguchi disease, this mutation also may be related to the pathogenesis of autosomal recessive retinitis pigmentosa. This phenomenon may provide evidence of variable expressivity of the mutation in the arrestin gene.

Mutation allele burden remains unchanged in chronic myelomonocytic leukaemia responding to hypomethylating agents

DOE PAGES

Merlevede, Jane; Droin, Nathalie; Qin, Tingting; ...

2016-02-24

The cytidine analogues azacytidine and 5-aza-2’-deoxycytidine (decitabine) are commonly used to treat myelodysplastic syndromes, with or without a myeloproliferative component. It remains unclear whether the response to these hypomethylating agents results from a cytotoxic or an epigenetic effect. In this study, we address this question in chronic myelomonocytic leukaemia. We describe a comprehensive analysis of the mutational landscape of these tumours, combining whole-exome and whole-genome sequencing. We identify an average of 14 ± 5 somatic mutations in coding sequences of sorted monocyte DNA and the signatures of three mutational processes. Serial sequencing demonstrates that the response to hypomethylating agents ismore » associated with changes in DNA methylation and gene expression, without any decrease in the mutation allele burden, nor prevention of new genetic alteration occurence. Lastly, our findings indicate that cytosine analogues restore a balanced haematopoiesis without decreasing the size of the mutated clone, arguing for a predominantly epigenetic effect.« less

A Novel Mutation in a Kazakh Family with X-Linked Alport Syndrome

PubMed Central

Rakhimova, Saule E.; Nigmatullina, Nazym B.; Momynaliev, Kuvat T.; Ramanculov, Yerlan M.

2015-01-01

Alport syndrome is a genetic condition that results in hematuria, progressive renal impairment, hearing loss, and occasionally lenticonus and retinopathy. Approximately 80% of Alport syndrome cases are caused by X-linked mutations in the COL4A5 gene encoding type IV collagen. The objective of this study was to define the SNP profiles for COL4A5 in patients with hereditary nephritis and hematuria. For this, we examined four subjects from one Kazakh family clinically affected with X-linked Alport syndrome due to COL4A5 gene mutations. All 51 exons of the COL4A5 gene were screened by linkage analysis and direct DNA sequencing, resulting in the identification of a novel mutation (G641E) in exon 25. The mutation was found only in two affected family individuals but was not present in healthy family members or 200 unrelated healthy controls. This result demonstrates that this novel mutation is pathogenic and has meaningful implications for the diagnosis of patients with Alport syndrome. PMID:26168235

A Novel Mutation in a Kazakh Family with X-Linked Alport Syndrome.

PubMed

Baikara, Barshagul T; Zholdybayeva, Elena V; Rakhimova, Saule E; Nigmatullina, Nazym B; Momynaliev, Kuvat T; Ramanculov, Yerlan M

2015-01-01

Alport syndrome is a genetic condition that results in hematuria, progressive renal impairment, hearing loss, and occasionally lenticonus and retinopathy. Approximately 80% of Alport syndrome cases are caused by X-linked mutations in the COL4A5 gene encoding type IV collagen. The objective of this study was to define the SNP profiles for COL4A5 in patients with hereditary nephritis and hematuria. For this, we examined four subjects from one Kazakh family clinically affected with X-linked Alport syndrome due to COL4A5 gene mutations. All 51 exons of the COL4A5 gene were screened by linkage analysis and direct DNA sequencing, resulting in the identification of a novel mutation (G641E) in exon 25. The mutation was found only in two affected family individuals but was not present in healthy family members or 200 unrelated healthy controls. This result demonstrates that this novel mutation is pathogenic and has meaningful implications for the diagnosis of patients with Alport syndrome.

Mutation allele burden remains unchanged in chronic myelomonocytic leukaemia responding to hypomethylating agents

PubMed Central

Merlevede, Jane; Droin, Nathalie; Qin, Tingting; Meldi, Kristen; Yoshida, Kenichi; Morabito, Margot; Chautard, Emilie; Auboeuf, Didier; Fenaux, Pierre; Braun, Thorsten; Itzykson, Raphael; de Botton, Stéphane; Quesnel, Bruno; Commes, Thérèse; Jourdan, Eric; Vainchenker, William; Bernard, Olivier; Pata-Merci, Noemie; Solier, Stéphanie; Gayevskiy, Velimir; Dinger, Marcel E.; Cowley, Mark J.; Selimoglu-Buet, Dorothée; Meyer, Vincent; Artiguenave, François; Deleuze, Jean-François; Preudhomme, Claude; Stratton, Michael R.; Alexandrov, Ludmil B.; Padron, Eric; Ogawa, Seishi; Koscielny, Serge; Figueroa, Maria; Solary, Eric

2016-01-01

The cytidine analogues azacytidine and 5-aza-2'-deoxycytidine (decitabine) are commonly used to treat myelodysplastic syndromes, with or without a myeloproliferative component. It remains unclear whether the response to these hypomethylating agents results from a cytotoxic or an epigenetic effect. In this study, we address this question in chronic myelomonocytic leukaemia. We describe a comprehensive analysis of the mutational landscape of these tumours, combining whole-exome and whole-genome sequencing. We identify an average of 14±5 somatic mutations in coding sequences of sorted monocyte DNA and the signatures of three mutational processes. Serial sequencing demonstrates that the response to hypomethylating agents is associated with changes in DNA methylation and gene expression, without any decrease in the mutation allele burden, nor prevention of new genetic alteration occurence. Our findings indicate that cytosine analogues restore a balanced haematopoiesis without decreasing the size of the mutated clone, arguing for a predominantly epigenetic effect. PMID:26908133

p53 mutation and expression in lymphoma.

PubMed Central

Adamson, D. J.; Thompson, W. D.; Dawson, A. A.; Bennett, B.; Haites, N. E.

1995-01-01

Mutation and abnormal expression of p53 was studied in 38 lymphomas [five Hodgkin's disease and 33 non-Hodgkin's lymphoma (NHL)]. CM1 polyclonal antibody was used to detect overexpression of p53. Three missense mutations were characterised in three cases of NHL after screening exons 5-8 of p53 of all the tumours with single-strand conformation polymorphism (SSCP) analysis. Only two out of three tumours with a missense mutation showed abnormal expression of p53 as measured by CM1. Conversely, seven out of nine tumours with positive CM1 staining had no point mutation demonstrated. Overexpression of p53 in the cases of NHL occurred in three out of twenty four low-grade tumours and five out of nine high-grade tumours (Kiel classification). The results suggest that abnormalities of p53 are commoner in high-grade than low-grade NHL, and that positive immunocytochemistry cannot be used to determine which tumours have mutations of p53. Images Figure 1 Figure 2 PMID:7599045

Germline mutations in PMS2 and MLH1 in individuals with solitary loss of PMS2 expression in colorectal carcinomas from the Colon Cancer Family Registry Cohort.

PubMed

Rosty, Christophe; Clendenning, Mark; Walsh, Michael D; Eriksen, Stine V; Southey, Melissa C; Winship, Ingrid M; Macrae, Finlay A; Boussioutas, Alex; Poplawski, Nicola K; Parry, Susan; Arnold, Julie; Young, Joanne P; Casey, Graham; Haile, Robert W; Gallinger, Steven; Le Marchand, Loïc; Newcomb, Polly A; Potter, John D; DeRycke, Melissa; Lindor, Noralane M; Thibodeau, Stephen N; Baron, John A; Win, Aung Ko; Hopper, John L; Jenkins, Mark A; Buchanan, Daniel D

2016-02-19

Immunohistochemistry for DNA mismatch repair proteins is used to screen for Lynch syndrome in individuals with colorectal carcinoma (CRC). Although solitary loss of PMS2 expression is indicative of carrying a germline mutation in PMS2, previous studies reported MLH1 mutation in some cases. We determined the prevalence of MLH1 germline mutations in a large cohort of individuals with a CRC demonstrating solitary loss of PMS2 expression. This cohort study included 88 individuals affected with a PMS2-deficient CRC from the Colon Cancer Family Registry Cohort. Germline PMS2 mutation analysis (long-range PCR and multiplex ligation-dependent probe amplification) was followed by MLH1 mutation testing (Sanger sequencing and multiplex ligation-dependent probe amplification). Of the 66 individuals with complete mutation screening, we identified a pathogenic PMS2 mutation in 49 (74%), a pathogenic MLH1 mutation in 8 (12%) and a MLH1 variant of uncertain clinical significance predicted to be damaging by in silico analysis in 3 (4%); 6 (9%) carried variants likely to have no clinical significance. Missense point mutations accounted for most alterations (83%; 9/11) in MLH1. The MLH1 c.113A> G p.Asn38Ser mutation was found in 2 related individuals. One individual who carried the MLH1 intronic mutation c.677+3A>G p.Gln197Argfs*8 leading to the skipping of exon 8, developed 2 tumours, both of which retained MLH1 expression. A substantial proportion of CRCs with solitary loss of PMS2 expression are associated with a deleterious MLH1 germline mutation supporting the screening for MLH1 in individuals with tumours of this immunophenotype, when no PMS2 mutation has been identified. 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/

Germline mutations in PMS2 and MLH1 in individuals with solitary loss of PMS2 expression in colorectal carcinomas from the Colon Cancer Family Registry Cohort

PubMed Central

Rosty, Christophe; Clendenning, Mark; Walsh, Michael D; Eriksen, Stine V; Southey, Melissa C; Winship, Ingrid M; Macrae, Finlay A; Boussioutas, Alex; Parry, Susan; Arnold, Julie; Young, Joanne P; Casey, Graham; Haile, Robert W; Gallinger, Steven; Le Marchand, Loïc; Newcomb, Polly A; Potter, John D; DeRycke, Melissa; Lindor, Noralane M; Thibodeau, Stephen N; Baron, John A; Win, Aung Ko; Hopper, John L; Jenkins, Mark A; Buchanan, Daniel D

2016-01-01

Objectives Immunohistochemistry for DNA mismatch repair proteins is used to screen for Lynch syndrome in individuals with colorectal carcinoma (CRC). Although solitary loss of PMS2 expression is indicative of carrying a germline mutation in PMS2, previous studies reported MLH1 mutation in some cases. We determined the prevalence of MLH1 germline mutations in a large cohort of individuals with a CRC demonstrating solitary loss of PMS2 expression. Design This cohort study included 88 individuals affected with a PMS2-deficient CRC from the Colon Cancer Family Registry Cohort. Germline PMS2 mutation analysis (long-range PCR and multiplex ligation-dependent probe amplification) was followed by MLH1 mutation testing (Sanger sequencing and multiplex ligation-dependent probe amplification). Results Of the 66 individuals with complete mutation screening, we identified a pathogenic PMS2 mutation in 49 (74%), a pathogenic MLH1 mutation in 8 (12%) and a MLH1 variant of uncertain clinical significance predicted to be damaging by in silico analysis in 3 (4%); 6 (9%) carried variants likely to have no clinical significance. Missense point mutations accounted for most alterations (83%; 9/11) in MLH1. The MLH1 c.113A> G p.Asn38Ser mutation was found in 2 related individuals. One individual who carried the MLH1 intronic mutation c.677+3A>G p.Gln197Argfs*8 leading to the skipping of exon 8, developed 2 tumours, both of which retained MLH1 expression. Conclusions A substantial proportion of CRCs with solitary loss of PMS2 expression are associated with a deleterious MLH1 germline mutation supporting the screening for MLH1 in individuals with tumours of this immunophenotype, when no PMS2 mutation has been identified. PMID:26895986

Crystal structure and functional interpretation of the erythrocyte spectrin tetramerization domain complex

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

Ipsaro, Jonathan J.; Harper, Sandra L.; Messick, Troy E.

2010-09-07

As the principal component of the membrane skeleton, spectrin confers integrity and flexibility to red cell membranes. Although this network involves many interactions, the most common hemolytic anemia mutations that disrupt erythrocyte morphology affect the spectrin tetramerization domains. Although much is known clinically about the resulting conditions (hereditary elliptocytosis and pyropoikilocytosis), the detailed structural basis for spectrin tetramerization and its disruption by hereditary anemia mutations remains elusive. Thus, to provide further insights into spectrin assembly and tetramer site mutations, a crystal structure of the spectrin tetramerization domain complex has been determined. Architecturally, this complex shows striking resemblance to multirepeat spectrinmore » fragments, with the interacting tetramer site region forming a central, composite repeat. This structure identifies conformational changes in {alpha}-spectrin that occur upon binding to {beta}-spectrin, and it reports the first structure of the {beta}-spectrin tetramerization domain. Analysis of the interaction surfaces indicates an extensive interface dominated by hydrophobic contacts and supplemented by electrostatic complementarity. Analysis of evolutionarily conserved residues suggests additional surfaces that may form important interactions. Finally, mapping of hereditary anemia-related mutations onto the structure demonstrate that most, but not all, local hereditary anemia mutations map to the interacting domains. The potential molecular effects of these mutations are described.« less

Crystal Structure and Functional Interpretation of the Erythrocyte spectrin Tetramerization Domain Complex

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

J Ipsaro; S Harper; T Messick

2011-12-31

As the principal component of the membrane skeleton, spectrin confers integrity and flexibility to red cell membranes. Although this network involves many interactions, the most common hemolytic anemia mutations that disrupt erythrocyte morphology affect the spectrin tetramerization domains. Although much is known clinically about the resulting conditions (hereditary elliptocytosis and pyropoikilocytosis), the detailed structural basis for spectrin tetramerization and its disruption by hereditary anemia mutations remains elusive. Thus, to provide further insights into spectrin assembly and tetramer site mutations, a crystal structure of the spectrin tetramerization domain complex has been determined. Architecturally, this complex shows striking resemblance to multirepeat spectrinmore » fragments, with the interacting tetramer site region forming a central, composite repeat. This structure identifies conformational changes in {alpha}-spectrin that occur upon binding to {beta}-spectrin, and it reports the first structure of the {beta}-spectrin tetramerization domain. Analysis of the interaction surfaces indicates an extensive interface dominated by hydrophobic contacts and supplemented by electrostatic complementarity. Analysis of evolutionarily conserved residues suggests additional surfaces that may form important interactions. Finally, mapping of hereditary anemia-related mutations onto the structure demonstrate that most, but not all, local hereditary anemia mutations map to the interacting domains. The potential molecular effects of these mutations are described.« less

TET2 Mutations Are Associated with Specific 5-Methylcytosine and 5-Hydroxymethylcytosine Profiles in Patients with Chronic Myelomonocytic Leukemia

PubMed Central

Pérez, Cristina; Martínez-Calle, Nicolas; Martín-Subero, José Ignacio; Segura, Victor; Delabesse, Eric; Fernandez-Mercado, Marta; Garate, Leire; Alvarez, Sara; Rifon, José; Varea, Sara; Boultwood, Jacqueline; Wainscoat, James S.; Cigudosa, Juan Cruz; Calasanz, María José; Cross, Nicholas C. P.

2012-01-01

Chronic myelomonocytic leukemia (CMML) has recently been associated with a high incidence of diverse mutations in genes such as TET2 or EZH2 that are implicated in epigenetic mechanisms. We have performed genome-wide DNA methylation arrays and mutational analysis of TET2, IDH1, IDH2, EZH2 and JAK2 in a group of 24 patients with CMML. 249 genes were differentially methylated between CMML patients and controls. Using Ingenuity pathway analysis, we identified enrichment in a gene network centered around PLC, JNK and ERK suggesting that these pathways, whose deregulation has beenrecently described in CMML, are affected by epigenetic mechanisms. Mutations of TET2, JAK2 and EZH2 were found in 15 patients (65%), 4 patients (17%) and 1 patient (4%) respectively while no mutations in the IDH1 and IDH2 genes were identified. Interestingly, patients with wild type TET2 clustered separately from patients with TET2 mutations, showed a higher degree of hypermethylation and were associated with higher risk karyotypes. Our results demonstrate the presence of aberrant DNA methylation in CMML and identifies TET2 mutant CMML as a biologically distinct disease subtype with a different epigenetic profile. PMID:22328940

Molecular analysis of pericentrin gene (PCNT) in a series of 24 Seckel/microcephalic osteodysplastic primordial dwarfism type II (MOPD II) families.

PubMed

Willems, M; Geneviève, D; Borck, G; Baumann, C; Baujat, G; Bieth, E; Edery, P; Farra, C; Gerard, M; Héron, D; Leheup, B; Le Merrer, M; Lyonnet, S; Martin-Coignard, D; Mathieu, M; Thauvin-Robinet, C; Verloes, A; Colleaux, L; Munnich, A; Cormier-Daire, V

2010-12-01

Microcephalic osteodysplastic primordial dwarfism type II (MOPD II, MIM 210720) and Seckel syndrome (SCKL, MIM 210600) belong to the primordial dwarfism group characterised by intrauterine growth retardation, severe proportionate short stature, and pronounced microcephaly. MOPD II is distinct from SCKL by more severe growth retardation, radiological abnormalities, and absent or mild mental retardation. Seckel syndrome is associated with defective ATR dependent DNA damage signalling. In 2008, loss-of-function mutations in the pericentrin gene (PCNT) have been identified in 28 patients, including 3 SCKL and 25 MOPDII cases. This gene encodes a centrosomal protein which plays a key role in the organisation of mitotic spindles. The aim of this study was to analyse PCNT in a large series of SCKL-MOPD II cases to further define the clinical spectrum associated with PCNT mutations. Among 18 consanguineous families (13 SCKL and 5 MOPDII) and 6 isolated cases (3 SCKL and 3 MOPD II), 13 distinct mutations were identified in 5/16 SCKL and 8/8 MOPDII including five stop mutations, five frameshift mutations, two splice site mutations, and one apparent missense mutation affecting the last base of exon 19. Moreover, we demonstrated that this latter mutation leads to an abnormal splicing with a predicted premature termination of translation. The clinical analysis of the 5 SCKL cases with PCNT mutations showed that they all presented minor skeletal changes and clinical features compatible with MOPDII diagnosis. It is therefore concluded that, despite variable severity, MOPDII is a genetically homogeneous condition due to loss-of-function of pericentrin.

Snowball: resampling combined with distance-based regression to discover transcriptional consequences of a driver mutation

PubMed Central

Xu, Yaomin; Guo, Xingyi; Sun, Jiayang; Zhao, Zhongming

2015-01-01

Motivation: Large-scale cancer genomic studies, such as The Cancer Genome Atlas (TCGA), have profiled multidimensional genomic data, including mutation and expression profiles on a variety of cancer cell types, to uncover the molecular mechanism of cancerogenesis. More than a hundred driver mutations have been characterized that confer the advantage of cell growth. However, how driver mutations regulate the transcriptome to affect cellular functions remains largely unexplored. Differential analysis of gene expression relative to a driver mutation on patient samples could provide us with new insights in understanding driver mutation dysregulation in tumor genome and developing personalized treatment strategies. Results: Here, we introduce the Snowball approach as a highly sensitive statistical analysis method to identify transcriptional signatures that are affected by a recurrent driver mutation. Snowball utilizes a resampling-based approach and combines a distance-based regression framework to assign a robust ranking index of genes based on their aggregated association with the presence of the mutation, and further selects the top significant genes for downstream data analyses or experiments. In our application of the Snowball approach to both synthesized and TCGA data, we demonstrated that it outperforms the standard methods and provides more accurate inferences to the functional effects and transcriptional dysregulation of driver mutations. Availability and implementation: R package and source code are available from CRAN at http://cran.r-project.org/web/packages/DESnowball, and also available at http://bioinfo.mc.vanderbilt.edu/DESnowball/. Contact: zhongming.zhao@vanderbilt.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25192743

Homozygous EDNRB mutation in a patient with Waardenburg syndrome type 1.

PubMed

Morimoto, Noriko; Mutai, Hideki; Namba, Kazunori; Kaneko, Hiroki; Kosaki, Rika; Matsunaga, Tatsuo

2018-04-01

To examine and expand the genetic spectrum of Waardenburg syndrome type 1 (WS1). Clinical features related to Waardenburg syndrome (WS) were examined in a five-year old patient. Mutation analysis of genes related to WS was performed in the proband and her parents. Molecular modeling of EDNRB and the p.R319W mutant was conducted to predict the pathogenicity of the mutation. The proband showed sensorineural hearing loss, heterochromia iridis, and dystopia canthorum, fulfilling the clinical criteria of WS1. Genetic analyses revealed that the proband had no mutation in PAX3 which has been known as the cause of WS1, but had a homozygous missense mutation (p.R319W) in endothelin receptor type B (EDNRB) gene. The asymptomatic parents had the mutation in a heterozygote state. This mutation has been previously reported in a heterozygous state in a patient with Hirschsprung's disease unaccompanied by WS, but the patient and her parents did not show any symptoms in gastrointestinal tract. Molecular modeling of EDNRB with the p.R319W mutation demonstrated reduction of the positively charged surface area in this region, which might reduce binding ability of EDNRB to G protein and lead to abnormal signal transduction underlying the WS phenotype. Our findings suggested that autosomal recessive mutation in EDNRB may underlie a part of WS1 with the current diagnostic criteria, and supported that Hirschsprung's disease is a multifactorial genetic disease which requires additional factors. Further molecular analysis is necessary to elucidate the gene interaction and to reappraise the current WS classification. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of TCT, a novel knockdown resistance allele mutation and analysis of resistance detection methods in the voltage-gated Na⁺ channel of Culex pipiens pallens from Shandong Province, China.

PubMed

Liu, Hong-Mei; Cheng, Peng; Huang, Xiaodan; Dai, Yu-Hua; Wang, Hai-Fang; Liu, Li-Juan; Zhao, Yu-Qiang; Wang, Huai-Wei; Gong, Mao-Qing

2013-02-01

The present study aimed to investigate deltamethrin resistance in Culex pipiens pallens (C. pipiens pallens) mosquitoes and its correlation with knockdown resistance (kdr) mutations. In addition, mosquito‑resistance testing methods were analyzed. Using specific primers in polymerase chain reaction (PCR) and allele-specific (AS)-PCR, kdr gene sequences isolated from wild C. pipiens pallens mosquitoes were sequenced. Linear regression analysis was used to determine the correlation between the mutations and deltamethrin resistance. A kdr allelic gene was cloned and sequenced. Analysis of the DNA sequences revealed the presence of two point mutations at the L1014 residue in the IIS6 transmembrane segment of the voltage‑gated sodium channel (VGSC): L1014F, TTA→TTT, replacing a leucine (L) with a phenylalanine (F); L1014S, TTA→TCA, replacing leucine (L) with serine (S). Two alternative kdr-like mutations, L1014F and L1014S, were identified to be positively correlated with the deltamethrin-resistant phenotype. In addition a novel mutation, TCT, was identified in the VGSC of C. pipiens pallens. PCR and AS-PCR yielded consistent results with respect to mosquito resistance. However, the detection rate of PCR was higher than that of AS-PCR. Further studies are required to determine the specific resistance mechanism. PCR and AS-PCR demonstrated suitability for mosquito resistance field tests, however, the former method may be superior to the latter.

The Energy Landscape Analysis of Cancer Mutations in Protein Kinases

PubMed Central

Dixit, Anshuman; Verkhivker, Gennady M.

2011-01-01

The growing interest in quantifying the molecular basis of protein kinase activation and allosteric regulation by cancer mutations has fueled computational studies of allosteric signaling in protein kinases. In the present study, we combined computer simulations and the energy landscape analysis of protein kinases to characterize the interplay between oncogenic mutations and locally frustrated sites as important catalysts of allostetric kinase activation. While structurally rigid kinase core constitutes a minimally frustrated hub of the catalytic domain, locally frustrated residue clusters, whose interaction networks are not energetically optimized, are prone to dynamic modulation and could enable allosteric conformational transitions. The results of this study have shown that the energy landscape effect of oncogenic mutations may be allosteric eliciting global changes in the spatial distribution of highly frustrated residues. We have found that mutation-induced allosteric signaling may involve a dynamic coupling between structurally rigid (minimally frustrated) and plastic (locally frustrated) clusters of residues. The presented study has demonstrated that activation cancer mutations may affect the thermodynamic equilibrium between kinase states by allosterically altering the distribution of locally frustrated sites and increasing the local frustration in the inactive form, while eliminating locally frustrated sites and restoring structural rigidity of the active form. The energy landsape analysis of protein kinases and the proposed role of locally frustrated sites in activation mechanisms may have useful implications for bioinformatics-based screening and detection of functional sites critical for allosteric regulation in complex biomolecular systems. PMID:21998754

Implementation of Amplicon Parallel Sequencing Leads to Improvement of Diagnosis and Therapy of Lung Cancer Patients.

PubMed

König, Katharina; Peifer, Martin; Fassunke, Jana; Ihle, Michaela A; Künstlinger, Helen; Heydt, Carina; Stamm, Katrin; Ueckeroth, Frank; Vollbrecht, Claudia; Bos, Marc; Gardizi, Masyar; Scheffler, Matthias; Nogova, Lucia; Leenders, Frauke; Albus, Kerstin; Meder, Lydia; Becker, Kerstin; Florin, Alexandra; Rommerscheidt-Fuss, Ursula; Altmüller, Janine; Kloth, Michael; Nürnberg, Peter; Henkel, Thomas; Bikár, Sven-Ernö; Sos, Martin L; Geese, William J; Strauss, Lewis; Ko, Yon-Dschun; Gerigk, Ulrich; Odenthal, Margarete; Zander, Thomas; Wolf, Jürgen; Merkelbach-Bruse, Sabine; Buettner, Reinhard; Heukamp, Lukas C

2015-07-01

The Network Genomic Medicine Lung Cancer was set up to rapidly translate scientific advances into early clinical trials of targeted therapies in lung cancer performing molecular analyses of more than 3500 patients annually. Because sequential analysis of the relevant driver mutations on fixated samples is challenging in terms of workload, tissue availability, and cost, we established multiplex parallel sequencing in routine diagnostics. The aim was to analyze all therapeutically relevant mutations in lung cancer samples in a high-throughput fashion while significantly reducing turnaround time and amount of input DNA compared with conventional dideoxy sequencing of single polymerase chain reaction amplicons. In this study, we demonstrate the feasibility of a 102 amplicon multiplex polymerase chain reaction followed by sequencing on an Illumina sequencer on formalin-fixed paraffin-embedded tissue in routine diagnostics. Analysis of a validation cohort of 180 samples showed this approach to require significantly less input material and to be more reliable, robust, and cost-effective than conventional dideoxy sequencing. Subsequently, 2657 lung cancer patients were analyzed. We observed that comprehensive biomarker testing provided novel information in addition to histological diagnosis and clinical staging. In 2657 consecutively analyzed lung cancer samples, we identified driver mutations at the expected prevalence. Furthermore we found potentially targetable DDR2 mutations at a frequency of 3% in both adenocarcinomas and squamous cell carcinomas. Overall, our data demonstrate the utility of systematic sequencing analysis in a clinical routine setting and highlight the dramatic impact of such an approach on the availability of therapeutic strategies for the targeted treatment of individual cancer patients.

Novel methods to enhance single strand conformation polymorphism (SSCP) senstivity and efficiency: Application to mutation detection in cystic fibrosis (CF)

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

Hagstrom, D.J.; Snow, K.; Yuan, Z.

1994-09-01

For single gene defects in which there are a variety of mutations with significant frequencies, it is a challenge to find an efficient and sensitive method for mutation detection. For example, although 70% to 75% of CF chromosomes in a North American Caucasian population have the mutation {delta}F508, more than 400 mutations (mostly single base pair substitutions) are represented on the remaining chromosomes. SSCP analysis is a relatively straightforward procedure and therefore suitable for routine use in a clinical laboratory. However, previous reports have demonstrated suboptimal sensitivity rates in screening for mutations. We have developed a novel set of conditionsmore » which greatly enhances sensitivity and efficiency of SSCP. Our protocol incorporates multiplex PCR, stepping of wattages during electrophoresis and increased salt concentration at the anode relative to the gel. To screen for mutations in the CFTR gene, three multiplex PCR reactions are performed using identical thermocycler parameters. Sizes of PCR products range from 441 bp to 196 bp: size differences of > 30 bp are necessary to ensure separation during electrophoresis. All PCR products are separated by electrophoresis at room temperature on a single gel containing 8% (37.5:1) polyacrylamide, 5% glycerol and 1x TBE. Using an anode buffer with increased salt (2x TBE) sharpens smaller sized bands, and stepping watts from 5W to 20W during electrophoresis enhances sensitivity. Positive controls were used to demonstrate that mutations could be detected. Other mutations or polymorphisms were verified by cycle sequencing of PCR products or by alternative PCR-based assays for the more common mutations. Thus, using 3 PCR reactions per patient and one gel condition, we are able to achieve a CF mutation detection rate of approximately 90% in a North American Caucasian population.« less

Clinicopathological analysis of endometrial carcinomas harboring somatic POLE exonuclease domain mutations.

PubMed

Hussein, Yaser R; Weigelt, Britta; Levine, Douglas A; Schoolmeester, J Kenneth; Dao, Linda N; Balzer, Bonnie L; Liles, Georgia; Karlan, Beth; Köbel, Martin; Lee, Cheng-Han; Soslow, Robert A

2015-04-01

The Cancer Genome Atlas described four major genomic groups of endometrial carcinomas, including a POLE ultramutated subtype comprising ∼10% of endometrioid adenocarcinoma, characterized by POLE exonuclease domain mutations, ultrahigh somatic mutation rates, and favorable outcome. Our aim was to examine the morphological and clinicopathological features of ultramutated endometrial carcinomas harboring somatic POLE exonuclease domain mutations. Hematoxylin and eosin slides and pathology reports for 8/17 POLE-mutated endometrial carcinomas described in the Cancer Genome Atlas study were studied; for the remaining cases, virtual whole slide images publicly available at cBioPortal (www.cbioportal.org) were examined. A second cohort of eight POLE mutated endometrial carcinomas from University of Calgary was also studied. Median age was 55 years (range 33-87 years). Nineteen patients presented as stage I, 1 stage II, and 5 stage III. The majority of cases (24 of the 25) demonstrated defining morphological features of endometrioid differentiation. The studied cases were frequently high grade (60%) and rich in tumor-infiltrating lymphocytes and/or peri-tumoral lymphocytes (84%); many tumors showed morphological heterogeneity (52%) and ambiguity (16%). Foci demonstrating severe nuclear atypia led to concern for serous carcinoma in 28% of cases. At the molecular level, the majority of the Cancer Genome Atlas POLE-mutated tumors were microsatellite stable (65%), and TP53 mutations were present in 35% of cases. They also harbored mutations in PTEN (94%), FBXW7 (82%), ARID1A (76%), and PIK3CA (71%). All patients from both cohorts were alive without disease, and none of the patients developed recurrence at the time of follow-up (median 33 months; range 2-102 months). In conclusion, the recognition of ultramutated endometrial carcinomas with POLE exonuclease domain mutation is important given their favorable outcome. Our histopathological review revealed that these tumors are commonly high grade, have obvious lymphocytic infiltrates, and can show ambiguous morphology. As they frequently harbor TP53 mutations, it is important not to misclassify them as serous carcinoma.

Individual crypt genetic heterogeneity and the origin of metaplastic glandular epithelium in human Barrett’s oesophagus

PubMed Central

Leedham, S J; Preston, S L; McDonald, S A C; Elia, G; Bhandari, P; Poller, D; Harrison, R; Novelli, M R; Jankowski, J A; Wright, N A

2008-01-01

Objectives: Current models of clonal expansion in human Barrett’s oesophagus are based upon heterogenous, flow-purified biopsy analysis taken at multiple segment levels. Detection of identical mutation fingerprints from these biopsy samples led to the proposal that a mutated clone with a selective advantage can clonally expand to fill an entire Barrett’s segment at the expense of competing clones (selective sweep to fixation model). We aimed to assess clonality at a much higher resolution by microdissecting and genetically analysing individual crypts. The histogenesis of Barrett’s metaplasia and neo-squamous islands has never been demonstrated. We investigated the oesophageal gland squamous ducts as the source of both epithelial sub-types. Methods: Individual crypts across Barrett’s biopsy and oesophagectomy blocks were dissected. Determination of tumour suppressor gene loss of heterozygosity patterns, p16 and p53 point mutations were carried out on a crypt-by-crypt basis. Cases of contiguous neo-squamous islands and columnar metaplasia with oesophageal squamous ducts were identified. Tissues were isolated by laser capture microdissection and genetically analysed. Results: Individual crypt dissection revealed mutation patterns that were masked in whole biopsy analysis. Dissection across oesophagectomy specimens demonstrated marked clonal heterogeneity, with multiple independent clones present. We identified a p16 point mutation arising in the squamous epithelium of the oesophageal gland duct, which was also present in a contiguous metaplastic crypt, whereas neo-squamous islands arising from squamous ducts were wild-type with respect to surrounding Barrett’s dysplasia. Conclusions: By studying clonality at the crypt level we demonstrate that Barrett’s heterogeneity arises from multiple independent clones, in contrast to the selective sweep to fixation model of clonal expansion previously described. We suggest that the squamous gland ducts situated throughout the oesophagus are the source of a progenitor cell that may be susceptible to gene mutation resulting in conversion to Barrett’s metaplastic epithelium. Additionally, these data suggest that wild-type ducts may be the source of neo-squamous islands. PMID:18305067

In silico investigation of molecular effects caused by missense mutations in creatine transporter protein

NASA Astrophysics Data System (ADS)

Zhang, Zhe; Schwatz, Charles; Alexov, Emil

2011-03-01

Creatine transporter (CT) protein, which is encoded by SLC6A8 gene, is essential for taking up the creatine in the cell, which in turn plays a key role in the spatial and temporal maintenance of energy in skeletal and cardiac muscle cells. It was shown that some missense mutations in CT cause mental retardation, while others are harmless non-synonymous single nucleoside polymorphism (nsSNP). Currently fifteen missense mutations in CT are known, among which twelve are disease-causing. Sequence analysis reveals that there is no clear trend distinguishing disease-causing from harmless missense mutations. Because of that, we built 3D model of the CT using highly homologous template and use the model to investigate the effects of mutations of CT stability and hydrogen bond network. It is demonstrated that disease-causing mutations affect the folding free energy and ionization states of titratable group in much greater extend as compared with harmless mutations. Supported by grants from NLM, NIH, grant numbers 1R03LM009748 and 1R03LM009748-S1.

Twenty novel mutations in BCKDHA, BCKDHB and DBT genes in a cohort of 52 Saudi Arabian patients with maple syrup urine disease.

PubMed

Imtiaz, Faiqa; Al-Mostafa, Abeer; Allam, Rabab; Ramzan, Khushnooda; Al-Tassan, Nada; Tahir, Asma I; Al-Numair, Nouf S; Al-Hamed, Mohamed H; Al-Hassnan, Zuhair; Al-Owain, Mohammad; Al-Zaidan, Hamad; Al-Amoudi, Mohammad; Qari, Alya; Balobaid, Ameera; Al-Sayed, Moeenaldeen

2017-06-01

Maple syrup urine disease (MSUD), an autosomal recessive inborn error of metabolism due to defects in the branched-chain α-ketoacid dehydrogenase (BCKD) complex, is commonly observed among other inherited metabolic disorders in the kingdom of Saudi Arabia. This report presents the results of mutation analysis of three of the four genes encoding the BCKD complex in 52 biochemically diagnosed MSUD patients originating from Saudi Arabia. The 25 mutations (20 novel) detected spanned across the entire coding regions of the BCKHDA , BCKDHB and DBT genes. There were no mutations found in the DLD gene in this cohort of patients. Prediction effects, conservation and modelling of novel mutations demonstrated that all were predicted to be disease-causing. All mutations presented in a homozygous form and we did not detect the presence of a "founder" mutation in any of three genes. In addition, prenatal molecular genetic testing was successfully carried out on chorionic villus samples or amniocenteses in 10 expectant mothers with affected children with MSUD, molecularly characterized by this study.

Functional analysis of mutations in a severe congenital neutropenia syndrome caused by glucose-6-phosphatase-β deficiency

PubMed Central

Lin, Su Ru; Pan, Chi-Jiunn; Mansfield, Brian C.; Chou, Janice Yang

2016-01-01

Glucose-6-phosphatase-β (G6Pase-β or G6PC3) deficiency is characterized by neutropenia and dysfunction in both neutrophils and macrophages. G6Pase-β is an enzyme embedded in the endoplasmic reticulum membrane that catalyzes the hydrolysis of glucose-6-phosphate (G6P) to glucose and phosphate. To date, 33 separate G6PC3 mutations have been identified in G6Pase-β-deficient patients but only the p.R253H and p.G260R missense mutations have been characterized functionally for pathogenicity. Here we functionally characterize 16 of the 19 known missense mutations using a sensitive assay, based on a recombinant adenoviral vector-mediated expression system, to demonstrate pathogenicity. Fourteen missense mutations completely abolish G6Pase-β enzymatic activity while the p.S139I and p.R189Q mutations retain 49% and 45%, respectively of wild type G6Pase-β activity. A database of residual enzymatic activity retained by the G6Pase-β mutations will serve as a reference for evaluating genotype-phenotype relationships. PMID:25492228

Substitution of aspartate for glycine 103 of the type II collagen triple helical domain: Identification of the minimal mutation which can produce Kniest dysplasia

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

Wilkin, D.J.; Rimoin, D.L.; Cohn, D.H.

1994-09-01

Kniest dysplasia is an autosomal dominant chondrodysplasia which results from mutations in the gene for type II collagen, COL2A1. Characteristics of the disorder include a short trunk and extremities, mid-face hypoplasia, cleft palate, myopia, retinal detachment, and hearing loss. Recently, deletions of all or part of exon 12 have been identified in individuals with Kniest dysplasia, suggesting that mutations within this region of the protein may primarily result in the Kniest dysplasia phenotype. We used SSCP to analyze an amplified genomic DNA fragment containing exon 12 from 7 individuals with Kniest dysplasia. An abnormality was identified in one patient. DNAmore » sequence analysis demonstrated that the patient was heterozygous for a G to A transition that implied substitution of glycine{sup 103} of the triple helix by aspartate. The mutation was not observed in DNA from either of the proband`s parents. Protein microsequencing demonstrated expression of the abnormal allele in the proband`s cartilage, indicating that the Kniest phenotype results from the presence of abnormal type II collagen molecules in the extracellular matrix. These data demonstrate the minimal mutation which can produce Kniest dysplasia and further support the hypothesis that alteration of a domain which includes the region encoded by exon 12 in the type II collagen protein leads to this disorder. Experiments designed to identify specific effects that mutations in this region have on intermolecular interactions among abnormal type II collagen molecules and other components of the cartilage extracellular matrix may clarify the underlying pathophysiology of Kniest dysplasia.« less

Sequence analysis of the L protein of the Ebola 2014 outbreak: Insight into conserved regions and mutations.

PubMed

Ayub, Gohar; Waheed, Yasir

2016-06-01

The 2014 Ebola outbreak was one of the largest that have occurred; it started in Guinea and spread to Nigeria, Liberia and Sierra Leone. Phylogenetic analysis of the current virus species indicated that this outbreak is the result of a divergent lineage of the Zaire ebolavirus. The L protein of Ebola virus (EBOV) is the catalytic subunit of the RNA‑dependent RNA polymerase complex, which, with VP35, is key for the replication and transcription of viral RNA. Earlier sequence analysis demonstrated that the L protein of all non‑segmented negative‑sense (NNS) RNA viruses consists of six domains containing conserved functional motifs. The aim of the present study was to analyze the presence of these motifs in 2014 EBOV isolates, highlight their function and how they may contribute to the overall pathogenicity of the isolates. For this purpose, 81 2014 EBOV L protein sequences were aligned with 475 other NNS RNA viruses, including Paramyxoviridae and Rhabdoviridae viruses. Phylogenetic analysis of all EBOV outbreak L protein sequences was also performed. Analysis of the amino acid substitutions in the 2014 EBOV outbreak was conducted using sequence analysis. The alignment demonstrated the presence of previously conserved motifs in the 2014 EBOV isolates and novel residues. Notably, all the mutations identified in the 2014 EBOV isolates were tolerant, they were pathogenic with certain examples occurring within previously determined functional conserved motifs, possibly altering viral pathogenicity, replication and virulence. The phylogenetic analysis demonstrated that all sequences with the exception of the 2014 EBOV sequences were clustered together. The 2014 EBOV outbreak has acquired a great number of mutations, which may explain the reasons behind this unprecedented outbreak. Certain residues critical to the function of the polymerase remain conserved and may be targets for the development of antiviral therapeutic agents.

Expanding sialidosis spectrum by genome-wide screening: NEU1 mutations in adult-onset myoclonus.

PubMed

Canafoglia, Laura; Robbiano, Angela; Pareyson, Davide; Panzica, Ferruccio; Nanetti, Lorenzo; Giovagnoli, Anna Rita; Venerando, Anna; Gellera, Cinzia; Franceschetti, Silvana; Zara, Federico

2014-06-03

To identify the genetic cause of a familial form of late-onset action myoclonus in 2 unrelated patients. Both probands had 2 siblings displaying a similar disorder. Extensive laboratory examinations, including biochemical assessment for urine sialic acid in the 2 probands, were negative. Exome sequencing was performed in the probands using an Illumina platform. Segregation analysis of putative mutations was performed in all family members by standard Sanger sequencing protocols. NEU1 mutations were detected in 3 siblings of each family with prominent cortical myoclonus presenting in the third decade of life and having a mild and slowly progressive course. They did not have macular cherry-red spot and their urinary sialic acid excretion was within normal values. Genetic analysis demonstrated a homozygous mutation in family 1 (c.200G>T, p.S67I) and 2 compound heterozygous mutations in family 2 (c.679G>A, p.G227R; c.913C>T, p.R305C). Our observation indicates that sialidosis should be suspected and the NEU1 gene analyzed in patients with isolated action myoclonus presenting in adulthood in the absence of other typical clinical and laboratory findings. © 2014 American Academy of Neurology.

A Novel Mutation of the Arginine Vasopressin Receptor 2 Gene in a Patient with Congenital Nephrogenic Diabetes Insipidus

PubMed Central

Tajima, Asako; Miyata, Ichiro; Katayama, Akira; Toyoda, Shigeru; Eto, Yoshikatsu

2005-01-01

We have identified a novel mutation of the arginine vasopressin receptor 2 (AVPR2) gene in a case of congenital X-linked nephrogenic diabetes insipidus (NDI). The patient was a 2-mo-old Japanese boy with persistent fever and failure to thrive. He was diagnosed as having congenital NDI by clinical and laboratory findings. Molecular analysis demonstrated that he was hemizygous for a G to C transversion in exon 2 of the AVPR2 gene which resulted in a glycine to arginine substitution (G107R) at the 107th codon of the first extracellular loop. His mother was heterozygous for the same mutation. We speculated that the G107R mutation would interfere with the binding capacity of the AVPR2, since G107R is located near F105 and R106, both of which are crucial for ligand binding. In cases of X-linked NDI, mutations in the AVPR2 gene are distributed widely. Thus, DNA analysis throughout the gene is of clinical value for the identification of female carriers, and it also gives precise information for genetic counseling. PMID:24790307

Whole-Genome and Epigenomic Landscapes of Etiologically Distinct Subtypes of Cholangiocarcinoma

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

Jusakul, Apinya; Cutcutache, Ioana; Yong, Chern Han

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy exhibiting high incidence in countries with endemic liver-fluke infection. We analysed 489 CCAs from 10 countries, combining whole-genome (71 cases), targeted/exome, copy-number, gene expression, and DNA methylation information. Integrative clustering defined four CCA clusters - Fluke- Positive CCAs (Clusters 1/2) are enriched in ERBB2 amplifications and TP53 mutations, conversely Fluke-Negative CCAs (Clusters 3/4) exhibit high copy-number alterations and PD-1/PD-L2 expression, or epigenetic mutations (IDH1/2, BAP1) and FGFR/PRKA-related gene rearrangements. Whole-genome analysis highlighted FGFR2 3’UTR deletion as a mechanism of FGFR2 upregulation. Integration of non-coding promoter mutations with protein-DNA binding profiles demonstrates pervasive modulation ofmore » H3K27me3-associated sites in CCA. Clusters 1 and 4 exhibit distinct DNA hypermethylation patterns targeting either CpG islands or shores - mutation signature and subclonality analysis suggests that these reflect different mutational pathways. Lastly, our results exemplify how genetics, epigenetics and environmental carcinogens can interplay across different geographies to generate distinct molecular subtypes of cancer.« less

Whole-Genome and Epigenomic Landscapes of Etiologically Distinct Subtypes of Cholangiocarcinoma

DOE PAGES

Jusakul, Apinya; Cutcutache, Ioana; Yong, Chern Han; ...

2017-06-30

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy exhibiting high incidence in countries with endemic liver-fluke infection. We analysed 489 CCAs from 10 countries, combining whole-genome (71 cases), targeted/exome, copy-number, gene expression, and DNA methylation information. Integrative clustering defined four CCA clusters - Fluke- Positive CCAs (Clusters 1/2) are enriched in ERBB2 amplifications and TP53 mutations, conversely Fluke-Negative CCAs (Clusters 3/4) exhibit high copy-number alterations and PD-1/PD-L2 expression, or epigenetic mutations (IDH1/2, BAP1) and FGFR/PRKA-related gene rearrangements. Whole-genome analysis highlighted FGFR2 3’UTR deletion as a mechanism of FGFR2 upregulation. Integration of non-coding promoter mutations with protein-DNA binding profiles demonstrates pervasive modulation ofmore » H3K27me3-associated sites in CCA. Clusters 1 and 4 exhibit distinct DNA hypermethylation patterns targeting either CpG islands or shores - mutation signature and subclonality analysis suggests that these reflect different mutational pathways. Lastly, our results exemplify how genetics, epigenetics and environmental carcinogens can interplay across different geographies to generate distinct molecular subtypes of cancer.« less

Tietz/Waardenburg type 2A syndrome associated with posterior microphthalmos in two unrelated patients with novel MITF gene mutations.

PubMed

Cortés-González, Vianney; Zenteno, Juan Carlos; Guzmán-Sánchez, Martín; Giordano-Herrera, Verónica; Guadarrama-Vallejo, Dalia; Ruíz-Quintero, Narlly; Villanueva-Mendoza, Cristina

2016-12-01

Tietz syndrome and Waardenburg syndrome type 2A are allelic conditions caused by MITF mutations. Tietz syndrome is inherited in an autosomal dominant pattern and is characterized by congenital deafness and generalized skin, hair, and eye hypopigmentation, while Waardenburg syndrome type 2A typically includes variable degrees of sensorineural hearing loss and patches of de-pigmented skin, hair, and irides. In this paper, we report two unrelated families with MITF mutations. The first family showed an autosomal dominant pattern and variable expressivity. The second patient was isolated. MITF gene analysis in the first family demonstrated a c.648A>C heterozygous mutation in exon 8 c.648A>C; p. (R216S), while in the isolated patient, an apparently de novo heterozygous c.1183_1184insG truncating mutation was demonstrated in exon 10. All patients except one had bilateral reduced ocular anteroposterior axial length and a high hyperopic refractive error corresponding to posterior microphthalmos, features that have not been described as part of the disease. Our results suggest that posterior microphthalmos might be part of the clinical characteristics of Tietz/Waardenburg syndrome type 2A and expand both the clinical and molecular spectrum of the disease. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Autosomal Recessive Hypotrichosis with Woolly Hair Caused by a Mutation in the Keratin 25 Gene Expressed in Hair Follicles.

PubMed

Zernov, Nikolay V; Skoblov, Mikhail Y; Marakhonov, Andrey V; Shimomura, Yutaka; Vasilyeva, Tatyana A; Konovalov, Fedor A; Abrukova, Anna V; Zinchenko, Rena A

2016-06-01

Hypotrichosis is an abnormal condition characterized by decreased hair density and various defects in hair structure and growth patterns. In particular, in woolly hair, hypotrichosis is characterized by a tightly curled structure and abnormal growth. In this study, we present a detailed comparative examination of individuals affected by autosomal-recessive hypotrichosis (ARH), which distinguishes two types of ARH. Earlier, we demonstrated that exon 4 deletion in the lipase H gene caused an ARH (hypotrichosis 7; MIM: 604379) in populations of the Volga-Ural region of Russia. Screening for this mutation in all affected individuals revealed its presence only in the group with the hypotrichosis 7 phenotype. Other patients formed a separate group of woolly hair-associated ARH, with a homozygous missense mutation c.712G>T (p.Val238Leu) in a highly conserved position of type I keratin KRT25 (K25). Haplotype analysis indicated a founder effect. An expression study in the HaCaT cell line demonstrated a deleterious effect of the p.Val238Leu mutation on the formation of keratin intermediate filaments. Hence, we have identified a previously unreported missense mutation in the KRT25 gene causing ARH with woolly hair. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Brooke-Spiegler syndrome: report of 10 patients from 8 families with novel germline mutations: evidence of diverse somatic mutations in the same patient regardless of tumor type.

PubMed

Sima, Radek; Vanecek, Tomas; Kacerovska, Denisa; Trubac, Pavel; Cribier, Bernard; Rutten, Arno; Vazmitel, Marina; Spagnolo, Dominic V; Litvik, Radek; Vantuchova, Yvetta; Weyers, Wolfgang; Pearce, Robert L; Pearn, John; Michal, Michal; Kazakov, Dmitry V

2010-06-01

Brooke-Spiegler syndrome (BSS) is an inherited autosomal dominant disease characterized by the development of multiple adnexal cutaneous neoplasms including spiradenoma, cylindroma, spiradenocylindroma, and trichoepithelioma (cribriform trichoblastoma). BSS patients have various mutations in the CYLD gene, a tumor suppressor gene located on chromosome 16q. Our search of the literature revealed 51 germline CYLD mutations reported to date. Somatic CYLD mutations have rarely been investigated. We studied 10 patients from 8 families with BSS. Analysis of germline mutations of the CYLD gene was performed using either peripheral blood or nontumorous tissue. In addition, 19 formalin-fixed paraffin-embedded tumor samples were analyzed for somatic mutations, including loss of heterozygosity studies. A total of 38 tumors were available for histopathologic review. We have identified 8 novel germline mutations, all of which consisted of substitutions, deletions, and insertions/duplications and all except one led to premature stop codons. The substitution mutation in a single case was also predicted to disrupt protein function and seems causally implicated in tumor formation. We demonstrate for the first time that somatic events, loss of heterozygosity, or sequence mutations may differ among multiple neoplasms even of the same histologic type, occurring in the same patient.

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

PubMed

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

2015-10-01

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

Analysis of alkaptonuria (AKU) mutations and polymorphisms reveals that the CCC sequence motif is a mutational hot spot in the homogentisate 1,2 dioxygenase gene (HGO).

PubMed Central

Beltrán-Valero de Bernabé, D; Jimenez, F J; Aquaron, R; Rodríguez de Córdoba, S

1999-01-01

We recently showed that alkaptonuria (AKU) is caused by loss-of-function mutations in the homogentisate 1,2 dioxygenase gene (HGO). Herein we describe haplotype and mutational analyses of HGO in seven new AKU pedigrees. These analyses identified two novel single-nucleotide polymorphisms (INV4+31A-->G and INV11+18A-->G) and six novel AKU mutations (INV1-1G-->A, W60G, Y62C, A122D, P230T, and D291E), which further illustrates the remarkable allelic heterogeneity found in AKU. Reexamination of all 29 mutations and polymorphisms thus far described in HGO shows that these nucleotide changes are not randomly distributed; the CCC sequence motif and its inverted complement, GGG, are preferentially mutated. These analyses also demonstrated that the nucleotide substitutions in HGO do not involve CpG dinucleotides, which illustrates important differences between HGO and other genes for the occurrence of mutation at specific short-sequence motifs. Because the CCC sequence motifs comprise a significant proportion (34.5%) of all mutated bases that have been observed in HGO, we conclude that the CCC triplet is a mutational hot spot in HGO. PMID:10205262

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

PubMed

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

2007-01-01

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

Scanning the Effects of Ethyl Methanesulfonate on the Whole Genome of Lotus japonicus Using Second-Generation Sequencing Analysis

PubMed Central

Mohd-Yusoff, Nur Fatihah; Ruperao, Pradeep; Tomoyoshi, Nurain Emylia; Edwards, David; Gresshoff, Peter M.; Biswas, Bandana; Batley, Jacqueline

2015-01-01

Genetic structure can be altered by chemical mutagenesis, which is a common method applied in molecular biology and genetics. Second-generation sequencing provides a platform to reveal base alterations occurring in the whole genome due to mutagenesis. A model legume, Lotus japonicus ecotype Miyakojima, was chemically mutated with alkylating ethyl methanesulfonate (EMS) for the scanning of DNA lesions throughout the genome. Using second-generation sequencing, two individually mutated third-generation progeny (M3, named AM and AS) were sequenced and analyzed to identify single nucleotide polymorphisms and reveal the effects of EMS on nucleotide sequences in these mutant genomes. Single-nucleotide polymorphisms were found in every 208 kb (AS) and 202 kb (AM) with a bias mutation of G/C-to-A/T changes at low percentage. Most mutations were intergenic. The mutation spectrum of the genomes was comparable in their individual chromosomes; however, each mutated genome has unique alterations, which are useful to identify causal mutations for their phenotypic changes. The data obtained demonstrate that whole genomic sequencing is applicable as a high-throughput tool to investigate genomic changes due to mutagenesis. The identification of these single-point mutations will facilitate the identification of phenotypically causative mutations in EMS-mutated germplasm. PMID:25660167

Primary ciliary dyskinesia: improving the diagnostic approach

PubMed Central

Leigh, Margaret W.; Zariwala, Maimoona A.; Knowles, Michael R.

2009-01-01

Purpose of review The diagnosis of primary ciliary dyskinesia (PCD) has relied on analysis of ciliary motility and ultrastructure; however, these tests are not readily available and have not been standardized. Consequently, the diagnosis of PCD may be delayed or missed or made incorrectly. This review outlines the potential utility of new diagnostic tests, including measurement of nasal nitric oxide (NO) production and systematic analysis for mutations in gene encoding ciliary proteins. Recent findings Clinical manifestations of PCD have been expanded to include neonatal respiratory distress and heterotaxy. Measurement of nasal NO has emerged as a useful screening test for PCD based on the very low levels in PCD (approximately 1/10 of normal values). Genetic testing is emerging for PCD and demonstrates extensive genetic heterogeneity. Some genes and gene mutations involved in PCD have been defined. Approximately one third of PCD cases have identifiable gene mutations in one of 6 different genes. An international effort is focused on defining PCD-causing defects in other genes. Summary The incorporation of nasal NO measurement as a screening test to define probable PCD cases and gene mutation analysis to make a definitive diagnosis of PCD should enhance diagnostic evaluation of PCD. PMID:19300264

Evolutionary and genetic analysis of the VP2 gene of canine parvovirus.

PubMed

Li, Gairu; Ji, Senlin; Zhai, Xiaofeng; Zhang, Yuxiang; Liu, Jie; Zhu, Mengyan; Zhou, Jiyong; Su, Shuo

2017-07-17

Canine parvovirus (CPV) type 2 emerged in 1978 in the USA and quickly spread among dog populations all over the world with high morbidity. Although CPV is a DNA virus, its genomic substitution rate is similar to some RNA viruses. Therefore, it is important to trace the evolution of CPV to monitor the appearance of mutations that might affect vaccine effectiveness. Our analysis shows that the VP2 genes of CPV isolated from 1979 to 2016 are divided into six groups: GI, GII, GIII, GIV, GV, and GVI. Amino acid mutation analysis revealed several undiscovered important mutation sites: F267Y, Y324I, and T440A. Of note, the evolutionary rate of the CPV VP2 gene from Asia and Europe decreased. Codon usage analysis showed that the VP2 gene of CPV exhibits high bias with an ENC ranging from 34.93 to 36.7. Furthermore, we demonstrate that natural selection plays a major role compared to mutation pressure driving CPV evolution. There are few studies on the codon usage of CPV. Here, we comprehensively studied the genetic evolution, codon usage pattern, and evolutionary characterization of the VP2 gene of CPV. The novel findings revealing the evolutionary process of CPV will greatly serve future CPV research.

Development of an Ammonium Sulfate DNA Extraction Method for Obtaining Amplifiable DNA in a Small Number of Cells and Its Application to Clinical Specimens

PubMed Central

Oh, Seo Young; Kim, Wook Youn; Hwang, Tae Sook; Han, Hye Seung; Lim, So Dug; Kim, Wan Seop

2013-01-01

DNA extraction from microdissected cells has become essential for handling clinical specimens with advances in molecular pathology. Conventional methods have limitations for extracting amplifiable DNA from specimens containing a small number of cells. We developed an ammonium sulfate DNA extraction method (A) and compared it with two other methods (B and C). DNA quality and quantity, β-globin amplification, and detectability of two cancer associated gene mutations were evaluated. Method A showed the best DNA yield, particularly when the cell number was very low. Amplification of the β-globin gene using DNA from the SNU 790 cell line and papillary thyroid carcinoma (PTC) cells extracted with Method A demonstrated the strongest band. BRAF V600E mutation analysis using ethanol-fixed PTC cells from a patient demonstrated both a “T” peak increase and an adjacent “A” peak decrease when 25 and 50 cells were extracted, whereas mutant peaks were too low to be analyzed using the other two methods. EGFR mutation analysis using formalin-fixed paraffin-embedded lung cancer tissues demonstrated a mutant peak with Method A, whereas the mutant peak was undetectable with Methods B or C. Method A yielded the best DNA quantity and quality with outstanding efficiency, particularly when paucicellular specimens were used. PMID:23691506

Building toy models of proteins using coevolutionary information

NASA Astrophysics Data System (ADS)

Cheng, Ryan; Raghunathan, Mohit; Onuchic, Jose

2015-03-01

Recent developments in global statistical methodologies have advanced the analysis of large collections of protein sequences for coevolutionary information. Coevolution between amino acids in a protein arises from compensatory mutations that are needed to maintain the stability or function of a protein over the course of evolution. This gives rise to quantifiable correlations between amino acid positions within the multiple sequence alignment of a protein family. Here, we use Direct Coupling Analysis (DCA) to infer a Potts model Hamiltonian governing the correlated mutations in a protein family to obtain the sequence-dependent interaction energies of a toy protein model. We demonstrate that this methodology predicts residue-residue interaction energies that are consistent with experimental mutational changes in protein stabilities as well as other computational methodologies. Furthermore, we demonstrate with several examples that DCA could be used to construct a structure-based model that quantitatively agrees with experimental data on folding mechanisms. This work serves as a potential framework for generating models of proteins that are enriched by evolutionary data that can potentially be used to engineer key functional motions and interactions in protein systems. This research has been supported by the NSF INSPIRE award MCB-1241332 and by the CTBP sponsored by the NSF (Grant PHY-1427654).

A comprehensive analysis of clinical outcomes in lung cancer patients harboring a MET exon 14 skipping mutation compared to other driver mutations in an East Asian population.

PubMed

Gow, Chien-Hung; Hsieh, Min-Shu; Wu, Shang-Gin; Shih, Jin-Yuan

2017-01-01

Recurrent somatic splice-site alterations at MET exon 14 (MET Δ14 ), which result in exon skipping and MET proto-oncogene, receptor tyrosine kinase (MET) activation, have been characterised. However, their demographic features and clinical outcomes in East Asian lung cancer patients have yet to be determined. A one-step reverse transcription-polymerase chain reaction (RT-PCR), using RNA samples from 850 East Asian lung cancer patients, was performed in order to detect MET Δ14 and five other major driver mutations, including those in the EGFR, KRAS, ALK, HER2, and ROS1 genes. Immunohistochemistry (IHC) was used to confirm the overexpression of MET in patients harbouring the MET Δ14 mutation. We analysed the demographic data and clinical outcomes of MET Δ14 mutation positive lung cancer patients and compared them to those of MET Δ14 mutation negative lung cancer patients. In total, 27 lung adenocarcinoma (ADC) patients and 1 squamous cell carcinoma patient with the MET Δ14 mutation were identified. The overall incidence was 3.3% for lung cancer and 4.0% for lung ADC. IHC demonstrated that the majority of lung cancer patients harboring a MET Δ14 mutation exhibited a strong cytoplasmic expression of MET. MET Δ14 mutation positive patients were generally quite elderly individuals. Stage IV MET Δ14 mutation positive lung cancer patients receiving no specific anti-MET therapy were observed to have a similar overall survival (OS) compared to patients in the all negative group (P>0.05). In the multivariate analysis, mutation status was found not to be a major risk factor for OS in lung cancer patients without appropriate tyrosine kinase inhibitors treatment. The OS of MET Δ14 mutation positive lung cancer patients is comparable to that of the major driver gene mutation negative lung cancer patients. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Molecular biological analysis in a patient with multiple lung adenocarcinomas.

PubMed

Wakayama, Tomoshige; Hirata, Hirokuni; Suka, Shunsuke; Sato, Kozo; Tatewaki, Masamitsu; Souma, Ryosuke; Satoh, Hideyuki; Tamura, Motohiko; Matsumura, Yuji; Imada, Hiroki; Sugiyama, Kumiya; Arima, Masafumi; Kurasawa, Kazuhiro; Fukuda, Takeshi; Fukushima, Yasutsugu

2018-05-01

The utility of molecular biological analysis in lung adenocarcinoma has been demonstrated. Herein we report a rare case presenting as multiple lung adenocarcinomas with four different EGFR gene mutations detected in three lung tumors. After opacification was detected by routine chest X-ray, the patient, a 64-year-old woman, underwent chest computed tomography which revealed a right lung segment S4 ground-glass nodule (GGN). Follow-up computed tomography revealed a 42 mm GGN nodule with a 26 mm nodule (S6) and a 20 mm GGN (S10). Histopathology of resected specimens from the right middle and lower lobes revealed all three nodules were adenocarcinomas. Four EGFR mutations were detected; no three tumors had the same mutations. Molecular biological analysis is a promising tool for the diagnosis of primary tumors in patients with multiple lung carcinomas of the same histotype, enabling appropriate treatment. © 2018 The Authors. Thoracic Cancer published by China Lung Oncology Group and John Wiley & Sons Australia, Ltd.

Targeted DNA sequencing and in situ mutation analysis using mobile phone microscopy

PubMed Central

Kühnemund, Malte; Wei, Qingshan; Darai, Evangelia; Wang, Yingjie; Hernández-Neuta, Iván; Yang, Zhao; Tseng, Derek; Ahlford, Annika; Mathot, Lucy; Sjöblom, Tobias; Ozcan, Aydogan; Nilsson, Mats

2017-01-01

Molecular diagnostics is typically outsourced to well-equipped centralized laboratories, often far from the patient. We developed molecular assays and portable optical imaging designs that permit on-site diagnostics with a cost-effective mobile-phone-based multimodal microscope. We demonstrate that targeted next-generation DNA sequencing reactions and in situ point mutation detection assays in preserved tumour samples can be imaged and analysed using mobile phone microscopy, achieving a new milestone for tele-medicine technologies. PMID:28094784

VarWalker: Personalized Mutation Network Analysis of Putative Cancer Genes from Next-Generation Sequencing Data

PubMed Central

Jia, Peilin; Zhao, Zhongming

2014-01-01

A major challenge in interpreting the large volume of mutation data identified by next-generation sequencing (NGS) is to distinguish driver mutations from neutral passenger mutations to facilitate the identification of targetable genes and new drugs. Current approaches are primarily based on mutation frequencies of single-genes, which lack the power to detect infrequently mutated driver genes and ignore functional interconnection and regulation among cancer genes. We propose a novel mutation network method, VarWalker, to prioritize driver genes in large scale cancer mutation data. VarWalker fits generalized additive models for each sample based on sample-specific mutation profiles and builds on the joint frequency of both mutation genes and their close interactors. These interactors are selected and optimized using the Random Walk with Restart algorithm in a protein-protein interaction network. We applied the method in >300 tumor genomes in two large-scale NGS benchmark datasets: 183 lung adenocarcinoma samples and 121 melanoma samples. In each cancer, we derived a consensus mutation subnetwork containing significantly enriched consensus cancer genes and cancer-related functional pathways. These cancer-specific mutation networks were then validated using independent datasets for each cancer. Importantly, VarWalker prioritizes well-known, infrequently mutated genes, which are shown to interact with highly recurrently mutated genes yet have been ignored by conventional single-gene-based approaches. Utilizing VarWalker, we demonstrated that network-assisted approaches can be effectively adapted to facilitate the detection of cancer driver genes in NGS data. PMID:24516372

VarWalker: personalized mutation network analysis of putative cancer genes from next-generation sequencing data.

PubMed

Jia, Peilin; Zhao, Zhongming

2014-02-01

A major challenge in interpreting the large volume of mutation data identified by next-generation sequencing (NGS) is to distinguish driver mutations from neutral passenger mutations to facilitate the identification of targetable genes and new drugs. Current approaches are primarily based on mutation frequencies of single-genes, which lack the power to detect infrequently mutated driver genes and ignore functional interconnection and regulation among cancer genes. We propose a novel mutation network method, VarWalker, to prioritize driver genes in large scale cancer mutation data. VarWalker fits generalized additive models for each sample based on sample-specific mutation profiles and builds on the joint frequency of both mutation genes and their close interactors. These interactors are selected and optimized using the Random Walk with Restart algorithm in a protein-protein interaction network. We applied the method in >300 tumor genomes in two large-scale NGS benchmark datasets: 183 lung adenocarcinoma samples and 121 melanoma samples. In each cancer, we derived a consensus mutation subnetwork containing significantly enriched consensus cancer genes and cancer-related functional pathways. These cancer-specific mutation networks were then validated using independent datasets for each cancer. Importantly, VarWalker prioritizes well-known, infrequently mutated genes, which are shown to interact with highly recurrently mutated genes yet have been ignored by conventional single-gene-based approaches. Utilizing VarWalker, we demonstrated that network-assisted approaches can be effectively adapted to facilitate the detection of cancer driver genes in NGS data.

Parkinsonism Associated with Glucocerebrosidase Mutation

PubMed Central

Sunwoo, Mun-Kyung; Kim, Seung-Min; Lee, Sarah

2011-01-01

Background Gaucher's disease is an autosomal recessive, lysosomal storage disease caused by mutations of the β-glucocerebrosidase gene (GBA). There is increasing evidence that GBA mutations are a genetic risk factor for the development of Parkinson's disease (PD). We report herein a family of Koreans exhibiting parkinsonism-associated GBA mutations. Case Report A 44-year-old woman suffering from slowness and paresthesia of the left arm for the previous 1.5years, visited our hospital to manage known invasive ductal carcinoma. During a preoperative evaluation, she was diagnosed with Gaucher's disease and double mutations of S271G and R359X in GBA. Parkinsonian features including low amplitude postural tremors, rigidity, bradykinesia and shuffling gait were observed. Genetic analysis also revealed that her older sister, who had also been diagnosed with PD and had been taking dopaminergic drugs for 8-years, also possessed a heterozygote R359X mutation in GBA. 18F-fluoropropylcarbomethoxyiodophenylnortropane positron-emission tomography in these patients revealed decreased uptake of dopamine transporter in the posterior portion of the bilateral putamen. Conclusions This case study demonstrates Korean familial cases of PD with heterozygote mutation of GBA, further supporting the association between PD and GBA mutation. PMID:21779299

A rare connexin 26 mutation in a patient with a forme fruste of keratitis-ichthyosis-deafness (KID) syndrome.

PubMed

Neoh, Ching Yin; Chen, Huijia; Ng, See Ket; Lane, Ellen Birgitte; Common, John Edmund Armourer

2009-10-01

Keratitis-ichthyosis-deafness (KID) syndrome is a rare ectodermal dysplasia characterized by generalized erythrokeratotic plaques, sensorineural hearing loss, and vascularizing keratitis. Cutaneous changes and hearing loss typically present in early childhood, whereas ocular symptoms present later. Mutations in the connexin (Cx) 26 gene, GJB2, are now established to underlie many of the affected cases, with the majority of patients harboring the p.D50N mutation. A rare patient demonstrating features of incomplete KID syndrome associated with an uncommon Cx26 gene mutation is described. The patient presented late in adolescence with partial features of KID syndrome. There was limited cutaneous involvement and the rare association of cystic acne. Both hearing impairment and ophthalmic involvement were mild in severity. Genetic mutation analysis revealed a previously described, rare mutation in GJB2, resulting in a glycine to arginine change at codon 12 (p.G12R). This report describes a patient exhibiting characteristics suggestive of a late-onset, incomplete form of KID syndrome with the GJB2 mutation (p.G12R). The p.G12R mutation has only been described in one other patient with KID syndrome, whose clinical presentation was not characterized.

Familial early-onset dementia with tau intron 10 + 16 mutation with clinical features similar to those of Alzheimer disease.

PubMed

Doran, Mark; du Plessis, Daniel G; Ghadiali, Eric J; Mann, David M A; Pickering-Brown, Stuart; Larner, Andrew J

2007-10-01

Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) owing to the tau intron 10 + 16 mutation usually occurs with a prototypical frontotemporal dementia phenotype with prominent disinhibition and affective disturbances. To report a new FTDP-17 pedigree with the tau intron 10 + 16 mutation demonstrating a clinical phenotype suggestive of Alzheimer disease. Case reports. Regional neuroscience centers in northwest England. Patients We examined 4 members of a kindred in which 8 individuals were affected in 3 generations. All 4 patients reported memory difficulty. Marked anomia was also present, but behavioral disturbances were conspicuously absent in the early stages of disease. All patients had an initial clinical diagnosis of Alzheimer disease. No mutations were found in the presenilin or amyloid precursor protein genes. Pathologic examination of the proband showed features typical of FTDP-17, and tau gene analysis showed the intron 10 + 16 mutation. This pedigree illustrates the phenotypic variability of tau intron 10 + 16 mutations. In pedigrees with a clinical diagnosis of Alzheimer disease but without presenilin or amyloid precursor protein gene mutations, tau gene mutations may be found.

Novel NTRK1 mutations cause hereditary sensory and autonomic neuropathy type IV: demonstration of a founder mutation in the Turkish population.

PubMed

Tüysüz, Beyhan; Bayrakli, Fatih; DiLuna, Michael L; Bilguvar, Kaya; Bayri, Yasar; Yalcinkaya, Cengiz; Bursali, Aysegul; Ozdamar, Elif; Korkmaz, Baris; Mason, Christopher E; Ozturk, Ali K; Lifton, Richard P; State, Matthew W; Gunel, Murat

2008-05-01

Hereditary sensory and autonomic neuropathy type IV (HSAN IV), or congenital insensitivity to pain with anhidrosis, is an autosomal recessive disorder characterized by insensitivity to noxious stimuli, anhidrosis from deinnervated sweat glands, and delayed mental and motor development. Mutations in the neurotrophic tyrosine kinase receptor type 1 (NTRK1), a receptor in the neurotrophin signaling pathway phosphorylated in response to nerve growth factor, are associated with this disorder. We identified six families from Northern Central Turkey with HSAN IV. We screened the NTRK1 gene for mutations in these families. Microsatellite and single nucleotide polymorphism (SNP) markers on the Affymetrix 250K chip platform were used to determine the haplotypes for three families harboring the same mutation. Screening for mutations in the NTRK1 gene demonstrated one novel frameshift mutation, two novel nonsense mutations, and three unrelated kindreds with the same splice-site mutation. Genotyping of the three families with the identical splice-site mutation revealed that they share the same haplotype. This report broadens the spectrum of mutations in NTRK1 that cause HSAN IV and demonstrates a founder mutation in the Turkish population.

Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding

PubMed Central

Koenig, Patrick; Lee, Chingwei V.; Walters, Benjamin T.; Janakiraman, Vasantharajan; Stinson, Jeremy; Patapoff, Thomas W.; Fuh, Germaine

2017-01-01

Somatic mutations within the antibody variable domains are critical to the immense capacity of the immune repertoire. Here, via a deep mutational scan, we dissect how mutations at all positions of the variable domains of a high-affinity anti-VEGF antibody G6.31 impact its antigen-binding function. The resulting mutational landscape demonstrates that large portions of antibody variable domain positions are open to mutation, and that beneficial mutations can be found throughout the variable domains. We determine the role of one antigen-distal light chain position 83, demonstrating that mutation at this site optimizes both antigen affinity and thermostability by modulating the interdomain conformational dynamics of the antigen-binding fragment. Furthermore, by analyzing a large number of human antibody sequences and structures, we demonstrate that somatic mutations occur frequently at position 83, with corresponding domain conformations observed for G6.31. Therefore, the modulation of interdomain dynamics represents an important mechanism during antibody maturation in vivo. PMID:28057863

Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer

PubMed Central

Muroni, Maria Rosaria; Sanges, Francesca; Sotgiu, Giovanni; Ena, Sara; Pira, Giovanna; Murgia, Luciano; Manca, Alessandra; Uras, Maria Gabriela; Sarobba, Maria Giuseppina; Urru, Silvana; De Miglio, Maria Rosaria

2015-01-01

Background Triple Negative Breast Cancer (TNBC) accounts for 12–24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20–40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data. Materials and Methods PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components. Results PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC. Conclusions Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies. PMID:26540293

Analysis of PIK3CA Mutations and Activation Pathways in Triple Negative Breast Cancer.

PubMed

Cossu-Rocca, Paolo; Orrù, Sandra; Muroni, Maria Rosaria; Sanges, Francesca; Sotgiu, Giovanni; Ena, Sara; Pira, Giovanna; Murgia, Luciano; Manca, Alessandra; Uras, Maria Gabriela; Sarobba, Maria Giuseppina; Urru, Silvana; De Miglio, Maria Rosaria

2015-01-01

Triple Negative Breast Cancer (TNBC) accounts for 12-24% of all breast carcinomas, and shows worse prognosis compared to other breast cancer subtypes. Molecular studies demonstrated that TNBCs are a heterogeneous group of tumors with different clinical and pathologic features, prognosis, genetic-molecular alterations and treatment responsivity. The PI3K/AKT is a major pathway involved in the regulation of cell survival and proliferation, and is the most frequently altered pathway in breast cancer, apparently with different biologic impact on specific cancer subtypes. The most common genetic abnormality is represented by PIK3CA gene activating mutations, with an overall frequency of 20-40%. The aims of our study were to investigate PIK3CA gene mutations on a large series of TNBC, to perform a wider analysis on genetic alterations involving PI3K/AKT and BRAF/RAS/MAPK pathways and to correlate the results with clinical-pathologic data. PIK3CA mutation analysis was performed by using cobas® PIK3CA Mutation Test. EGFR, AKT1, BRAF, and KRAS genes were analyzed by sequencing. Immunohistochemistry was carried out to identify PTEN loss and to investigate for PI3K/AKT pathways components. PIK3CA mutations were detected in 23.7% of TNBC, whereas no mutations were identified in EGFR, AKT1, BRAF, and KRAS genes. Moreover, we observed PTEN loss in 11.3% of tumors. Deregulation of PI3K/AKT pathways was revealed by consistent activation of pAKT and p-p44/42 MAPK in all PIK3CA mutated TNBC. Our data shows that PIK3CA mutations and PI3K/AKT pathway activation are common events in TNBC. A deeper investigation on specific TNBC genomic abnormalities might be helpful in order to select patients who would benefit from current targeted therapy strategies.

Multi-omics Based Identification of Specific Biochemical Changes Associated With PfKelch13-Mutant Artemisinin-Resistant Plasmodium falciparum.

PubMed

Siddiqui, Ghizal; Srivastava, Anubhav; Russell, Adrian S; Creek, Darren J

2017-05-01

The emergence of artemisinin resistance in the malaria parasite Plasmodium falciparum poses a major threat to the control and elimination of malaria. Certain point mutations in the propeller domain of PfKelch13 are associated with resistance, but PfKelch13 mutations do not always result in clinical resistance. The underlying mechanisms associated with artemisinin resistance are poorly understood, and the impact of PfKelch13 mutations on cellular biochemistry is not defined. This study aimed to identify global biochemical differences between PfKelch13-mutant artemisinin-resistant and -sensitive strains of P. falciparum by combining liquid chromatography-mass spectrometry (LC-MS)-based proteomics, peptidomics, and metabolomics. Proteomics analysis found both PfKelch13 mutations examined to be specifically associated with decreased abundance of PfKelch13 protein. Metabolomics analysis demonstrated accumulation of glutathione and its precursor, gamma-glutamylcysteine, and significant depletion of 1 other putative metabolite in resistant strains. Peptidomics analysis revealed lower abundance of several endogenous peptides derived from hemoglobin (HBα and HBβ) in the artemisinin-resistant strains. PfKelch13 mutations associated with artemisinin resistance lead to decreased abundance of PfKelch13 protein, decreased hemoglobin digestion, and enhanced glutathione production. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Detection of Activating Estrogen Receptor Gene (ESR1) Mutations in Single Circulating Tumor Cells.

PubMed

Paolillo, Carmela; Mu, Zhaomei; Rossi, Giovanna; Schiewer, Matthew J; Nguyen, Thomas; Austin, Laura; Capoluongo, Ettore; Knudsen, Karen; Cristofanilli, Massimo; Fortina, Paolo

2017-10-15

Purpose: Early detection is essential for treatment plans before onset of metastatic disease. Our purpose was to demonstrate feasibility to detect and monitor estrogen receptor 1 ( ESR1 ) gene mutations at the single circulating tumor cell (CTC) level in metastatic breast cancer (MBC). Experimental Design: We used a CTC molecular characterization approach to investigate heterogeneity of 14 hotspot mutations in ESR1 and their correlation with endocrine resistance. Combining the CellSearch and DEPArray technologies allowed recovery of 71 single CTCs and 12 WBC from 3 ER-positive MBC patients. Forty CTCs and 12 WBC were subjected to whole genome amplification by MALBAC and Sanger sequencing. Results: Among 3 selected patients, 2 had an ESR1 mutation (Y537). One showed two different ESR1 variants in a single CTC and another showed loss of heterozygosity. All mutations were detected in matched cell-free DNA (cfDNA). Furthermore, one had 2 serial blood samples analyzed and showed changes in both cfDNA and CTCs with emergence of mutations in ESR1 (Y537S and T570I), which has not been reported previously. Conclusions: CTCs are easily accessible biomarkers to monitor and better personalize management of patients with previously demonstrated ER-MBC who are progressing on endocrine therapy. We showed that single CTC analysis can yield important information on clonal heterogeneity and can be a source of discovery of novel and potential driver mutations. Finally, we also validate a workflow for liquid biopsy that will facilitate early detection of ESR1 mutations, the emergence of endocrine resistance and the choice of further target therapy. Clin Cancer Res; 23(20); 6086-93. ©2017 AACR . ©2017 American Association for Cancer Research.

An "ex vivo model" contributing to the diagnosis and evaluation of new drugs in cystic fibrosis.

PubMed

Di Lullo, A M; Scorza, M; Amato, F; Comegna, M; Raia, V; Maiuri, L; Ilardi, G; Cantone, E; Castaldo, G; Iengo, M

2017-06-01

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. About 2000 mutations have been described so far. We setup an ex vivo model of human nasal epithelial cells (HNECs) to study CF patients testing the effect of novel mutations and molecular therapies. We performed sampling (by brushing), followed by culture and analysis of HNECs using a series of molecular techniques. We performed 50 brushings from CF patients and controls. Using cultured cells, we: i) demonstrated the widely heterogeneous CFTR expression in patients and in controls; ii) defined the splicing effect of a CFTR mutation; iii) assessed the CFTR gating activity in patients bearing different mutations; iv) demonstrated that butyrate significantly enhances CFTR expression. Based on our data, we can conclude: 1) HNEC brushing is performed without anaesthesia and is well tolerated in all CF patients (children and adults); 2) HNECs can be preserved for up to 48 hours before culture allowings multicentre studies; 3) HNECs culture can be considered a suitable model to study the molecular effects of new CFTR gene mutations and/or uncertain meaning specific mutations of carriers; 4) an ex vivo model of HNECs may be used to evaluate, before human use, the effect of new drugs on patients' cells bearing specific CFTR mutations; 5) the methodology is adequate for a quantitative measurement, by fluorescence, of the CFTR gating activity of the HNECs from patients with different genotypes identifying: a) CF patients bearing two severe mutations with an activity < 10% (compared to controls - 100%); b) CF patients bearing at least a mild mutation with an activity of 10-20%; c) CF carriers (heterozygous subjects) with an activity between 40-70%. © Copyright by Società Italiana di Otorinolaringologia e Chirurgia Cervico-Facciale, Rome, Italy.

Associations between mutations and a VNTR in the human phenylalanine hydroxylase gene

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

Goltsov, A.A.; Eisensmith, R.C.; Woo, S.L.C.

1992-09-01

The HindIII RFLP in the human phenylalanine hydroxylase (PAH) gene is caused by the presence of an AT-rich (70%) minisatellite region. This region contains various multiples of 30-bp tandem repeats and is located 3 kb downstream of the final exon of the gene. PCR-mediated amplification of this region from haplotyped PAH chromosomes indicates that the previously reported 4.0-kb HindIII allele contains three of these repeats, while the 4.4-kb HindIII allele contains 12 of these repeats. The 4.2-kb HindIII fragment can contain six, seven, eight, or nine copies of this repeat. These variations permit more detailed analysis of mutant haplotypes 1,more » 5, 6, and, possibly, others. Kindred analysis in phenylketonuria families demonstrates Mendelian segregation of these VNTR alleles, as well as associations between theses alleles and certain PAH mutations. The R261Q mutation, associated with haplotype 1, is associated almost exclusively with an allele containing eight repeats; the R408W mutation, when occurring on a haplotype 1 background, may also be associated with the eight-repeat VNTR allele. Other PAH mutations associated with haplotype 1, R252W and P281L, do not appear to segregate with specific VNTR alleles. The IVS-10 mutation, when associated with haplotype 6, is associated exclusively with an allele containing seven repeats. The combined use of this VNTR system and the existing RFLP haplotype system will increase the performance of prenatal diagnostic tests based on haplotype analysis. In addition, this VNTR may prove useful in studies concerning the origins and distributions of PAH mutations in different human populations. 32 refs., 3 figs., 3 tabs.« less

Cardio‐facio‐cutaneous and Noonan syndromes due to mutations in the RAS/MAPK signalling pathway: genotype–phenotype relationships and overlap with Costello syndrome

PubMed Central

Nava, Caroline; Hanna, Nadine; Michot, Caroline; Pereira, Sabrina; Pouvreau, Nathalie; Niihori, Tetsuya; Aoki, Yoko; Matsubara, Yoichi; Arveiler, Benoit; Lacombe, Didier; Pasmant, Eric; Parfait, Béatrice; Baumann, Clarisse; Héron, Delphine; Sigaudy, Sabine; Toutain, Annick; Rio, Marlène; Goldenberg, Alice; Leheup, Bruno; Verloes, Alain; Cavé, Hélène

2007-01-01

Cardio‐facio‐cutaneous (CFC) syndrome, Noonan syndrome (NS), and Costello syndrome (CS) are clinically related developmental disorders that have been recently linked to mutations in the RAS/MEK/ERK signalling pathway. This study was a mutation analysis of the KRAS, BRAF, MEK1 and MEK2 genes in a total of 130 patients (40 patients with a clinical diagnosis of CFC, 20 patients without HRAS mutations from the French Costello family support group, and 70 patients with NS without PTPN11 or SOS1 mutations). BRAF mutations were found in 14/40 (35%) patients with CFC and 8/20 (40%) HRAS‐negative patients with CS. KRAS mutations were found in 1/40 (2.5%) patients with CFC, 2/20 (10%) HRAS‐negative patients with CS and 4/70 patients with NS (5.7%). MEK1 mutations were found in 4/40 patients with CFC (10%), 4/20 (20%) HRAS‐negative patients with CS and 3/70 (4.3%) patients with NS, and MEK2 mutations in 4/40 (10%) patients with CFC. Analysis of the major phenotypic features suggests significant clinical overlap between CS and CFC. The phenotype associated with MEK mutations seems less severe, and is compatible with normal mental development. Features considered distinctive for CS were also found to be associated with BRAF or MEK mutations. Because of its particular cancer risk, the term “Costello syndrome” should only be used for patients with proven HRAS mutation. These results confirm that KRAS is a minor contributor to NS and show that MEK is involved in some cases of NS, demonstrating a phenotypic continuum between the clinical entities. Although some associated features appear to be characteristic of a specific gene, no simple rule exists to distinguish NS from CFC easily. PMID:17704260

Mutation rate evolution in replicator dynamics.

PubMed

Allen, Benjamin; Rosenbloom, Daniel I Scholes

2012-11-01

The mutation rate of an organism is itself evolvable. In stable environments, if faithful replication is costless, theory predicts that mutation rates will evolve to zero. However, positive mutation rates can evolve in novel or fluctuating environments, as analytical and empirical studies have shown. Previous work on this question has focused on environments that fluctuate independently of the evolving population. Here we consider fluctuations that arise from frequency-dependent selection in the evolving population itself. We investigate how the dynamics of competing traits can induce selective pressure on the rates of mutation between these traits. To address this question, we introduce a theoretical framework combining replicator dynamics and adaptive dynamics. We suppose that changes in mutation rates are rare, compared to changes in the traits under direct selection, so that the expected evolutionary trajectories of mutation rates can be obtained from analysis of pairwise competition between strains of different rates. Depending on the nature of frequency-dependent trait dynamics, we demonstrate three possible outcomes of this competition. First, if trait frequencies are at a mutation-selection equilibrium, lower mutation rates can displace higher ones. Second, if trait dynamics converge to a heteroclinic cycle-arising, for example, from "rock-paper-scissors" interactions-mutator strains succeed against non-mutators. Third, in cases where selection alone maintains all traits at positive frequencies, zero and nonzero mutation rates can coexist indefinitely. Our second result suggests that relatively high mutation rates may be observed for traits subject to cyclical frequency-dependent dynamics.

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

St-Louis, M.; Poudrier, J.; Phaneuf, D.

The deficiency of fumarylacetoacetate hydrolase, the last enzyme in the tyrosine catabolic pathway is the cause of hereditary tyrosinemia type I (HT1), an autosomal recessive disease. The disease has been reported worldwide. The incidence is much higher in two clusters: the Saguenay- Lac St-Jean region (Quebec, Canada) and in Scandinavia. Seven mutations have been reported in the last two years. Here we describe two new missense mutations identified by direct sequencing of PCR products in two HT1 patients, a Norwegian (patient No. 1) and a French-Canadian (patient No. 2). The first mutation consists of a G to A transition atmore » position 337 of the FAH gene which predicts a change from glycine to serine (G337S). The second mutation is an A to G transition at position 381 which predicts a change from arginine to glycine (R381G). Patient No. 1 seems heterozygous for the G337S mutation and for a splice mutation (IVS12+5G{r_arrow}A) which was previously described. Patient No. 2 was also found heterozygous for the R381G mutation and for a rare nonsense mutation (E357X) already reported. In vitro transcription and translation were performed on mutant cDNA to demonstrate the responsibility of these two mutations in causing the decreased amount of FAH detected by Western blot analysis.« less

Disease Mutations in Rab7 Result in Unregulated Nucleotide Exchange and Inappropriate Activation

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

B McCray; E Skordalakes; J Taylor

2011-12-31

Rab GTPases are molecular switches that orchestrate vesicular trafficking, maturation and fusion by cycling between an active, GTP-bound form, and an inactive, GDP-bound form. The activity cycle is coupled to GTP hydrolysis and is tightly controlled by regulatory proteins. Missense mutations of the GTPase Rab7 cause a dominantly inherited axonal degeneration known as Charcot-Marie-Tooth type 2B through an unknown mechanism. We present the 2.8 A crystal structure of GTP-bound L129F mutant Rab7 which reveals normal conformations of the effector binding regions and catalytic site, but an alteration to the nucleotide binding pocket that is predicted to alter GTP binding. Throughmore » extensive biochemical analysis, we demonstrate that disease-associated mutations in Rab7 do not lead to an intrinsic GTPase defect, but permit unregulated nucleotide exchange leading to both excessive activation and hydrolysis-independent inactivation. Consistent with augmented activity, mutant Rab7 shows significantly enhanced interaction with a subset of effector proteins. In addition, dynamic imaging demonstrates that mutant Rab7 is abnormally retained on target membranes. However, we show that the increased activation of mutant Rab7 is counterbalanced by unregulated, GTP hydrolysis-independent membrane cycling. Notably, disease mutations are able to rescue the membrane cycling of a GTPase-deficient mutant. Thus, we demonstrate that disease mutations uncouple Rab7 from the spatial and temporal control normally imposed by regulatory proteins and cause disease not by a gain of novel toxic function, but by misregulation of native Rab7 activity.« less

Copy Number Variants and Exome Sequencing Analysis in Six Pairs of Chinese Monozygotic Twins Discordant for Congenital Heart Disease.

PubMed

Xu, Yuejuan; Li, Tingting; Pu, Tian; Cao, Ruixue; Long, Fei; Chen, Sun; Sun, Kun; Xu, Rang

2017-12-01

Congenital heart disease (CHD) is one of the most common birth defects. More than 200 susceptibility loci have been identified for CHDs, yet a large part of the genetic risk factors remain unexplained. Monozygotic (MZ) twins are thought to be completely genetically identical; however, discordant phenotypes have been found in MZ twins. Recent studies have demonstrated genetic differences between MZ twins. We aimed to test whether copy number variants (CNVs) and/or genetic mutation differences play a role in the etiology of CHDs by using single nucleotide polymorphism (SNP) genotyping arrays and whole exome sequencing of twin pairs discordant for CHDs. Our goal was to identify mutations present only in the affected twins, which could identify novel candidates for CHD susceptibility loci. We present a comprehensive analysis for the CNVs and genetic mutation results of the selected individuals but detected no consistent differences within the twin pairs. Our study confirms that chromosomal structure or genetic mutation differences do not seem to play a role in the MZ twins discordant for CHD.

Common founder mutation in the LDL receptor gene causing familial hypercholesterolaemia in the Icelandic population.

PubMed

Gudnason, V; Sigurdsson, G; Nissen, H; Humphries, S E

1997-01-01

Haplotype analysis in 18 apparently unrelated families with familial hypercholesterolaemia (FH) in Iceland has identified at least five different chromosomes cosegregating with hypercholesterolaemia. The most common haplotype was identified in 11 of the 18 families, indicating a responsible for FH in the Icelandic population. By using single-strand conformation polymorphism (SSCP) and direct sequencing of amplified DNA, we identified a novel mutation (a T to a C) in the second nucleotide in the 5' part of intron 4 in the LDL receptor gene. This mutation was present in approximately 60% of the FH families (10/18), supporting the prediction of a common founder. These families could be traced to a common ancestor in half of the cases by going back no further than the eighteenth century. The mutation was predicted to affect correct splicing of exon 4, and analysis at the cellular level demonstrated an abnormal mRNA containing intron 4 sequence in lymphoblastoid cells from a patient carrying this mutation. Translation of the mRNA would lead to a premature stop codon and a truncated nonfunctional protein of 285 amino acids. The novel sequence change created a new restriction site for the restriction endonuclease NlaIII, and using this assay, 29 unrelated individuals with possible FH attending a lipid clinic for treatment were examined for this mutation. Two individuals in this group of patients were found to be carriers of this mutation, supporting the suggestion of a founder mutation. Using this assay for the detection of FH in the Icelandic population should identify > 60% of these individuals.

Hydrops fetalis and pulmonary lymphangiectasia due to FOXC2 mutation: an autosomal dominant hereditary lymphedema syndrome with variable expression.

PubMed

de Bruyn, Gwendolyn; Casaer, Alexandra; Devolder, Katrien; Van Acker, Geert; Logghe, Hilde; Devriendt, Koen; Cornette, Luc

2012-03-01

Non-immune hydrops fetalis may find its origin within genetically determined lymphedema syndromes, caused by mutations in FOXC2 and SOX-18. We describe a newborn girl, diagnosed with non-immune hydrops fetalis at a gestational age of 30 weeks. Family history revealed the presence of an autosomal dominant late-onset form of lymphedema of the lower limbs in her father, associated with an aberrant implantation of the eyelashes in some individuals. The newborn, hydropic girl suffered from severe pulmonary lymphangiectasia, resulting in terminal respiratory failure at the age of 3 months. Genetic analysis in both the father and the newborn girl demonstrated a heterozygous FOXC2 mutation, i.e., c.939C>A, p.Tyr313X. Her two older sisters are currently asymptomatic and the parents decided not to test them for the FOXC2 mutation. Patients with a mutation in the FOXC2 transcription factor usually show lower limb lymphedema with onset at or after puberty, together with distichiasis. However, the eye manifestations can be very mild and easily overlooked. The association between FOXC2 mutation and neonatal hydrops resulting in terminal respiratory failure is not reported so far. Therefore, in sporadic patients diagnosed with non-immune hydrops fetalis, lymphangiogenic genes should be systematically screened for mutations. In addition, all cases of fetal edema must prompt a thorough analysis of the familial pedigree, in order to detect familial patterns and to facilitate adequate antenatal counseling.

Assessment of cytology based molecular analysis to guide targeted therapy in advanced non-small-cell lung cancer.

PubMed

Li, Wenbin; Zhang, Zhihui; Guo, Lei; Qiu, Tian; Ling, Yun; Cao, Jian; Guo, Huiqin; Zhao, Huan; Li, Lin; Ying, Jianming

2016-02-16

To investigate the use of molecular testing on cytological specimens in selecting advanced non-small cell lung cancer (NSCLC) patients who are adequate for targeted treatment, a total of 137 NSCLC cases were analyzed by fluorescence in situ hybridization (FISH) for anaplastic lymphoma kinase (ALK) rearrangements, and Epidermal growth factor receptor (EGFR), kirsten rat sarcoma viral oncogene homolog (KRAS) mutations were evaluated by quantitative real-time PCR (qRT-PCR) platform combining amplification refractory mutation system (ARMS) primers and TaqMan probes. Cytological specimens included 91 fine-needle aspirates, 5 fibreoptic bronchoscopic derived samples and 41 pleural effusions. Among 137 NSCLCs analyzed for ALK FISH, 16 (11.7%, of 137) were detected to harbor ALK rearrangement. FISH positive cases were all defined as adenocarcinoma (ADC) histologic subtype and the FNA samples showed the highest ALK positive rate (13.2%, 12/91). Of the 9 ALK FISH positive patients who received crizotinib treatment, 8 (88.9%) patients exhibited tumor regression. In addition, 60 (44.8%, of 134) cases were found to harbor EGFR mutations and 22 patients with EGFR sensitive mutations who received gefitinib or erlotinib treatment showed a median PFS of 16.0 months. Mutations of KRAS occurred in 8 (6.0%, of 134) cases and this was mutually exclusive from EGFR mutation. Our results demonstrated that ALK FISH and EGFR, KRAS mutational analysis on cytological specimens are sensitive methods for screening advanced stage NSCLC patients who are adequate for targeted treatment.

Cis-regulatory somatic mutations and gene-expression alteration in B-cell lymphomas.

PubMed

Mathelier, Anthony; Lefebvre, Calvin; Zhang, Allen W; Arenillas, David J; Ding, Jiarui; Wasserman, Wyeth W; Shah, Sohrab P

2015-04-23

With the rapid increase of whole-genome sequencing of human cancers, an important opportunity to analyze and characterize somatic mutations lying within cis-regulatory regions has emerged. A focus on protein-coding regions to identify nonsense or missense mutations disruptive to protein structure and/or function has led to important insights; however, the impact on gene expression of mutations lying within cis-regulatory regions remains under-explored. We analyzed somatic mutations from 84 matched tumor-normal whole genomes from B-cell lymphomas with accompanying gene expression measurements to elucidate the extent to which these cancers are disrupted by cis-regulatory mutations. We characterize mutations overlapping a high quality set of well-annotated transcription factor binding sites (TFBSs), covering a similar portion of the genome as protein-coding exons. Our results indicate that cis-regulatory mutations overlapping predicted TFBSs are enriched in promoter regions of genes involved in apoptosis or growth/proliferation. By integrating gene expression data with mutation data, our computational approach culminates with identification of cis-regulatory mutations most likely to participate in dysregulation of the gene expression program. The impact can be measured along with protein-coding mutations to highlight key mutations disrupting gene expression and pathways in cancer. Our study yields specific genes with disrupted expression triggered by genomic mutations in either the coding or the regulatory space. It implies that mutated regulatory components of the genome contribute substantially to cancer pathways. Our analyses demonstrate that identifying genomically altered cis-regulatory elements coupled with analysis of gene expression data will augment biological interpretation of mutational landscapes of cancers.

Genomic analysis of the origins and evolution of multicentric diffuse lower-grade gliomas.

PubMed

Hayes, Josie; Yu, Yao; Jalbert, Llewellyn E; Mazor, Tali; Jones, Lindsey E; Wood, Matthew D; Walsh, Kyle M; Bengtsson, Henrik; Hong, Chibo; Oberndorfer, Stefan; Roetzer, Thomas; Smirnov, Ivan V; Clarke, Jennifer L; Aghi, Manish K; Chang, Susan M; Nelson, Sarah J; Woehrer, Adelheid; Phillips, Joanna J; Solomon, David A; Costello, Joseph F

2018-04-09

Rare multicentric lower-grade gliomas (LGGs) represent a unique opportunity to study the heterogeneity among distinct tumor foci in a single patient and to infer their origins and parallel patterns of evolution. In this study, we integrate clinical features, histology, and immunohistochemistry for 4 patients with multicentric LGG, arising both synchronously and metachronously. For 3 patients we analyze the phylogeny of the lesions using exome sequencing, including one case with a total of 8 samples from the 2 lesions. One patient was diagnosed with multicentric isocitrate dehydrogenase 1 (IDH1) mutated diffuse astrocytomas harboring distinct IDH1 mutations, R132H and R132C; the latter mutation has been associated with Li-Fraumeni syndrome, which was subsequently confirmed in the patient's germline DNA and shown in additional cases with The Cancer Genome Atlas data. In another patient, phylogenetic analysis of synchronously arising grade II and grade III diffuse astrocytomas demonstrated a single shared mutation, IDH1 R132H, and revealed convergent evolution via non-overlapping mutations in ATRX and TP53. In 2 cases, there was divergent evolution of IDH1-mutated and 1p/19q-codeleted oligodendroglioma and IDH1-mutated and 1p/19q-intact diffuse astrocytoma, occurring synchronously in one case and metachronously in a second. Each tumor in multicentric LGG cases may arise independently or may diverge very early in their development, presenting as genetically and histologically distinct tumors. Comprehensive sampling of these lesions can therefore significantly alter diagnosis and management. Additionally, somatic IDH1 R132C mutation in either multicentric or solitary LGG identifies unsuspected germline TP53 mutation, validating the limited number of published cases.

Mutational And Structural Studies Of The PixD BLUF Output Signal That Affects Light-Regulated Interactions With PixE

PubMed Central

Yuan, Hua; Dragnea, Vladimira; Wu, Qiong; Gardner, Kevin H.; Bauer, Carl E.

2011-01-01

PixD (Slr1694) is a BLUF (blue-light using FAD) photoreceptor used by the cyanobacterium Synechocystis sp. PCC6803 to control phototaxis toward blue light. In this study we probe the involvement of a conserved Tyr8-Gln50-Met93 triad in promoting an output signal upon blue light excitation of the bound flavin. Analysis of acrylamide quenching of Trp91 fluorescence shows that the side chain of this residue remains partially solvent exposed in both the lit and dark states. Mutational analysis demonstrates that substitution mutations at Tyr8 and Gln50 result in the loss of the photocycle while a mutation of Met93 does not appreciably disturb the formation of the light excited state and only minimally accelerates its decay from 5.7 s to 4.5 s. However, mutations in Tyr8, Gln50 and Met93 disrupt the ability of PixD dimers to interact with PixE to form a higher ordered PixD10-PixE5 complex, which is indicative of a lit conformational state. Solution NMR spectroscopy and X-ray crystallographic analyses confirm that a Tyr8 to Phe mutation is locked in a pseudo light excited state revealing flexible areas in PixD that likely constitute part of an output signal upon light excitation of wild type PixD. PMID:21688827

Whole-Genome and Epigenomic Landscapes of Etiologically Distinct Subtypes of Cholangiocarcinoma.

PubMed

Jusakul, Apinya; Cutcutache, Ioana; Yong, Chern Han; Lim, Jing Quan; Huang, Mi Ni; Padmanabhan, Nisha; Nellore, Vishwa; Kongpetch, Sarinya; Ng, Alvin Wei Tian; Ng, Ley Moy; Choo, Su Pin; Myint, Swe Swe; Thanan, Raynoo; Nagarajan, Sanjanaa; Lim, Weng Khong; Ng, Cedric Chuan Young; Boot, Arnoud; Liu, Mo; Ong, Choon Kiat; Rajasegaran, Vikneswari; Lie, Stefanus; Lim, Alvin Soon Tiong; Lim, Tse Hui; Tan, Jing; Loh, Jia Liang; McPherson, John R; Khuntikeo, Narong; Bhudhisawasdi, Vajaraphongsa; Yongvanit, Puangrat; Wongkham, Sopit; Totoki, Yasushi; Nakamura, Hiromi; Arai, Yasuhito; Yamasaki, Satoshi; Chow, Pierce Kah-Hoe; Chung, Alexander Yaw Fui; Ooi, London Lucien Peng Jin; Lim, Kiat Hon; Dima, Simona; Duda, Dan G; Popescu, Irinel; Broet, Philippe; Hsieh, Sen-Yung; Yu, Ming-Chin; Scarpa, Aldo; Lai, Jiaming; Luo, Di-Xian; Carvalho, André Lopes; Vettore, André Luiz; Rhee, Hyungjin; Park, Young Nyun; Alexandrov, Ludmil B; Gordân, Raluca; Rozen, Steven G; Shibata, Tatsuhiro; Pairojkul, Chawalit; Teh, Bin Tean; Tan, Patrick

2017-10-01

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy exhibiting high incidence in countries with endemic liver-fluke infection. We analyzed 489 CCAs from 10 countries, combining whole-genome (71 cases), targeted/exome, copy-number, gene expression, and DNA methylation information. Integrative clustering defined 4 CCA clusters-fluke-positive CCAs (clusters 1/2) are enriched in ERBB2 amplifications and TP53 mutations; conversely, fluke-negative CCAs (clusters 3/4) exhibit high copy-number alterations and PD-1 / PD-L2 expression, or epigenetic mutations ( IDH1/2, BAP1 ) and FGFR / PRKA -related gene rearrangements. Whole-genome analysis highlighted FGFR2 3' untranslated region deletion as a mechanism of FGFR2 upregulation. Integration of noncoding promoter mutations with protein-DNA binding profiles demonstrates pervasive modulation of H3K27me3-associated sites in CCA. Clusters 1 and 4 exhibit distinct DNA hypermethylation patterns targeting either CpG islands or shores-mutation signature and subclonality analysis suggests that these reflect different mutational pathways. Our results exemplify how genetics, epigenetics, and environmental carcinogens can interplay across different geographies to generate distinct molecular subtypes of cancer. Significance: Integrated whole-genome and epigenomic analysis of CCA on an international scale identifies new CCA driver genes, noncoding promoter mutations, and structural variants. CCA molecular landscapes differ radically by etiology, underscoring how distinct cancer subtypes in the same organ may arise through different extrinsic and intrinsic carcinogenic processes. Cancer Discov; 7(10); 1116-35. ©2017 AACR. This article is highlighted in the In This Issue feature, p. 1047 . ©2017 American Association for Cancer Research.

Androgen Receptor Functional Analyses by High Throughput Imaging: Determination of Ligand, Cell Cycle, and Mutation-Specific Effects

PubMed Central

Szafran, Adam T.; Szwarc, Maria; Marcelli, Marco; Mancini, Michael A.

2008-01-01

Background Understanding how androgen receptor (AR) function is modulated by exposure to steroids, growth factors or small molecules can have important mechanistic implications for AR-related disease therapies (e.g., prostate cancer, androgen insensitivity syndrome, AIS), and in the analysis of environmental endocrine disruptors. Methodology/Principal Findings We report the development of a high throughput (HT) image-based assay that quantifies AR subcellular and subnuclear distribution, and transcriptional reporter gene activity on a cell-by-cell basis. Furthermore, simultaneous analysis of DNA content allowed determination of cell cycle position and permitted the analysis of cell cycle dependent changes in AR function in unsynchronized cell populations. Assay quality for EC50 coefficients of variation were 5–24%, with Z' values reaching 0.91. This was achieved by the selective analysis of cells expressing physiological levels of AR, important because minor over-expression resulted in elevated nuclear speckling and decreased transcriptional reporter gene activity. A small screen of AR-binding ligands, including known agonists, antagonists, and endocrine disruptors, demonstrated that nuclear translocation and nuclear “speckling” were linked with transcriptional output, and specific ligands were noted to differentially affect measurements for wild type versus mutant AR, suggesting differing mechanisms of action. HT imaging of patient-derived AIS mutations demonstrated a proof-of-principle personalized medicine approach to rapidly identify ligands capable of restoring multiple AR functions. Conclusions/Significance HT imaging-based multiplex screening will provide a rapid, systems-level analysis of compounds/RNAi that may differentially affect wild type AR or clinically relevant AR mutations. PMID:18978937

Analysis of NCAM helps identify unusual phenotypes of hereditary inclusion-body myopathy.

PubMed

Broccolini, A; Gidaro, T; Tasca, G; Morosetti, R; Rodolico, C; Ricci, E; Mirabella, M

2010-07-20

Hereditary inclusion-body myopathy or distal myopathy with rimmed vacuoles (h-IBM/DMRV) is due to mutations of the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene, which codes for an enzyme of the sialic acid biosynthetic pathway. By Western blot (WB) analysis, we have previously shown that in h-IBM/DMRV muscle, the neural cell adhesion molecule (NCAM) has increased electrophoretic mobility that reflects reduced sialylation of the protein. To identify patients with h-IBM/DMRV with atypical clinical or pathologic phenotype using NCAM analysis and the possible cellular mechanism associated with the overall abnormal sialylation of NCAM observed in this disorder. WB analysis of NCAM was performed on muscle biopsies of 84 patients with an uncharacterized muscle disorder who were divided in the following 2 groups: 1) 46 patients with a proximal muscle weakness in whom the main limb-girdle muscular dystrophy syndromes had been ruled out; and 2) 38 patients with a distal distribution of weakness in whom a neurogenic affection had been excluded. Patients in whom a reduced sialylation of NCAM was suspected were studied for the presence of GNE mutations. In 3 patients, we found that NCAM had increased electrophoretic mobility, thus suggesting an abnormal sialylation of the protein. The genetic study demonstrated that they all carried pathogenic GNE mutations. Further studies demonstrated that hyposialylated NCAM, showing increased electrophoretic mobility on WB, is expressed by nonregenerating fibers in h-IBM/DMRV muscle. WB analysis of NCAM may be instrumental in the identification of h-IBM/DMRV with atypical clinical or pathologic features.

Role of an archaeal PitA transporter in the copper and arsenic resistance of Metallosphaera sedula, an extreme thermoacidophile.

PubMed

McCarthy, Samuel; Ai, Chenbing; Wheaton, Garrett; Tevatia, Rahul; Eckrich, Valerie; Kelly, Robert; Blum, Paul

2014-10-01

Thermoacidophilic archaea, such as Metallosphaera sedula, are lithoautotrophs that occupy metal-rich environments. In previous studies, an M. sedula mutant lacking the primary copper efflux transporter, CopA, became copper sensitive. In contrast, the basis for supranormal copper resistance remained unclear in the spontaneous M. sedula mutant, CuR1. Here, transcriptomic analysis of copper-shocked cultures indicated that CuR1 had a unique regulatory response to metal challenge corresponding to the upregulation of 55 genes. Genome resequencing identified 17 confirmed mutations unique to CuR1 that were likely to change gene function. Of these, 12 mapped to genes with annotated function associated with transcription, metabolism, or transport. These mutations included 7 nonsynonymous substitutions, 4 insertions, and 1 deletion. One of the insertion mutations mapped to pseudogene Msed_1517 and extended its reading frame an additional 209 amino acids. The extended mutant allele was identified as a homolog of Pho4, a family of phosphate symporters that includes the bacterial PitA proteins. Orthologs of this allele were apparent in related extremely thermoacidophilic species, suggesting M. sedula naturally lacked this gene. Phosphate transport studies combined with physiologic analysis demonstrated M. sedula PitA was a low-affinity, high-velocity secondary transporter implicated in copper resistance and arsenate sensitivity. Genetic analysis demonstrated that spontaneous arsenate-resistant mutants derived from CuR1 all underwent mutation in pitA and nonselectively became copper sensitive. Taken together, these results point to archaeal PitA as a key requirement for the increased metal resistance of strain CuR1 and its accelerated capacity for copper bioleaching. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Role of an Archaeal PitA Transporter in the Copper and Arsenic Resistance of Metallosphaera sedula, an Extreme Thermoacidophile

PubMed Central

McCarthy, Samuel; Ai, Chenbing; Wheaton, Garrett; Tevatia, Rahul; Eckrich, Valerie; Kelly, Robert

2014-01-01

Thermoacidophilic archaea, such as Metallosphaera sedula, are lithoautotrophs that occupy metal-rich environments. In previous studies, an M. sedula mutant lacking the primary copper efflux transporter, CopA, became copper sensitive. In contrast, the basis for supranormal copper resistance remained unclear in the spontaneous M. sedula mutant, CuR1. Here, transcriptomic analysis of copper-shocked cultures indicated that CuR1 had a unique regulatory response to metal challenge corresponding to the upregulation of 55 genes. Genome resequencing identified 17 confirmed mutations unique to CuR1 that were likely to change gene function. Of these, 12 mapped to genes with annotated function associated with transcription, metabolism, or transport. These mutations included 7 nonsynonymous substitutions, 4 insertions, and 1 deletion. One of the insertion mutations mapped to pseudogene Msed_1517 and extended its reading frame an additional 209 amino acids. The extended mutant allele was identified as a homolog of Pho4, a family of phosphate symporters that includes the bacterial PitA proteins. Orthologs of this allele were apparent in related extremely thermoacidophilic species, suggesting M. sedula naturally lacked this gene. Phosphate transport studies combined with physiologic analysis demonstrated M. sedula PitA was a low-affinity, high-velocity secondary transporter implicated in copper resistance and arsenate sensitivity. Genetic analysis demonstrated that spontaneous arsenate-resistant mutants derived from CuR1 all underwent mutation in pitA and nonselectively became copper sensitive. Taken together, these results point to archaeal PitA as a key requirement for the increased metal resistance of strain CuR1 and its accelerated capacity for copper bioleaching. PMID:25092032

A single mutation in the GSTe2 gene allows tracking of metabolically based insecticide resistance in a major malaria vector

PubMed Central

2014-01-01

Background Metabolic resistance to insecticides is the biggest threat to the continued effectiveness of malaria vector control. However, its underlying molecular basis, crucial for successful resistance management, remains poorly characterized. Results Here, we demonstrate that the single amino acid change L119F in an upregulated glutathione S-transferase gene, GSTe2, confers high levels of metabolic resistance to DDT in the malaria vector Anopheles funestus. Genome-wide transcription analysis revealed that GSTe2 was the most over-expressed detoxification gene in DDT and permethrin-resistant mosquitoes from Benin. Transgenic expression of GSTe2 in Drosophila melanogaster demonstrated that over-transcription of this gene alone confers DDT resistance and cross-resistance to pyrethroids. Analysis of GSTe2 polymorphism established that the point mutation is tightly associated with metabolic resistance to DDT and its geographical distribution strongly correlates with DDT resistance patterns across Africa. Functional characterization of recombinant GSTe2 further supports the role of the L119F mutation, with the resistant allele being more efficient at metabolizing DDT than the susceptible one. Importantly, we also show that GSTe2 directly metabolizes the pyrethroid permethrin. Structural analysis reveals that the mutation confers resistance by enlarging the GSTe2 DDT-binding cavity, leading to increased DDT access and metabolism. Furthermore, we show that GSTe2 is under strong directional selection in resistant populations, and a restriction of gene flow is observed between African regions, enabling the prediction of the future spread of this resistance. Conclusions This first DNA-based metabolic resistance marker in mosquitoes provides an essential tool to track the evolution of resistance and to design suitable resistance management strategies. PMID:24565444

Prevalence and clinical significance of mediator complex subunit 12 mutations in 362 Han Chinese samples with uterine leiomyoma.

PubMed

Wu, Juan; Zou, Yang; Luo, Yong; Guo, Jiu-Bai; Liu, Fa-Ying; Zhou, Jiang-Yan; Zhang, Zi-Yu; Wan, Lei; Huang, Ou-Ping

2017-07-01

Uterine leiomyomas (ULs) are the most common gynecological benign tumors originating from the myometrium. Prevalent mutations in the mediator complex subunit 12 (MED12) gene have been identified in ULs, and functional evidence has revealed that these mutations may promote the development of ULs. However, whether MED12 mutations are associated with certain clinical characteristics in ULs remains largely unknown. In the present study, the potential mutations of MED12 and its paralogous gene, mediator complex subunit 12-like (MED12L), were screened in 362 UL tumors from Han Chinese patients. A total of 158 out of 362 UL tumors (43.6%) were identified as harboring MED12 somatic mutations, and the majority of these mutations were restricted to the 44th residue. MED12 mutations were also observed in 2 out of 145 (1.4%) adjacent control myometrium. Furthermore, the mutation spectrum of MED12 in the concurrent leiomyomas was noticeably different. Correlation analysis of MED12 mutations with the available clinical features indicated that patients with mutated MED12 tended to have smaller cervical diameters. By contrast, no MED12L mutation was identified in the present samples. In summary, the present study demonstrated the presence of prevalent MED12 somatic mutations in UL samples, and the MED12 mutation was associated with smaller cervical diameters. The low mutation frequency of MED12 in adjacent control myometrium indicated that MED12 mutation may be an early event in the pathogenesis of ULs. Furthermore, MED12 mutation status in concurrent tumors from multiple leiomyomas supported several prior observations that the majority of these tumors arose independently.

Proven germline mosaicism in a father of two children with CHARGE syndrome.

PubMed

Pauli, S; Pieper, L; Häberle, J; Grzmil, P; Burfeind, P; Steckel, M; Lenz, U; Michelmann, H W

2009-05-01

CHARGE syndrome is an autosomal dominant malformation syndrome caused by mutations in the CHD7 gene. The majority of cases are sporadic and only few familial cases have been reported. In these families, mosaicism in one parent, as well as parent- to-child transmission of a CHD7 mutation, has been described. In some further cases, germline mosaicism has been suggested. Here, we report the first case in which germline mosaicism could be demonstrated in a father of two affected children with CHARGE syndrome. The truncating mutation c.7302dupA in exon 34 of the CHD7 gene was found in both affected children but was not detected in parental lymphocytes. However, in DNA extracted from the father's spermatozoa, the c.7302dupA mutation could be identified. Furthermore, mutation analysis of DNA isolated from 59 single spermatozoa revealed that the c.7302dupA mutation occurs in 16 spermatozoa, confirming germline mosaicism in the father of the affected children. This result has a high impact for genetic counselling of the family and for their recurrence risk in further pregnancies.

A novel Phex mutation with defective glycosylation causes hypophosphatemia and rickets in mice.

PubMed

Xiong, Xiwen; Qi, Xin; Ge, Xiaomei; Gu, Pengyu; Zhao, Jing; Zhao, Qingshun; Gao, Xiang

2008-01-01

N-ethyl-N-nitrosourea (ENU) mutagenesis is a phenotype-driven approach with potential to assign function to every locus in the mouse genome. In this article, we describe a new mutation, Pug, as a mouse model for X-linked hypophosphatemic rickets (XLH) in human. Mice carrying the Pug mutation exhibit abnormal phenotypes including growth retardation, hypophosphatemia and decreased bone mineral density (BMD). The new mutation was mapped to X-chromosome between 65.4 cM and 66.6 cM, where Phex gene resides. Sequence analysis revealed a unique T-to-C transition mutation resulting in Phe-to-Ser substitution at amino acid 80 of PHEX protein. In vitro studies of Pug mutation demonstrated that PHEX(pug) was incompletely glycosylated and sequestrated in the endoplasmic reticulum region of cell, whereas wild-type PHEX could be fully glycosylated and transported to the plasma membrane to exert its function as an endopeptidase. Taken together, the Pug mutant directly confirms the role of Phex in phosphate homeostasis and normal skeletal development and may serves as a new disease model of human hypophosphatemic rickets.

Recurrent hotspot mutations in HRAS Q61 and PI3K-AKT pathway genes as drivers of breast adenomyoepitheliomas.

PubMed

Geyer, Felipe C; Li, Anqi; Papanastasiou, Anastasios D; Smith, Alison; Selenica, Pier; Burke, Kathleen A; Edelweiss, Marcia; Wen, Huei-Chi; Piscuoglio, Salvatore; Schultheis, Anne M; Martelotto, Luciano G; Pareja, Fresia; Kumar, Rahul; Brandes, Alissa; Fan, Dan; Basili, Thais; Da Cruz Paula, Arnaud; Lozada, John R; Blecua, Pedro; Muenst, Simone; Jungbluth, Achim A; Foschini, Maria P; Wen, Hannah Y; Brogi, Edi; Palazzo, Juan; Rubin, Brian P; Ng, Charlotte K Y; Norton, Larry; Varga, Zsuzsanna; Ellis, Ian O; Rakha, Emad A; Chandarlapaty, Sarat; Weigelt, Britta; Reis-Filho, Jorge S

2018-05-08

Adenomyoepithelioma of the breast is a rare tumor characterized by epithelial-myoepithelial differentiation, whose genetic underpinning is largely unknown. Here we show through whole-exome and targeted massively parallel sequencing analysis that whilst estrogen receptor (ER)-positive adenomyoepitheliomas display PIK3CA or AKT1 activating mutations, ER-negative adenomyoepitheliomas harbor highly recurrent codon Q61 HRAS hotspot mutations, which co-occur with PIK3CA or PIK3R1 mutations. In two- and three-dimensional cell culture models, forced expression of HRAS Q61R in non-malignant ER-negative breast epithelial cells with or without a PIK3CA H1047R somatic knock-in results in transformation and the acquisition of the cardinal features of adenomyoepitheliomas, including the expression of myoepithelial markers, a reduction in E-cadherin expression, and an increase in AKT signaling. Our results demonstrate that adenomyoepitheliomas are genetically heterogeneous, and qualify mutations in HRAS, a gene whose mutations are vanishingly rare in common-type breast cancers, as likely drivers of ER-negative adenomyoepitheliomas.

Novel mutation in ABCC6 gene in a Japanese pedigree with pseudoxanthoma elasticum and retinitis pigmentosa.

PubMed

Yoshida, S; Honda, M; Yoshida, A; Nakao, S; Goto, Y; Nakamura, T; Fujisawa, K; Ishibashi, T

2005-02-01

To report a novel mutation of the ABCC6 gene in a Japanese family that had a case of pseudoxanthoma elasticum (PXE) another with PXE and retinitis pigmentosa. Ophthalmologic examinations were performed, and the ABCC6 gene was analysed by direct genomic sequencing. Fundus examinations of the 48-year-old proband disclosed angioid streaks and a peud'orange appearance of the retina of the both eyes, whereas both of his 25- and 20-year-old daughters had pigmentary degeneration and angioid streaks. In the sibilings, the mixed cone-rod ERG was almost nondetectable, whereas that of the proband was well-preserved. Molecular genetic analysis revealed that the proband has a homozygous nonsense mutation at the 595 bp in the ABCC6, and the siblings were heterozygous for the same mutation. This mutation was not detected in Japanese subjects in the JSNP database (http://snp.ims.u-tokyo.ac.jp/). Our results demonstrated an association between a novel mutation in the ABCC6 gene and PXE in a Japanese family.

Lifetime exercise intolerance with lactic acidosis as key manifestation of novel compound heterozygous ACAD9 mutations causing complex I deficiency.

PubMed

Schrank, Bertold; Schoser, Benedikt; Klopstock, Thomas; Schneiderat, Peter; Horvath, Rita; Abicht, Angela; Holinski-Feder, Elke; Augustis, Sarunas

2017-05-01

We report a 36-year-old female having lifetime exercise intolerance and lactic acidosis with nausea associated with novel compound heterozygous Acyl-CoA dehydrogenase 9 gene (ACAD9) mutations (p.Ala390Thr and p.Arg518Cys). ACAD9 is an assembly factor for the mitochondrial respiratory chain complex I. ACAD9 mutations are recognized as frequent causes of complex I deficiency. Our patient presented with exercise intolerance, rapid fatigue, and nausea since early childhood. Mild physical workload provoked the occurrence of nausea and vomiting repeatedly. Her neurological examination, laboratory findings and muscle biopsy demonstrated no abnormalities. A bicycle spiroergometry provoked significant lactic acidosis during and following exercise pointing towards a mitochondrial disorder. Subsequently, the analysis of respiratory chain enzyme activities in muscle revealed severe isolated complex I deficiency. Candidate gene sequencing revealed two novel heterozygous ACAD9 mutations. This patient report expands the mutational and phenotypic spectrum of diseases associated with mutations in ACAD9. Copyright © 2017 Elsevier B.V. All rights reserved.

Transmembrane Domains of NS2B Contribute to both Viral RNA Replication and Particle Formation in Japanese Encephalitis Virus.

PubMed

Li, Xiao-Dan; Deng, Cheng-Lin; Ye, Han-Qing; Zhang, Hong-Lei; Zhang, Qiu-Yan; Chen, Dong-Dong; Zhang, Pan-Tao; Shi, Pei-Yong; Yuan, Zhi-Ming; Zhang, Bo

2016-06-15

Flavivirus nonstructural protein 2B (NS2B) is a transmembrane protein that functions as a cofactor for viral NS3 protease. The cytoplasmic region (amino acids 51 to 95) alone of NS2B is sufficient for NS3 protease activity, whereas the role of transmembrane domains (TMDs) remains obscure. Here, we demonstrate for the first time that flavivirus NS2B plays a critical role in virion assembly. Using Japanese encephalitis virus (JEV) as a model, we performed a systematic mutagenesis at the flavivirus conserved residues within the TMDs of NS2B. As expected, some mutations severely attenuated (L38A and R101A) or completely destroyed (G12L) viral RNA synthesis. Interestingly, two mutations (G37L and P112A) reduced viral RNA synthesis and blocked virion assembly. None of the mutations affected NS2B-NS3 protease activity. Because mutations G37L and P112A affected virion assembly, we selected revertant viruses for these two mutants. For mutant G37L, replacement with G37F, G37H, G37T, or G37S restored virion assembly. For mutant P112A, insertion of K at position K127 (leading to K127KK) of NS2B rescued virion assembly. A biomolecular fluorescent complementation (BiFC) analysis demonstrated that (i) mutation P112A selectively weakened NS2B-NS2A interaction and (ii) the adaptive mutation K127KK restored NS2B-NS2A interaction. Collectively, our results demonstrate that, in addition to being a cofactor for NS3 protease, flavivirus NS2B also functions in viral RNA replication, as well as virion assembly. Many flaviviruses are important human pathogens. Understanding the molecular mechanisms of the viral infection cycle is essential for vaccine and antiviral development. In this study, we demonstrate that the TMDs of JEV NS2B participate in both viral RNA replication and virion assembly. A viral genetic study and a BiFC assay demonstrated that interaction between NS2B and NS2A may participate in modulating viral assembly in the flavivirus life cycle. Compensatory-mutation analysis confirmed that there was a correlation between viral assembly and NS2B-NS2A interaction. TMDs of NS2B may serve as novel antiviral targets to prevent flavivirus infection, and the structure determination of NS2B will help us to understand the functional mechanism of NS2B in viral RNA replication and assembly. The results have uncovered a new function of flavivirus NS2B in virion assembly, possibly through interaction with the NS2A protein. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

FGFR3 mutation causes abnormal membranous ossification in achondroplasia.

PubMed

Di Rocco, Federico; Biosse Duplan, Martin; Heuzé, Yann; Kaci, Nabil; Komla-Ebri, Davide; Munnich, Arnold; Mugniery, Emilie; Benoist-Lasselin, Catherine; Legeai-Mallet, Laurence

2014-06-01

FGFR3 gain-of-function mutations lead to both chondrodysplasias and craniosynostoses. Achondroplasia (ACH), the most frequent dwarfism, is due to an FGFR3-activating mutation which results in impaired endochondral ossification. The effects of the mutation on membranous ossification are unknown. Fgfr3(Y367C/+) mice mimicking ACH and craniofacial analysis of patients with ACH and FGFR3-related craniosynostoses provide an opportunity to address this issue. Studying the calvaria and skull base, we observed abnormal cartilage and premature fusion of the synchondroses leading to modifications of foramen magnum shape and size in Fgfr3(Y367C/+) mice, ACH and FGFR3-related craniosynostoses patients. Partial premature fusion of the coronal sutures and non-ossified gaps in frontal bones were also present in Fgfr3(Y367C/+) mice and ACH patients. Our data provide strong support that not only endochondral ossification but also membranous ossification is severely affected in ACH. Demonstration of the impact of FGFR3 mutations on craniofacial development should initiate novel pharmacological and surgical therapeutic approaches.

Comprehensive molecular profiling of 718 Multiple Myelomas reveals significant differences in mutation frequencies between African and European descent cases

PubMed Central

Christofferson, Austin; Aldrich, Jessica; Jewell, Scott; Kittles, Rick A.; Derome, Mary; Craig, David Wesley; Carpten, John D.

2017-01-01

Multiple Myeloma (MM) is a plasma cell malignancy with significantly greater incidence and mortality rates among African Americans (AA) compared to Caucasians (CA). The overall goal of this study is to elucidate differences in molecular alterations in MM as a function of self-reported race and genetic ancestry. Our study utilized somatic whole exome, RNA-sequencing, and correlated clinical data from 718 MM patients from the Multiple Myeloma Research Foundation CoMMpass study Interim Analysis 9. Somatic mutational analyses based upon self-reported race corrected for ancestry revealed significant differences in mutation frequency between groups. Of interest, BCL7A, BRWD3, and AUTS2 demonstrate significantly higher mutation frequencies among AA cases. These genes are all involved in translocations in B-cell malignancies. Moreover, we detected a significant difference in mutation frequency of TP53 and IRF4 with frequencies higher among CA cases. Our study provides rationale for interrogating diverse tumor cohorts to best understand tumor genomics across populations. PMID:29166413

Activation of tyrosine kinases by mutation of the gatekeeper threonine

PubMed Central

Azam, Mohammad; Seeliger, Markus A; Gray, Nathanael S; Kuriyan, John; Daley, George Q

2008-01-01

Protein kinases targeted by small-molecule inhibitors develop resistance through mutation of the ‘gatekeeper’ threonine residue of the active site. Here we show that the gatekeeper mutation in the cellular forms of c-ABL, c-SRC, platelet-derived growth factor receptor-α and -β, and epidermal growth factor receptor activates the kinase and promotes malignant transformation of BaF3 cells. Structural analysis reveals that a network of hydrophobic interactions—the hydrophobic spine—characteristic of the active kinase conformation is stabilized by the gatekeeper substitution. Substitution of glycine for the residues constituting the spine disrupts the hydrophobic connectivity and inactivates the kinase. Furthermore, a small-molecule inhibitor that maximizes complementarity with the dismantled spine (compound 14) inhibits the gatekeeper mutation of BCR-ABL-T315I. These results demonstrate that mutation of the gatekeeper threonine is a common mechanism of activation for tyrosine kinases and provide structural insights to guide the development of next-generation inhibitors. PMID:18794843

Structure of the human MSH2 locus and analysis of two Muir-Torre kindreds for msh2 mutations

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

Kolodner, R.D.; Lipford, J.; Kane, M.F.

1994-12-01

Hereditary nonpolyposis colorectal carcinoma (HNPCC) is a major cancer susceptibility syndrome known to be caused by inheritance of mutations in genes such as hMSH2 and hMLH1, which encode components of a DNA mismatch repair system. The MSH2 genomic locus has been cloned and shown to cover {approximately}73 kb of genomic DNA and to contain 16 exons. The sequence of all of the intron-exon junctions has been determined and used to develop methods for analyzing each MSH2 exon for mutations. These methods have been used to analyze two large HNPCC kindreds exhibiting features of the Muir-Torre syndrome and demonstrate that cancermore » susceptibility is due to the inheritance of a frameshift mutation in the MSH2 gene in one family and a nonsense mutation in the MSH2 gene in the other family. 59 refs., 5 figs., 1 tab.« less

Hypomorphic mutation in mouse Nppc gene causes retarded bone growth due to impaired endochondral ossification

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

Tsuji, Takehito; Kondo, Eri; Yasoda, Akihiro

2008-11-07

Long bone abnormality (lbab/lbab) is a spontaneous mutant mouse characterized by dwarfism with shorter long bones. A missense mutation was reported in the Nppc gene, which encodes C-type natriuretic peptide (CNP), but it has not been confirmed whether this mutation is responsible for the dwarf phenotype. To verify that the mutation causes the dwarfism of lbab/lbab mice, we first investigated the effect of CNP in lbab/lbab mice. By transgenic rescue with chondrocyte-specific expression of CNP, the dwarf phenotype in lbab/lbab mice was completely compensated. Next, we revealed that CNP derived from the lbab allele retained only slight activity to inducemore » cGMP production through its receptor. Histological analysis showed that both proliferative and hypertrophic zones of chondrocytes in the growth plate of lbab/lbab mice were markedly reduced. Our results demonstrate that lbab/lbab mice have a hypomorphic mutation in the Nppc gene that is responsible for dwarfism caused by impaired endochondral ossification.« less

Analysis of the Afrikaner mutation in exon 9 of the low-density lipoprotein receptor gene in a large Dutch kindred suffering from familial hypercholesterolaemia.

PubMed

Defesche, J C; Lansberg, P J; Reymer, P W; Lamping, R J; Kastelein, J J

1993-02-01

Familial hypercholesterolaemia (FH) is the most common genetic metabolic disorder, affecting about 1 in 500 persons in the general population. With novel techniques, it is possible to identify the molecular defects underlying FH in the gene coding for the low-density lipoprotein (LDL) receptor, thereby confirming the diagnosis of FH. In this study we present a large family with a specific mutation in exon 9 of the LDL-receptor gene (an Afrikaner mutation) and we demonstrate that by a large-scale case-finding study in this family, carriers of such a mutation can be detected. Of 63 family members, 13 were shown to be at risk for cardiovascular disease as judged by their lipoprotein profile or the presence of the Afrikaner mutation. Two persons were detected, affected with a dyslipidaemia other than FH. Medical management was initiated in order to reduce the high cardiovascular risk associated with this disorder.

Different mutation patterns of mitochondrial DNA displacement-loop in hepatocellular carcinomas induced by N-nitrosodiethylamine and a choline-deficient l-amino acid-defined diet in rats.

PubMed

Onishi, Mariko; Sokuza, Yui; Nishikawa, Tomoki; Mori, Chiharu; Uwataki, Kimiko; Honoki, Kanya; Tsujiuchi, Toshifumi

2007-10-12

Mutations of the mitochondria DNA (mtDNA) displacement loop (D-loop) were investigated to clarify different changes of exogenous and endogenous liver carcinogenesis in rats. We induced hepatocellular carcinomas (HCCs) in rats with N-nitrosodiethylamine (DEN) and a choline-deficient l-amino acid-defined (CDAA) diet. DNAs were extracted from 10 HCCs induced by DEN and 10 HCCs induced by the CDAA diet. To identify mutations in mtDNA D-loop, polymerase chain reaction (PCR)-single strand conformation polymorphism (SSCP) analysis, followed by nucleotide sequencing, was performed. Mutations were detected in 5 out of 10 HCCs (50%) induced by DEN. Four out of 5 mutations were G/C to A/T transitions at positions 15707, 15717, 15930, and 16087, and one T/A to C/G transition at position 15559. By contrast, no mutations were found in 10 HCCs induced by the CDAA diet. These results demonstrated that mutations in mtDNA D-loop occur in rat HCCs induced by DEN but not by the CDAA diet, suggesting that mtDNA D-loop is a target of exogenous liver carcinogenesis in rats.

Diploid yeast cells yield homozygous spontaneous mutations

NASA Technical Reports Server (NTRS)

Esposito, M. S.; Bruschi, C. V.; Brushi, C. V. (Principal Investigator)

1993-01-01

A leucine-requiring hybrid of Saccharomyces cerevisiae, homoallelic at the LEU1 locus (leu1-12/leu1-12) and heterozygous for three chromosome-VII genetic markers distal to the LEU1 locus, was employed to inquire: (1) whether spontaneous gene mutation and mitotic segregation of heterozygous markers occur in positive nonrandom association and (2) whether homozygous LEU1/LEU1 mutant diploids are generated. The results demonstrate that gene mutation of leu1-12 to LEU1 and mitotic segregation of heterozygous chromosome-VII markers occur in strong positive nonrandom association, suggesting that the stimulatory DNA lesion is both mutagenic and recombinogenic. In addition, genetic analysis of diploid Leu+ revertants revealed that approximately 3% of mutations of leu1-12 to LEU1 result in LEU1/LEU1 homozygotes. Red-white sectored Leu+ colonies exhibit genotypes that implicate post-replicational chromatid breakage and exchange near the site of leu1-12 reversion, chromosome loss, and subsequent restitution of diploidy, in the sequence of events leading to mutational homozygosis. By analogy, diploid cell populations can yield variants homozygous for novel recessive gene mutations at biologically significant rates. Mutational homozygosis may be relevant to both carcinogenesis and the evolution of asexual diploid organisms.

A Groupwise Association Test for Rare Mutations Using a Weighted Sum Statistic

PubMed Central

Madsen, Bo Eskerod; Browning, Sharon R.

2009-01-01

Resequencing is an emerging tool for identification of rare disease-associated mutations. Rare mutations are difficult to tag with SNP genotyping, as genotyping studies are designed to detect common variants. However, studies have shown that genetic heterogeneity is a probable scenario for common diseases, in which multiple rare mutations together explain a large proportion of the genetic basis for the disease. Thus, we propose a weighted-sum method to jointly analyse a group of mutations in order to test for groupwise association with disease status. For example, such a group of mutations may result from resequencing a gene. We compare the proposed weighted-sum method to alternative methods and show that it is powerful for identifying disease-associated genes, both on simulated and Encode data. Using the weighted-sum method, a resequencing study can identify a disease-associated gene with an overall population attributable risk (PAR) of 2%, even when each individual mutation has much lower PAR, using 1,000 to 7,000 affected and unaffected individuals, depending on the underlying genetic model. This study thus demonstrates that resequencing studies can identify important genetic associations, provided that specialised analysis methods, such as the weighted-sum method, are used. PMID:19214210

Farewell to GBM-O: Genomic and transcriptomic profiling of glioblastoma with oligodendroglioma component reveals distinct molecular subgroups.

PubMed

Hinrichs, Benjamin H; Newman, Scott; Appin, Christina L; Dunn, William; Cooper, Lee; Pauly, Rini; Kowalski, Jeanne; Rossi, Michael R; Brat, Daniel J

2016-01-13

Glioblastoma with oligodendroglioma component (GBM-O) was recognized as a histologic pattern of glioblastoma (GBM) by the World Health Organization (WHO) in 2007 and is distinguished by the presence of oligodendroglioma-like differentiation. To better understand the genetic underpinnings of this morphologic entity, we performed a genome-wide, integrated copy number, mutational and transcriptomic analysis of eight (seven primary, primary secondary) cases. Three GBM-O samples had IDH1 (p.R132H) mutations; two of these also demonstrated 1p/19q co-deletion and had a proneural transcriptional profile, a molecular signature characteristic of oligodendroglioma. The additional IDH1 mutant tumor lacked 1p/19q co-deletion, harbored a TP53 mutation, and overall, demonstrated features most consistent with IDH mutant (secondary) GBM. Finally, five tumors were IDH wild-type (IDHwt) and had chromosome seven gains, chromosome 10 losses, and homozygous 9p deletions (CDKN2A), alterations typical of IDHwt (primary) GBM. IDHwt GBM-Os also demonstrated EGFR and PDGFRA amplifications, which correlated with classical and proneural expression subtypes, respectively. Our findings demonstrate that GBM-O is composed of three discrete molecular subgroups with characteristic mutations, copy number alterations and gene expression patterns. Despite displaying areas that morphologically resemble oligodendroglioma, the current results indicate that morphologically defined GBM-O does not correspond to a particular genetic signature, but rather represents a collection of genetically dissimilar entities. Ancillary testing, especially for IDH and 1p/19q, should be used for determining these molecular subtypes.

Diagnosis of autosomal dominant polycystic kidney disease using efficient PKD1 and PKD2 targeted next-generation sequencing.

PubMed

Trujillano, Daniel; Bullich, Gemma; Ossowski, Stephan; Ballarín, José; Torra, Roser; Estivill, Xavier; Ars, Elisabet

2014-09-01

Molecular diagnostics of autosomal dominant polycystic kidney disease (ADPKD) relies on mutation screening of PKD1 and PKD2, which is complicated by extensive allelic heterogeneity and the presence of six highly homologous sequences of PKD1. To date, specific sequencing of PKD1 requires laborious long-range amplifications. The high cost and long turnaround time of PKD1 and PKD2 mutation analysis using conventional techniques limits its widespread application in clinical settings. We performed targeted next-generation sequencing (NGS) of PKD1 and PKD2. Pooled barcoded DNA patient libraries were enriched by in-solution hybridization with PKD1 and PKD2 capture probes. Bioinformatics analysis was performed using an in-house developed pipeline. We validated the assay in a cohort of 36 patients with previously known PKD1 and PKD2 mutations and five control individuals. Then, we used the same assay and bioinformatics analysis in a discovery cohort of 12 uncharacterized patients. We detected 35 out of 36 known definitely, highly likely, and likely pathogenic mutations in the validation cohort, including two large deletions. In the discovery cohort, we detected 11 different pathogenic mutations in 10 out of 12 patients. This study demonstrates that laborious long-range PCRs of the repeated PKD1 region can be avoided by in-solution enrichment of PKD1 and PKD2 and NGS. This strategy significantly reduces the cost and time for simultaneous PKD1 and PKD2 sequence analysis, facilitating routine genetic diagnostics of ADPKD.

An application of LOH analysis for detecting the genetic influences of space environmental radiation

NASA Astrophysics Data System (ADS)

Yatagai, F.; Umebayashi, Y.; Honma, M.; Abe, T.; Suzuki, H.; Shimazu, T.; Ishioka, N.; Iwaki, M.

To detect the genetic influence of space environmental radiation at the chromosome level we proposed an application of loss of heterozygosity LOH analysis system for the mutations induced in human lymphoblastoid TK6 cells Surprisingly we succeeded the mutation detection in the frozen dells which were exposed to a low-dose 10 cGy of carbon-ion beam irradiation Mutation assays were performed within a few days or after about one month preservation at --80 r C following irradiation The results showed an increase in mutation frequency at the thymidine kinase TK gene locus 1 6-fold 2 5 X 10 -6 to 3 9 X 10 -6 and 2 1-fold 2 5 X 10 -6 to 5 3 X 10 -6 respectively Although the relative distributions of mutation classes were not changed by the radiation exposure in either assay an interesting characteristic was detected using this LOH analysis system two TK locus markers and eleven microsatellite loci spanning chromosome 17 The radiation-specific patterns of interstitial deletions were observed in the hemizygous LOH mutants which were considered as a result of end-joining repair of carbon ion-induced DNA double-strand breaks These results clearly demonstrate that this analysis can be used for the detection of low-dose ionizing radiation effects in the frozen cells In addition we performed so called adaptive response experiments in which TK6 cells were pre-irradiated with low-dose 2 5 sim 10 cGy of X-ray and then exposed to challenging dose 2Gy of X-rays Interestingly the

Mutational analysis of the myelin protein zero (MPZ) gene associated with Charcot-Marie-Tooth neuropathy type 1B

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

Roa, B.B.; Warner, L.E.; Lupski, J.R.

1994-09-01

The MPZ gene that maps to chromosome 1q22q23 encodes myelin protein zero, which is the most abundant peripheral nerve myelin protein that functions as a homophilic adhesion molecule in myelin compaction. Association of the MPZ gene with the dysmyelinating peripheral neuropathies Charcot-Marie-Tooth disease type 1B (CMT1B) and the more severe Dejerine-Sottas syndrome (DSS) was previously demonstrated by MPZ mutations identified in CMT1B and in rare DSS patients. In this study, the coding region of the MPZ gene was screened for mutations in a cohort of 74 unrelated patients with either CMT type 1 or DSS who do not carry themore » most common CMT1-associated molecular lesion of a 1.5 Mb DNA duplication on 17p11.2-p12. Heteroduplex analysis detected base mismatches in ten patients that were distributed over three exons of MPZ. Direct sequencing of PCR-amplified genomic DNA identified a de novo MPZ mutation associated with CMT1B that predicts an Ile(135)Thr substitution. This finding further confirms the role of MPZ in the CMT1B disease process. In addition, two polymorphisms were identified within the Gly(200) and Ser(228) codons that do not alter the respective amino acid residues. A fourth base mismatch in MPZ exon 3 detected by heteroduplex analysis is currently being characterized by direct sequence determination. Previously, four unrelated patients in this same cohort were found to have unique point mutations in the coding region of the PMP22 gene. The collective findings on CMT1 point mutations could suggest that regulatory region mutations, and possibly mutations in CMT gene(s) apart from the MPZ, PMP22 and Cx32 genes identified thus far, may prove to be significant for a number of CMT1 cases that do not involve DNA duplication.« less

Single-Molecule Counting of Point Mutations by Transient DNA Binding

NASA Astrophysics Data System (ADS)

Su, Xin; Li, Lidan; Wang, Shanshan; Hao, Dandan; Wang, Lei; Yu, Changyuan

2017-03-01

High-confidence detection of point mutations is important for disease diagnosis and clinical practice. Hybridization probes are extensively used, but are hindered by their poor single-nucleotide selectivity. Shortening the length of DNA hybridization probes weakens the stability of the probe-target duplex, leading to transient binding between complementary sequences. The kinetics of probe-target binding events are highly dependent on the number of complementary base pairs. Here, we present a single-molecule assay for point mutation detection based on transient DNA binding and use of total internal reflection fluorescence microscopy. Statistical analysis of single-molecule kinetics enabled us to effectively discriminate between wild type DNA sequences and single-nucleotide variants at the single-molecule level. A higher single-nucleotide discrimination is achieved than in our previous work by optimizing the assay conditions, which is guided by statistical modeling of kinetics with a gamma distribution. The KRAS c.34 A mutation can be clearly differentiated from the wild type sequence (KRAS c.34 G) at a relative abundance as low as 0.01% mutant to WT. To demonstrate the feasibility of this method for analysis of clinically relevant biological samples, we used this technology to detect mutations in single-stranded DNA generated from asymmetric RT-PCR of mRNA from two cancer cell lines.

Genetic heritage of the Old Order Mennonites of southeastern Pennsylvania.

PubMed

Puffenberger, E G

2003-08-15

The Old Order Mennonites of southeastern Pennsylvania are a religious isolate with origins in 16th-century Switzerland. The Swiss Mennonites immigrated to Pennsylvania over a 50-year period in the early 18th century. The history of this population in the United States provides insight into the increased incidence of several genetic diseases, most notably maple syrup urine disease (MSUD), Hirschsprung disease (HSCR), and congenital nephrotic syndrome. A comparison between the Old Order Mennonites and the Old Order Amish demonstrates the unique genetic heritage of each group despite a common religious and geographic history. Unexpectedly, several diseases in both groups demonstrate allelic and/or locus heterogeneity. The population genetics of the 1312T --> A BCKDHA gene mutation, which causes classical MSUD, are presented in detail. The incidence of MSUD in the Old Order Mennonites is estimated to be 1/358 births, yielding a corrected carrier frequency of 7.96% and a mutation allele frequency of 4.15%. Analysis of the population demonstrates that repeated cycles of sampling effects, population bottlenecks, and subsequent genetic drift were important in shaping the current allele frequencies. A linkage disequilibrium analysis of 1312T --> A mutation haplotypes is provided and discussed in the context of the known genealogical history of the population. Finally, data from microsatellite marker genotyping within the Old Order Mennonite population are provided that show a significant but modest decrease in genetic diversity and elevated levels of background linkage disequilibrium. Copyright 2003 Wiley-Liss, Inc.

Integrated genomic classification of melanocytic tumors of the central nervous system using mutation analysis, copy number alterations and DNA methylation profiling.

PubMed

Griewank, Klaus; Koelsche, Christian; van de Nes, Johannes A P; Schrimpf, Daniel; Gessi, Marco; Möller, Inga; Sucker, Antje; Scolyer, Richard A; Buckland, Michael E; Murali, Rajmohan; Pietsch, Torsten; von Deimling, Andreas; Schadendorf, Dirk

2018-06-11

In the central nervous system, distinguishing primary leptomeningeal melanocytic tumors from melanoma metastases and predicting their biological behavior solely using histopathologic criteria can be challenging. We aimed to assess the diagnostic and prognostic value of integrated molecular analysis. Targeted next-generation-sequencing, array-based genome-wide methylation analysis and BAP1 immunohistochemistry was performed on the largest cohort of central nervous system melanocytic tumors analyzed to date, incl. 47 primary tumors of the central nervous system, 16 uveal melanomas. 13 cutaneous melanoma metastasis and 2 blue nevus-like melanomas. Gene mutation, DNA-methylation and copy-number profiles were correlated with clinicopathological features. Combining mutation, copy-number and DNA-methylation profiles clearly distinguished cutaneous melanoma metastases from other melanocytic tumors. Primary leptomeningeal melanocytic tumors, uveal melanomas and blue nevus-like melanoma showed common DNA-methylation, copy-number alteration and gene mutation signatures. Notably, tumors demonstrating chromosome 3 monosomy and BAP1 alterations formed a homogeneous subset within this group. Integrated molecular profiling aids in distinguishing primary from metastatic melanocytic tumors of the central nervous system. Primary leptomeningeal melanocytic tumors, uveal melanoma and blue nevus-like melanoma share molecular similarity with chromosome 3 and BAP1 alterations markers of poor prognosis. Copyright ©2018, American Association for Cancer Research.

A reverse genetic approach identifies an ancestral frameshift mutation in RP1 causing recessive progressive retinal degeneration in European cattle breeds.

PubMed

Michot, Pauline; Chahory, Sabine; Marete, Andrew; Grohs, Cécile; Dagios, Dimitri; Donzel, Elise; Aboukadiri, Abdelhak; Deloche, Marie-Christine; Allais-Bonnet, Aurélie; Chambrial, Matthieu; Barbey, Sarah; Genestout, Lucie; Boussaha, Mekki; Danchin-Burge, Coralie; Fritz, Sébastien; Boichard, Didier; Capitan, Aurélien

2016-08-10

Domestication and artificial selection have resulted in strong genetic drift, relaxation of purifying selection and accumulation of deleterious mutations. As a consequence, bovine breeds experience regular outbreaks of recessive genetic defects which might represent only the tip of the iceberg since their detection depends on the observation of affected animals with distinctive symptoms. Thus, recessive mutations resulting in embryonic mortality or in non-specific symptoms are likely to be missed. The increasing availability of whole-genome sequences has opened new research avenues such as reverse genetics for their investigation. Our aim was to characterize the genetic load of 15 European breeds using data from the 1000 bull genomes consortium and prove that widespread harmful mutations remain to be detected. We listed 2489 putative deleterious variants (in 1923 genes) segregating at a minimal frequency of 5 % in at least one of the breeds studied. Gene enrichment analysis showed major enrichment for genes related to nervous, visual and auditory systems, and moderate enrichment for genes related to cardiovascular and musculoskeletal systems. For verification purposes, we investigated the phenotypic consequences of a frameshift variant in the retinitis pigmentosa-1 gene segregating in several breeds and at a high frequency (27 %) in Normande cattle. As described in certain human patients, clinical and histological examination revealed that this mutation causes progressive degeneration of photoreceptors leading to complete blindness in homozygotes. We established that the deleterious allele was even more frequent in the Normande breed before 1975 (>40 %) and has been progressively counter-selected likely because of its associated negative effect on udder morphology. Finally, using identity-by-descent analysis we demonstrated that this mutation resulted from a unique ancestral event that dates back to ~2800 to 4000 years. We provide a list of mutations that likely represent a substantial part of the genetic load of domestication in European cattle. We demonstrate that they accumulated non-randomly and that genes related to cognition and sensory functions are particularly affected. Finally, we describe an ancestral deleterious variant segregating in different breeds causing progressive retinal degeneration and irreversible blindness in adult animals.

Distinct mutations with different inheritance mode caused similar retinal dystrophies in one family: a demonstration of the importance of genetic annotations in complicated pedigrees.

PubMed

Chen, Xue; Sheng, Xunlun; Liu, Yani; Li, Zili; Sun, Xiantao; Jiang, Chao; Qi, Rui; Yuan, Shiqin; Wang, Xuhui; Zhou, Ge; Zhen, Yanyan; Xie, Ping; Liu, Qinghuai; Yan, Biao; Zhao, Chen

2018-05-29

Retinitis pigmentosa (RP) is the most common form of inherited retinal dystrophy presenting remarkable genetic heterogeneity. Genetic annotations would help with better clinical assessments and benefit gene therapy, and therefore should be recommended for RP patients. This report reveals the disease causing mutations in two RP pedigrees with confusing inheritance patterns using whole exome sequencing (WES). Twenty-five participants including eight patients from two families were recruited and received comprehensive ophthalmic evaluations. WES was applied for mutation identification. Bioinformatics annotations, intrafamilial co-segregation tests, and in silico analyses were subsequently conducted for mutation verification. All patients were clinically diagnosed with RP. The first family included two siblings born to parents with consanguineous marriage; however, no potential pathogenic variant was found shared by both patients. Further analysis revealed that the female patient carried a recurrent homozygous C8ORF37 p.W185*, while the male patient had hemizygous OFD1 p.T120A. The second family was found to segregate mutations in two genes, TULP1 and RP1. Two patients born to consanguineous marriage carried homozygous TULP1 p.R419W, while a recurrent heterozygous RP1 p.L762Yfs*17 was found in another four patients presenting an autosomal dominant inheritance pattern. Crystal structural analysis further indicated that the substitution from arginine to tryptophan at the highly conserved residue 419 of TULP1 could lead to the elimination of two hydrogen bonds between residue 419 and residues V488 and S534. All four genes, including C8ORF37, OFD1, TULP1 and RP1, have been previously implicated in RP etiology. Our study demonstrates the coexistence of diverse inheritance modes and mutations affecting distinct disease causing genes in two RP families with consanguineous marriage. Our data provide novel insights into assessments of complicated pedigrees, reinforce the genetic complexity of RP, and highlight the need for extensive molecular evaluations in such challenging families with diverse inheritance modes and mutations.

An autopsy case of leptomeningeal amyloidosis associated with transthyretin Gly47Arg mutation.

PubMed

Uehara, Takuya; Kakuda, Keita; Sumi-Akamaru, Hisae; Yamauchi, Amane; Mochizuki, Hideki; Naka, Takashi

2016-11-29

We report the case of a 47-year-old woman with a 4-year history of progressive numbness in the distal portions of both her lower limbs, diarrhea alternating with periods of constipation, and orthostatic syncope. She demonstrated sensory dominant neuropathy and dysautonomia including orthostatic hypotension, paralytic ileus, and urinary retention. A systemic mutation analysis revealed a G47R mutation in transthyretin (TTR). Her general condition was so poor that we could not perform active treatment. Her consciousness had been impaired for a few months. She died at the age of 47 due to multiple organ failure. An autopsy revealed amyloid deposits in the subarachnoid space of the brainstem and the spinal cord as well as in the peripheral nerve and other organs. To date, this is the first case in which a G47R mutation is associated with leptomeningeal amyloidosis.

Application of oligonucleotide array CGH to the simultaneous detection of a deletion in the nuclear TK2 gene and mtDNA depletion.

PubMed

Zhang, Shulin; Li, Fang-Yuan; Bass, Harold N; Pursley, Amber; Schmitt, Eric S; Brown, Blaire L; Brundage, Ellen K; Mardach, Rebecca; Wong, Lee-Jun

2010-01-01

Thymidine kinase 2 (TK2), encoded by the TK2 gene on chromosome 16q22, is one of the deoxyribonucleoside kinases responsible for the maintenance of mitochondrial deoxyribonucleotide pools. Defects in TK2 mainly cause a myopathic form of the mitochondrial DNA depletion syndrome (MDDS). Currently, only point mutations and small insertions and deletions have been reported in TK2 gene; gross rearrangements of TK2 gene and possible hepatic involvement in patients with TK2 mutations have not been described. We report a non-consanguineous Jordanian family with three deceased siblings due to mtDNA depletion. Sequence analysis of the father detected a heterozygous c.761T>A (p.I254N) mutation in his TK2 gene; however, point mutations in the mother were not detected. Subsequent gene dosage analysis using oligonucleotide array CGH identified an intragenic approximately 5.8-kb deletion encompassing the 5'UTR to intron 2 of her TK2 gene. Sequence analysis confirmed that the deletion spans c.1-495 to c.283-2899 of the TK2 gene (nucleotide 65,136,256-65,142,086 of chromosome 16). Analysis of liver and muscle specimens from one of the deceased infants in this family revealed compound heterozygosity for the paternal point mutation and maternal intragenic deletion. In addition, a significant reduction of the mtDNA content in liver and muscle was detected (10% and 20% of age- and tissue-matched controls, respectively). Prenatal diagnosis was performed in the third pregnancy. The fetus was found to carry both the point mutation and the deletion. This child died 6months after birth due to myopathy. A serum specimen demonstrated elevated liver transaminases in two of the infants from whom results were available. This report expands the mutation spectrum associated with TK2 deficiency. While the myopathic form of MDDS appears to be the main phenotype of TK2 mutations, liver dysfunction may also be a part of the mitochondrial depletion syndrome caused by TK2 gene defects.

CD38 expression and immunoglobulin variable region mutations are independent prognostic variables in chronic lymphocytic leukemia, but CD38 expression may vary during the course of the disease.

PubMed

Hamblin, Terry J; Orchard, Jenny A; Ibbotson, Rachel E; Davis, Zadie; Thomas, Peter W; Stevenson, Freda K; Oscier, David G

2002-02-01

Although the presence or absence of somatic mutations in the immunoglobulin variable region (IgV(H)) genes in chronic lymphocytic leukemia (B-CLL) identifies subtypes with very different prognoses, the assay is technically complex and unavailable to most laboratories. CD38 expression has been suggested as a surrogate marker for the 2 subtypes. IgV(H) mutations and CD38 expression in 145 patients with B-CLL with a long follow-up were compared. The 2 assays gave discordant results in 41 patients (28.3%). Multivariate analysis demonstrated that Binet stage, IgV(H) mutations and CD38 were independent prognostic indicators. Median survival time in patients whose cells had unmutated IgV(H) genes and expressed CD38 was 8 years; in those with mutated IgV(H) genes not expressing CD38, it was 26 years. For those with discordant results, median survival time was 15 years. Thus, although CD38 expression does not identify the same 2 subsets as IgV(H) mutations in CLL, it is an independent risk factor that can be used with IgV(H) mutations and clinical stage to select patients with B-CLL with the worst prognoses. Using cryopreserved cells taken at intervals during the course of the disease, however, changes of CD38 expression over time were demonstrated in 10 of 41 patients. Causes of the variation of CD38 expression require further study. Additional prospective studies are required for comparing CD38 expression with other prognostic factors and for taking sequential measurements during the course of the disease.

Role of key-regulator genes in melanoma susceptibility and pathogenesis among patients from South Italy.

PubMed

Casula, Milena; Muggiano, Antonio; Cossu, Antonio; Budroni, Mario; Caracò, Corrado; Ascierto, Paolo A; Pagani, Elena; Stanganelli, Ignazio; Canzanella, Sergio; Sini, Mariacristina; Palomba, Grazia; Palmieri, Giuseppe

2009-10-03

Several genetic alterations have been demonstrated to contribute to the development and progression of melanoma. In this study, we further investigated the impact of key-regulator genes in susceptibility and pathogenesis of such a disease. A large series (N = 846) of sporadic and familial cases originating from South Italy was screened for germline mutations in p16(CDKN2A), BRCA2, and MC1R genes by DHPLC analysis and automated DNA sequencing. Paired primary melanomas and lymph node metastases from same patients (N = 35) as well as melanoma cell lines (N = 18) were analyzed for somatic mutations in NRAS, BRAF, and p16(CDKN2A) genes. For melanoma susceptibility, investigations at germline level indicated that p16(CDKN2A) was exclusively mutated in 16/545 (2.9%) non-Sardinian patients, whereas BRCA2 germline mutations were observed in 4/91 (4.4%) patients from North Sardinia only. Two MC1R germline variants, Arg151Cys and Asp294His, were significantly associated with melanoma in Sardinia. Regarding genetic events involved in melanoma pathogenesis at somatic level, mutually-exclusive mutations of NRAS and BRAF genes were observed at quite same rate (about two thirds) in cultured and in vivo melanomas (either primary or metastatic lesions). Conversely, p16(CDKN2A) gene alterations were observed at increased rates moving from primary to metastatic melanomas and melanoma cell lines. Activation of the ERK gene product was demonstrated to be consistently induced by a combination of molecular alterations (NRAS/BRAF mutations and p16(CDKN2A) silencing). Our findings further clarified that: a) mutation prevalence in melanoma susceptibility genes may vary within each specific geographical area; b) multiple molecular events are accumulating during melanomagenesis.

Screening of SOD1, FUS and TARDBP genes in patients with amyotrophic lateral sclerosis in central-southern China.

PubMed

Hou, Lihua; Jiao, Bin; Xiao, Tingting; Zhou, Lu; Zhou, Zhifan; Du, Juan; Yan, Xinxiang; Wang, Junling; Tang, Beisha; Shen, Lu

2016-09-08

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting motor neurons of the brain, brainstem and spinal cord. To date, mutations in more than 30 genes have been linked to the pathogenesis of ALS. Among them, SOD1, FUS and TARDBP are ranked as the three most common genes associated with ALS. However, no mutation analysis has been reported in central-southern China. In this study, we sequenced SOD1, FUS and TARDBP in a central-southern Chinese cohort of 173 patients with ALS (15 familial ALS and 158 sporadic ALS) to detect mutations. As a result, five missense mutations in SOD1, namely, p.D101N, p.D101G, p.C111Y, p.N86S and p.V87A, were identified in three unrelated familial probands and three sporadic cases; two mutations in FUS were found in two unrelated familial probands, including an insertion mutation (p.P525_Y526insY) and a missense mutation (p.R521H); no variants of TARDBP were observed in patients. Therefore, SOD1 mutations were present in 20.0% of familial ALS patients and 1.9% of sporadic ALS patients, while FUS mutations were responsible for 13.3% of familial ALS cases, and TARDBP mutations were rare in either familial or sporadic ALS cases. This study broadens the known mutational spectrum in patients with ALS and further demonstrates the necessity for genetic screening in ALS patients from central-southern China.

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

PubMed Central

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

2012-01-01

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

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

PubMed

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

2012-08-01

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

Blinded study determination of high sensitivity and specificity microchip electrophoresis–SSCP/HA to detect mutations in the p53 gene

PubMed Central

Hestekin, Christa N.; Lin, Jennifer S.; Senderowicz, Lionel; Jakupciak, John P.; O’Connell, Catherine; Rademaker, Alfred; Barron, Annelise E.

2012-01-01

Knowledge of the genetic changes that lead to disease has grown and continues to grow at a rapid pace. However, there is a need for clinical devices that can be used routinely to translate this knowledge into the treatment of patients. Use in a clinical setting requires high sensitivity and specificity (>97%) in order to prevent misdiagnoses. Single strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are two DNA-based, complementary methods for mutation detection that are inexpensive and relatively easy to implement. However, both methods are most commonly detected by slab gel electrophoresis, which can be labor-intensive, time-consuming, and often the methods are unable to produce high sensitivity and specificity without the use of multiple analysis conditions. Here we demonstrate the first blinded study using microchip electrophoresis-SSCP/HA. We demonstrate the ability of microchip electrophoresis-SSCP/HA to detect with 98% sensitivity and specificity >100 samples from the p53 gene exons 5–9 in a blinded study in an analysis time of less than 10 minutes. PMID:22002021

A novel F11 mutation in a Korean pediatric patient with recurrent epistaxis.

PubMed

Kim, Juwon; Kim, Yoonjung; Shin, Seam; Lyu, Chuhl Joo; Choi, Jong Rak; Lee, Kyung-A

2013-06-01

Congenital FXI deficiency (hemophilia C) is a rare bleeding disorder that has been documented mostly in Ashkenazi Jews. Unlike other hemophilias, bleeding tendency varies considerably among individuals, and FXI deficiency rarely manifests as spontaneous bleeding. FXI deficiency is caused primarily by mutations in the F11 gene. Herein, we report a case of a 10-year-old boy with recurrent nose bleeding due to FXI deficiency who was confirmed to have a novel mutation in F11. A molecular analysis of DNA extracted from peripheral blood collected from the patient [FXI clotting activity (FXI:C): 11%] revealed compound heterozygous mutations, Q226X and L424F, in F11, consistent with the severe disease phenotype of the patient. Pedigree analysis showed that the patient received L424F from his father (FXI:C = 49%) and Q226X from the mother (FXI:C = 48%). The sister (FXI:C = 47%) of the patient only had L424F, presumably inherited from her father. Multiple sequence alignment demonstrated that L424 is highly conserved across mammals, indicating that it is important for the function of FXI. In-silico analysis indicated that replacement of L424 by phenylalanine had a detrimental influence on FXI, consistent with the severe phenotype of the patient. Compilation of FXI deficiency cases in east Asian populations would be of great value because different populations appear to have different F11 mutation spectra.

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.

Can robots patch-clamp as well as humans? Characterization of a novel sodium channel mutation

PubMed Central

Estacion, M; Choi, J S; Eastman, E M; Lin, Z; Li, Y; Tyrrell, L; Yang, Y; Dib-Hajj, S D; Waxman, S G

2010-01-01

Ion channel missense mutations cause disorders of excitability by changing channel biophysical properties. As an increasing number of new naturally occurring mutations have been identified, and the number of other mutations produced by molecular approaches such as in situ mutagenesis has increased, the need for functional analysis by patch-clamp has become rate limiting. Here we compare a patch-clamp robot using planar-chip technology with human patch-clamp in a functional assessment of a previously undescribed Nav1.7 sodium channel mutation, S211P, which causes erythromelalgia. This robotic patch-clamp device can increase throughput (the number of cells analysed per day) by 3- to 10-fold. Both modes of analysis show that the mutation hyperpolarizes activation voltage dependence (−8 mV by manual profiling, −11 mV by robotic profiling), alters steady-state fast inactivation so that it requires an additional Boltzmann function for a second fraction of total current (∼20% manual, ∼40% robotic), and enhances slow inactivation (hyperpolarizing shift −15 mV by human, −13 mV robotic). Manual patch-clamping demonstrated slower deactivation and enhanced (∼2-fold) ramp response for the mutant channel while robotic recording did not, possibly due to increased temperature and reduced signal-to-noise ratio on the robotic platform. If robotic profiling is used to screen ion channel mutations, we recommend that each measurement or protocol be validated by initial comparison to manual recording. With this caveat, we suggest that, if results are interpreted cautiously, robotic patch-clamp can be used with supervision and subsequent confirmation from human physiologists to facilitate the initial profiling of a variety of electrophysiological parameters of ion channel mutations. PMID:20123784

Effects of helix and fingertip mutations on the thermostability of xyn11A investigated by molecular dynamics simulations and enzyme activity assays.

PubMed

Sutthibutpong, Thana; Rattanarojpong, Triwit; Khunrae, Pongsak

2017-12-04

Local conformational changes and global unfolding pathways of wildtype xyn11A recombinant and its mutated structures were studied through a series of atomistic molecular dynamics (MD) simulations, along with enzyme activity assays at three incubation temperatures to investigate the effects of mutations at three different sites to the thermostability. The first mutation was to replace an unstable negatively charged residue at a surface beta turn near the active site (D32G) by a hydrophobic residue. The second mutation was to create a disulphide bond (S100C/N147C) establishing a strong connection between an alpha helix and a distal beta hairpin associated with the thermally sensitive Thumb loop, and the third mutation add an extra hydrogen bond (A155S) to the same alpha helix. From the MD simulations performed, MM/PBSA energy calculations of the unfolding energy were in a good agreement with the enzyme activities measured from the experiment, as all mutated structures demonstrated the improved thermostability, especially the S100C/N147C proved to be the most stable mutant both by the simulations and the experiment. Local conformational analysis at the catalytic sites and the xylan access region also suggested that mutated xyn11A structures could accommodate xylan binding. However, the analysis of global unfolding pathways showed that structural disruptions at the beta sheet regions near the N-terminal were still imminent. These findings could provide the insight on the molecular mechanisms underlying the enhanced thermostability due to mutagenesis and changes in the protein unfolding pathways for further protein engineering of the GH11 family xylanase enzymes.

A high-throughput analysis of the IDH1(R132H) protein expression in pituitary adenomas.

PubMed

Casar-Borota, Olivera; Øystese, Kristin Astrid Berland; Sundström, Magnus; Melchior, Linea; Popovic, Vera

2016-08-01

Inactivating mutations of isocitrate dehydrogenase (IDH) 1 and 2, mitochondrial enzymes participating in the Krebs tricarboxylic acid cycle play a role in the tumorigenesis of gliomas and also less frequently in acute myeloid leukemia and other malignancies. Inhibitors of mutant IDH1 and IDH2 may potentially be effective in the treatment of the IDH mutation driven tumors. Mutations in the succinate dehydrogenase, the other enzyme complex participating in the Krebs cycle and electron transfer of oxidative phosphorylation occur in the paragangliomas, gastrointestinal stromal tumors, and occasionally in the pituitary adenomas. We aimed to determine whether the IDH1(R132H) mutation, the most frequent IDH mutation in human malignancies, occurs in pituitary adenomas. We performed immunohistochemical analysis by using a monoclonal anti-IDH1(R132H) antibody on the tissue microarrays containing specimens from the pituitary adenomas of different hormonal types from 246 patients. In positive samples, the status of the IDH1 gene was further examined by molecular genetic analyses. In all but one patient, there was no expression of mutated IDH1(R132H) protein in the tumor cells by immunohistochemistry. Only one patient with a recurring clinically non-functioning gonadotroph adenoma demonstrated IDH1(R132H)-immunostaining in both the primary tumor and the recurrence. However, no mutation in the IDH1 gene was detected using different molecular genetic analyses. IDH1(R132H) mutation occurs only exceptionally in pituitary adenomas and does not play a role in their pathogenesis. Patients with pituitary adenomas do not seem to be candidates for treatment with the inhibitors of mutant IDH1.

CNGA3 mutations in two United Arab Emirates families with achromatopsia.

PubMed

Ahuja, Yachna; Kohl, Susanne; Traboulsi, Elias I

2008-07-10

ACHROMATOPSIA RESULTS FROM MUTATIONS IN ONE OF THREE GENES: cyclic nucleotide-gated channel, alpha-3 (CNGA3); cyclic nucleotide-gated channel, beta-3 (CNGB3); and guanine nucleotide-binding protein, alpha-transducing activity polypeptide 2 (GNAT2). We report the responsible mutations in two United Arab Emirates families who have this autosomal recessive disease. Clinical examinations were performed in seven patients from three nuclear families. Molecular genetic testing for common CNGA3 and CNGB3 mutations was undertaken using standard protocols. All patients were extremely light sensitive and had reduced visual acuity and no color perception. Fundus examinations did not show any visible abnormalities. After further pedigree analysis, two of the families were found to be linked through the paternal line. Two mutations in CNGA3 were identified: Arg283Trp and Gly397Val. Family A, the larger pedigree, had one branch in which two sisters and one brother were homozygous for the Gly397Val mutation and another branch in which a brother and sister were compound heterozygous for both aforenamed mutations. Family B, however, only had two brothers who were homozygous for the Arg283Trp mutation. Achromatopsia in these two United Arab Emirates families results from two different mutations in CNGA3. Two branches of the same pedigree had individuals with both homozygous and compound heterozygous disease, demonstrating a complex molecular pathology in this large family.

Full-length mutation search of the TP53 gene in acute myeloid leukemia has increased significance as a prognostic factor.

PubMed

Terada, Kazuki; Yamaguchi, Hiroki; Ueki, Toshimitsu; Usuki, Kensuke; Kobayashi, Yutaka; Tajika, Kenji; Gomi, Seiji; Kurosawa, Saiko; Miyadera, Keiki; Tokura, Taichiro; Omori, Ikuko; Marumo, Atushi; Fujiwara, Yusuke; Yui, Shunsuke; Ryotokuji, Takeshi; Osaki, Yoshiki; Arai, Kunihito; Kitano, Tomoaki; Kosaka, Fumiko; Wakita, Satoshi; Tamai, Hayato; Fukuda, Takahiro; Inokuchi, Koiti

2018-01-01

TP53 gene abnormality has been reported to be an unfavorable prognostic factor in acute myeloid leukemia (AML). However, almost all studies of TP53 gene abnormality so far have been limited to mutation searches in the DNA binding domain. As there have been few reports examining both mutation and deletion over the full-length of the TP53 gene, the clinical characteristics of TP53 gene abnormality have not yet been clearly established. In this study, TP53 gene mutation was observed in 7.3% of the total 412 de novo AML cases (33 mutations in 30 cases), with mutation outside the DNA binding domain in eight cases (27%). TP53 gene deletion was observed in 3.1% of 358 cases. All cases had monoallelic deletion with TP53 gene mutation on the opposite allele. Multivariate analysis demonstrated that TP53 gene mutation in the DNA binding domain and outside the DNA binding domain was an independent poor prognostic factor for overall survival and relapse-free survival among the total cohort and it is also an unfavorable prognostic factor in FLT3-ITD-negative AML cases aged 70 years or below with intermediate cytogenetic prognosis. In stratified treatment, full-length search for TP53 gene mutation is therefore very important.

Diffuse reticuloendothelial system involvement in type IV glycogen storage disease with a novel GBE1 mutation: a case report and review.

PubMed

Magoulas, Pilar L; El-Hattab, Ayman W; Roy, Angshumoy; Bali, Deeksha S; Finegold, Milton J; Craigen, William J

2012-06-01

Glycogen storage disease type IV is a rare autosomal recessive disorder of glycogen metabolism caused by mutations in the GBE1 gene that encodes the 1,4-alpha-glucan-branching enzyme 1. Its clinical presentation is variable, with the most common form presenting in early childhood with primary hepatic involvement. Histologic manifestations in glycogen storage disease type IV typically consist of intracytoplasmic non-membrane-bound inclusions containing abnormally branched glycogen (polyglucosan bodies) within hepatocytes and myocytes. We report a female infant with classic hepatic form of glycogen storage disease type IV who demonstrated diffuse reticuloendothelial system involvement with the spleen, bone marrow, and lymph nodes infiltrated by foamy histiocytes with intracytoplasmic polyglucosan deposits. Sequence analysis of the GBE1 gene revealed compound heterozygosity for a previously described frameshift mutation (c.1239delT) and a novel missense mutation (c.1279G>A) that is predicted to alter a conserved glycine residue. GBE enzyme analysis revealed no detectable activity. A review of the literature for glycogen storage disease type IV patients with characterized molecular defects and deficient enzyme activity reveals most GBE1 mutations to be missense mutations clustering in the catalytic enzyme domain. Individuals with the classic hepatic form of glycogen storage disease type IV tend to be compound heterozygotes for null and missense mutations. Although the extensive reticuloendothelial system involvement that was observed in our patient is not typical of glycogen storage disease type IV, it may be associated with severe enzymatic deficiency and a poor outcome. Copyright © 2012 Elsevier Inc. All rights reserved.

Functional studies of RYR1 mutations in the skeletal muscle ryanodine receptor using human RYR1 complementary DNA.

PubMed

Sato, Keisaku; Pollock, Neil; Stowell, Kathryn M

2010-06-01

Malignant hyperthermia is associated with mutations within the gene encoding the skeletal muscle ryanodine receptor, the calcium channel that releases Ca from sarcoplasmic reticulum stores triggering muscle contraction, and other metabolic activities. More than 200 variants have been identified in the ryanodine receptor, but only some of these have been shown to functionally affect the calcium channel. To implement genetic testing for malignant hyperthermia, variants must be shown to alter the function of the channel. A number of different ex vivo methods can be used to demonstrate functionality, as long as cells from human patients can be obtained and cultured from at least two unrelated families. Because malignant hyperthermia is an uncommon disorder and many variants seem to be private, including the newly identified H4833Y mutation, these approaches are limited. The authors cloned the human skeletal muscle ryanodine receptor complementary DNA and expressed both normal and mutated forms in HEK-293 cells and carried out functional analysis using ryanodine binding assays in the presence of a specific agonist, 4-chloro-m-cresol, and the antagonist Mg. Transiently expressed human ryanodine receptor proteins colocalized with an endoplasmic reticulum marker in HEK-293 cells. Ryanodine binding assays confirmed that mutations causing malignant hyperthermia resulted in a hypersensitive channel, while those causing central core disease resulted in a hyposensitive channel. The functional assays validate recombinant human skeletal muscle ryanodine receptor for analysis of variants and add an additional mutation (H4833Y) to the repertoire of mutations that can be used for the genetic diagnosis of malignant hyperthermia.

COLD-PCR Technologies in the Area of Personalized Medicine: Methodology and Applications.

PubMed

Mauger, Florence; How-Kit, Alexandre; Tost, Jörg

2017-06-01

Somatic mutations bear great promise for use as biomarkers for personalized medicine, but are often present only in low abundance in biological material and are therefore difficult to detect. Many assays for mutation analysis in cancer-related genes (hotspots) have been developed to improve diagnosis, prognosis, prediction of drug resistance, and monitoring of the response to treatment. Two major approaches have been developed: mutation-specific amplification methods and methods that enrich and detect mutations without prior knowledge on the exact location and identity of the mutation. CO-amplification at Lower Denaturation temperature Polymerase Chain Reaction (COLD-PCR) methods such as full-, fast-, ice- (improved and complete enrichment), enhanced-ice, and temperature-tolerant COLD-PCR make use of a critical temperature in the polymerase chain reaction to selectively denature wild-type-mutant heteroduplexes, allowing the enrichment of rare mutations. Mutations can subsequently be identified using a variety of laboratory technologies such as high-resolution melting, digital polymerase chain reaction, pyrosequencing, Sanger sequencing, or next-generation sequencing. COLD-PCR methods are sensitive, specific, and accurate if appropriately optimized and have a short time to results. A large variety of clinical samples (tumor DNA, circulating cell-free DNA, circulating cell-free fetal DNA, and circulating tumor cells) have been studied using COLD-PCR in many different applications including the detection of genetic changes in cancer and infectious diseases, non-invasive prenatal diagnosis, detection of microorganisms, or DNA methylation analysis. In this review, we describe in detail the different COLD-PCR approaches, highlighting their specificities, advantages, and inconveniences and demonstrating their use in different fields of biological and biomedical research.

Clinical impact of endometrial cancer stratified by genetic mutational profiles, POLE mutation, and microsatellite instability.

PubMed

Haruma, Tomoko; Nagasaka, Takeshi; Nakamura, Keiichiro; Haraga, Junko; Nyuya, Akihiro; Nishida, Takeshi; Goel, Ajay; Masuyama, Hisashi; Hiramatsu, Yuji

2018-01-01

The molecular characterization of endometrial cancer (EC) can facilitate identification of various tumor subtypes. Although EC patients with POLE mutations reproducibly demonstrate better prognosis, the outcome of patients with microsatellite instability (MSI) remains controversial. This study attempted to interrogate whether genetic stratification of EC can identify distinct subsets with prognostic significance. A cohort of 138 EC patients who underwent surgical resection with curative intent was enrolled. Sanger sequencing was used to evaluate mutations in the POLE and KRAS genes. MSI analysis was performed using four mononucleotide repeat markers and methylation status of the MLH1 promoter was measured by a fluorescent bisulfite polymerase chain reaction (PCR). Protein expression for mismatch repair (MMR) proteins was evaluated by immunohistochemistry (IHC). Extensive hypermethylation of the MLH1 promoter was observed in 69.6% ECs with MLH1 deficiency and 3.5% with MMR proficiency, but in none of the ECs with loss of other MMR genes (P < .0001). MSI-positive and POLE mutations were found in 29.0% and 8.7% EC patients, respectively. Our MSI analysis showed a sensitivity of 92.7% for EC patients with MMR deficiency, and a specificity of 97.9% for EC patients with MMR proficiency. In univariate and multivariate analyses, POLE mutations and MSI status was significantly associated with progression-free survival (P = 0.0129 and 0.0064, respectively) but not with endometrial cancer-specific survival. This study provides significant evidence that analyses of proofreading POLE mutations and MSI status based on mononucleotide repeat markers are potentially useful biomarkers to identify EC patients with better prognosis.

Mutations in RAB39B cause X-linked intellectual disability and early-onset Parkinson disease with α-synuclein pathology.

PubMed

Wilson, Gabrielle R; Sim, Joe C H; McLean, Catriona; Giannandrea, Maila; Galea, Charles A; Riseley, Jessica R; Stephenson, Sarah E M; Fitzpatrick, Elizabeth; Haas, Stefan A; Pope, Kate; Hogan, Kirk J; Gregg, Ronald G; Bromhead, Catherine J; Wargowski, David S; Lawrence, Christopher H; James, Paul A; Churchyard, Andrew; Gao, Yujing; Phelan, Dean G; Gillies, Greta; Salce, Nicholas; Stanford, Lynn; Marsh, Ashley P L; Mignogna, Maria L; Hayflick, Susan J; Leventer, Richard J; Delatycki, Martin B; Mellick, George D; Kalscheuer, Vera M; D'Adamo, Patrizia; Bahlo, Melanie; Amor, David J; Lockhart, Paul J

2014-12-04

Advances in understanding the etiology of Parkinson disease have been driven by the identification of causative mutations in families. Genetic analysis of an Australian family with three males displaying clinical features of early-onset parkinsonism and intellectual disability identified a ∼45 kb deletion resulting in the complete loss of RAB39B. We subsequently identified a missense mutation (c.503C>A [p.Thr168Lys]) in RAB39B in an unrelated Wisconsin kindred affected by a similar clinical phenotype. In silico and in vitro studies demonstrated that the mutation destabilized the protein, consistent with loss of function. In vitro small-hairpin-RNA-mediated knockdown of Rab39b resulted in a reduction in the density of α-synuclein immunoreactive puncta in dendritic processes of cultured neurons. In addition, in multiple cell models, we demonstrated that knockdown of Rab39b was associated with reduced steady-state levels of α-synuclein. Post mortem studies demonstrated that loss of RAB39B resulted in pathologically confirmed Parkinson disease. There was extensive dopaminergic neuron loss in the substantia nigra and widespread classic Lewy body pathology. Additional pathological features included cortical Lewy bodies, brain iron accumulation, tau immunoreactivity, and axonal spheroids. Overall, we have shown that loss-of-function mutations in RAB39B cause intellectual disability and pathologically confirmed early-onset Parkinson disease. The loss of RAB39B results in dysregulation of α-synuclein homeostasis and a spectrum of neuropathological features that implicate RAB39B in the pathogenesis of Parkinson disease and potentially other neurodegenerative disorders. Copyright © 2014 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Exome sequencing for simultaneous mutation screening in children with hemophagocytic lymphohistiocytosis.

PubMed

Mukda, Ekchol; Trachoo, Objoon; Pasomsub, Ekawat; Tiyasirichokchai, Rawiphorn; Iemwimangsa, Nareenart; Sosothikul, Darintr; Chantratita, Wasun; Pakakasama, Samart

2017-08-01

In the present study, we used exome sequencing to analyze PRF1, UNC13D, STX11, and STXBP2, as well as genes associated with primary immunodeficiency disease (RAB27A, LYST, AP3B1, SH2D1A, ITK, CD27, XIAP, and MAGT1) in Thai children with hemophagocytic lymphohistiocytosis (HLH). We performed mutation analysis of HLH-associated genes in 25 Thai children using an exome sequencing method. Genetic variations found within these target genes were compared to exome sequencing data from 133 healthy individuals. Variants identified with minor allele frequencies <5% and novel mutations were confirmed using Sanger sequencing. Exome sequencing data revealed 101 non-synonymous single nucleotide polymorphisms (SNPs) in all subjects. These SNPs were classified as pathogenic (n = 1), likely pathogenic (n = 16), variant of unknown significance (n = 12), or benign variant (n = 72). Homozygous, compound heterozygous, and double-gene heterozygous variants, involving mutations in PRF1 (n = 3), UNC13D (n = 2), STXBP2 (n = 3), LYST (n = 3), XIAP (n = 2), AP3B1 (n = 1), RAB27A (n = 1), and MAGT1 (n = 1), were demonstrated in 12 patients. Novel mutations were found in most patients in this study. In conclusion, exome sequencing demonstrated the ability to identify rare genetic variants in HLH patients. This method is useful in the detection of mutations in multi-gene associated diseases.

Comparative Genomic Analysis of Two Clonally Related Multidrug Resistant Mycobacterium tuberculosis by Single Molecule Real Time Sequencing.

PubMed

Leung, Kenneth Siu-Sing; Siu, Gilman Kit-Hang; Tam, Kingsley King-Gee; To, Sabrina Wai-Chi; Rajwani, Rahim; Ho, Pak-Leung; Wong, Samson Sai-Yin; Zhao, Wei W; Ma, Oliver Chiu-Kit; Yam, Wing-Cheong

2017-01-01

Background: Multidrug-resistant tuberculosis (MDR-TB) is posing a major threat to global TB control. In this study, we focused on two consecutive MDR-TB isolated from the same patient before and after the initiation of anti-TB treatment. To better understand the genomic characteristics of MDR-TB, Single Molecule Real-Time (SMRT) Sequencing and comparative genomic analyses was performed to identify mutations that contributed to the stepwise development of drug resistance and growth fitness in MDR-TB under in vivo challenge of anti-TB drugs. Result: Both pre-treatment and post-treatment strain demonstrated concordant phenotypic and genotypic susceptibility profiles toward rifampicin, pyrazinamide, streptomycin, fluoroquinolones, aminoglycosides, cycloserine, ethionamide, and para-aminosalicylic acid. However, although both strains carried identical missense mutations at rpoB S531L, inhA C-15T, and embB M306V, MYCOTB Sensititre assay showed that the post-treatment strain had 16-, 8-, and 4-fold elevation in the minimum inhibitory concentrations (MICs) toward rifabutin, isoniazid, and ethambutol respectively. The results have indicated the presence of additional resistant-related mutations governing the stepwise development of MDR-TB. Further comparative genomic analyses have identified three additional polymorphisms between the clinical isolates. These include a single nucleotide deletion at nucleotide position 360 of rv0888 in pre-treatment strain, and a missense mutation at rv3303c ( lpdA) V44I and a 6-bp inframe deletion at codon 67-68 in rv2071c ( cobM) in the post-treatment strain. Multiple sequence alignment showed that these mutations were occurring at highly conserved regions among pathogenic mycobacteria. Using structural-based and sequence-based algorithms, we further predicted that the mutations potentially have deleterious effect on protein function. Conclusion: This is the first study that compared the full genomes of two clonally-related MDR-TB clinical isolates during the course of anti-TB treatment. Our work has demonstrated the robustness of SMRT Sequencing in identifying mutations among MDR-TB clinical isolates. Comparative genome analysis also suggested novel mutations at rv0888, lpdA , and cobM that might explain the difference in antibiotic resistance and growth pattern between the two MDR-TB strains.

Detection of IDH1 R132H mutation in acute myeloid leukemia by mutation-specific immunohistochemistry.

PubMed

Byers, Richard; Hornick, Jason L; Tholouli, Eleni; Kutok, Jeffery; Rodig, Scott J

2012-01-01

IDH1 mutations are present but are uncommon in acute myeloid leukemia (AML) and although prognostically favorable in gliomas their clinical significance in AML is unclear. Some have associated IDH1 mutations with inferior outcome, whereas others found no association with prognosis. Complicating these analyses is the need to sequence IDH1 from leukemic blasts, which is technically challenging and not yet routine. Mutation-specific antibodies enable robust, cost-effective detection of mutations in routine biopsy samples. Immunohistochemistry for the R132H mutation-specific antibody was performed in a tissue microarray containing 159 cases of AML, detecting the R132H mutation in 7 cases (4.4%). Positivity was associated with intermediate risk cytogenetics. Our results demonstrate an association between the R132H IDH1 mutation and intermediate risk cytogenetics in AML, suggesting that R132H IDH1 mutation may be associated with improved clinical outcome and demonstrate the feasibility of using mutation-specific antibodies to genotype and subclassify AML.

Analysis of human MutS homolog 2 missense mutations in patients with colorectal cancer.

PubMed

Zhang, Xiaomei; Chen, Senqing; Yu, Jun; Zhang, Yuanying; Lv, Min; Zhu, Ming

2018-05-01

Germline mutations of DNA mismatch repair gene human MutS homolog 2 ( hMSH2 ) are associated with hereditary nonpolyposis colorectal cancer (HNPCC). A total of one-third of these mutations are missense mutations. Several hMSH2 missense mutations have been identified in patients in East Asia, although their function has not been evaluated. In the present study, the role of ten hMSH2 missense mutations in the pathogenesis of colorectal cancer was examined. The hMSH2/hMSH6 protein interaction system was established using yeast two-hybrid screening. Next, the missense mutations were analyzed for their ability to affect the protein interaction of hMSH2 with its partner hMSH6. Additionally, the Sorting Intolerant from Tolerant tool was applied to predict the effects of different amino acid substitutions. The results demonstrated that certain hMSH2 mutations (L173R and C199R) caused a significant functional change in the human hMutSα complex and were identified to be pathological mutations. The Y408C, D603Y, P696L and S703Y mutations partially affected interaction and partly affected the function of hMSH2. The remaining four variants, T8M, I169V, A370T and Q419K, may be non-functional polymorphisms or could affect protein function through other molecular mechanisms. The present study evaluated the functional consequences of previously unknown missense mutations in hMSH2 , and may contribute to improved clinical diagnosis and mutation screening of HNPCC.

Association of a novel point mutation in MSH2 gene with familial multiple primary cancers.

PubMed

Hu, Hai; Li, Hong; Jiao, Feng; Han, Ting; Zhuo, Meng; Cui, Jiujie; Li, Yixue; Wang, Liwei

2017-10-03

Multiple primary cancers (MPC) have been identified as two or more cancers without any subordinate relationship that occur either simultaneously or metachronously in the same or different organs of an individual. Lynch syndrome is an autosomal dominant genetic disorder that increases the risk of many types of cancers. Lynch syndrome patients who suffer more than two cancers can also be considered as MPC; patients of this kind provide unique resources to learn how genetic mutation causes MPC in different tissues. We performed a whole genome sequencing on blood cells and two tumor samples of a Lynch syndrome patient who was diagnosed with five primary cancers. The mutational landscape of the tumors, including somatic point mutations and copy number alternations, was characterized. We also compared Lynch syndrome with sporadic cancers and proposed a model to illustrate the mutational process by which Lynch syndrome progresses to MPC. We revealed a novel pathologic mutation on the MSH2 gene (G504 splicing) that associates with Lynch syndrome. Systematical comparison of the mutation landscape revealed that multiple cancers in the proband were evolutionarily independent. Integrative analysis showed that truncating mutations of DNA mismatch repair (MMR) genes were significantly enriched in the patient. A mutation progress model that included germline mutations of MMR genes, double hits of MMR system, mutations in tissue-specific driver genes, and rapid accumulation of additional passenger mutations was proposed to illustrate how MPC occurs in Lynch syndrome patients. Our findings demonstrate that both germline and somatic alterations are driving forces of carcinogenesis, which may resolve the carcinogenic theory of Lynch syndrome.

Insights into the pathogenesis of dominant retinitis pigmentosa associated with a D477G mutation in RPE65.

PubMed

Choi, Elliot H; Suh, Susie; Sander, Christopher L; Hernandez, Christian J Ortiz; Bulman, Elizabeth R; Khadka, Nimesh; Dong, Zhiqian; Shi, Wuxian; Palczewski, Krzysztof; Kiser, Philip D

2018-04-12

RPE65 is the essential trans-cis isomerase of the classical retinoid (visual) cycle. Mutations in RPE65 give rise to severe retinal dystrophies, most of which are associated with loss of protein function and recessive inheritance. The only known exception is a c.1430G>A (D477G) mutation that gives rise to dominant retinitis pigmentosa with delayed onset and choroidal and macular involvement. Position 477 is distant from functionally critical regions of RPE65. Hence, the mechanism of D477G pathogenicity remains unclear, although protein misfolding and aggregation mechanisms have been suggested. We characterized a D477G knock-in mouse model which exhibited mild age-dependent changes in retinal structure and function. Immunoblot analysis of protein extracts from the eyes of the knock-in mice demonstrated the presence of ubiquitinated RPE65 and reduced RPE65 expression. We observed an accumulation of retinyl esters in the knock-in mice as well as a delay in rhodopsin regeneration kinetics and diminished electroretinography responses, indicative of RPE65 functional impairment induced by the D477G mutation in vivo. However, a cell line expressing D477G RPE65 revealed protein expression levels, cellular localization, and retinoid isomerase activity comparable to cells expressing wild-type protein. Structural analysis of an RPE65 chimera suggested that the D477G mutation does not perturb protein folding or tertiary structure. Instead, the mutation generates an aggregation-prone surface that could induce cellular toxicity through abnormal complex formation as suggested by crystal packing analysis. These results indicate that a toxic gain-of-function induced by the D477G RPE65 substitution may play a role in the pathogenesis of this form of dominant retinitis pigmentosa.

Molecular analysis reveals a high mutation frequency in the first untranslated exon of the PPOX gene and largely excludes variegate porphyria in a subset of clinically affected Afrikaner families.

PubMed

Kotze, M J; De Villiers, J N; Groenewald, J Z; Rooney, R N; Loubser, O; Thiart, R; Oosthuizen, C J; van Niekerk, M M; Groenewald, I M; Retief, A E; Warnich, L

1998-10-01

A subset of probands from 11 South African families with clinical and/or biochemical features of variegate porphyria (VP), but without the known protoporphyrinogen oxidase (PPOX) gene defects identified previously in the South African population, were subjected to mutation analysis. Disease-related mutation(s) could not be identified after screening virtually the entire PPOX gene by heteroduplex single-strand conformation polymorphism analysis (HEX-SSCP), although three new sequence variants were detected in exon 1 of the gene in three normal controls. The presence of these single base changes at nucleotide positions 22 (C/G), 27 (C/A) and 127 (C/A), in addition to the known exon 1 polymorphisms I-26 and I-150, indicates that this untranslated region of the PPOX gene is particularly mutation-prone. Furthermore, microsatellite markers flanking the PPOX and alpha-1 antitrypsin (PI) gene, on chromosomes 1 and 14, respectively, were used to assess the probability of involvement of these loci in disease presentation. Common alleles transmitted from affected parent to affected child were determined where possible in the mutation-negative index cases. Allelic frequencies of these alleles were compared to findings in the normal population, but no predominant disease-associated allele could be identified. Co-segregation of a specific haplotype with the disease phenotype could also not be demonstrated in a large Afrikaner family. It is concluded that further studies are warranted to determine the genetic factor(s) underlying the autosomal dominant pattern of inheritance in molecularly uncharacterized cases showing clinical symptoms of an acute porphyria. Copyright 1998 Academic Press.

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.

CFTR mutation distribution among U.S. Hispanic and African American individuals: evaluation in cystic fibrosis patient and carrier screening populations.

PubMed

Sugarman, Elaine A; Rohlfs, Elizabeth M; Silverman, Lawrence M; Allitto, Bernice A

2004-01-01

We reviewed CFTR mutation distribution among Hispanic and African American individuals referred for CF carrier screening and compared mutation frequencies to those derived from CF patient samples. Results from CFTR mutation analyses received from January 2001 through September 2003, were analyzed for four populations: Hispanic individuals with a CF diagnosis (n = 159) or carrier screening indication (n = 15,333) and African American individuals with a CF diagnosis (n = 108) or carrier screening indication (n = 8,973). All samples were tested for the same 87 mutation panel. In the Hispanic population, 42 mutations were identified: 30 in the patient population (77.5% detection rate) and 33 among carrier screening referrals. Five mutations not included in the ACMG/ACOG carrier screening panel (3876delA, W1089X, R1066C, S549N, 1949del84) accounted for 7.55% detection in patients and 5.58% among carriers. Among African American referrals, 33 different mutations were identified: 21 in the patient population (74.4% detection) and 23 in the carrier screening population. Together, A559T and 711+5G>A were observed at a detection rate of 3.71% in CF patients and 6.38% in carriers. The mutation distribution seen in both the carrier screening populations reflected an increased frequency of mutations with variable expression such as D1152H, R117H, and L206W. A detailed analysis of CFTR mutation distribution in the Hispanic and African American patient and carrier screening populations demonstrates that a diverse group of mutations is most appropriate for diagnostic and carrier screening in these populations. To best serve the increasingly diverse U.S. population, ethnic-specific mutations should be included in mutation panels.

A comprehensive biophysical description of pairwise epistasis throughout an entire protein domain

PubMed Central

Olson, C. Anders; Wu, Nicholas C.; Sun, Ren

2014-01-01

SUMMARY Background Non-additivity in fitness effects from two or more mutations, termed epistasis, can result in compensation of deleterious mutations or negation of beneficial mutations. Recent evidence shows the importance of epistasis in individual evolutionary pathways. However, an unresolved question in molecular evolution is how often and how significantly fitness effects change in alternative genetic backgrounds. Results To answer this question we quantified the effects of all single mutations and double mutations between all positions in the IgG-binding domain of protein G (GB1). By observing the first two steps of all possible evolutionary pathways, this fitness profile enabled the characterization of the extent and magnitude of pairwise epistasis throughout an entire protein molecule. Furthermore, we developed a novel approach to quantitatively determine the effects of single mutations on structural stability (ΔΔGU). This enabled determination of the importance of stability effects in functional epistasis. Conclusions Our results illustrate common biophysical mechanisms for occurrences of positive and negative epistasis. Our results show pervasive positive epistasis within a conformationally dynamic network of residues. The stability analysis shows that significant negative epistasis, which is more common than positive epistasis, mostly occurs between combinations of destabilizing mutations. Furthermore, we show that although significant positive epistasis is rare, many deleterious mutations are beneficial in at least one alternative mutational background. The distribution of conditionally beneficial mutations throughout the domain demonstrates that the functional portion of sequence space can be significantly expanded by epistasis. PMID:25455030

Using Student Writing and Lexical Analysis to Reveal Student Thinking about the Role of Stop Codons in the Central Dogma

PubMed Central

Prevost, Luanna B.; Smith, Michelle K.; Knight, Jennifer K.

2016-01-01

Previous work has shown that students have persistent difficulties in understanding how central dogma processes can be affected by a stop codon mutation. To explore these difficulties, we modified two multiple-choice questions from the Genetics Concept Assessment into three open-ended questions that asked students to write about how a stop codon mutation potentially impacts replication, transcription, and translation. We then used computer-assisted lexical analysis combined with human scoring to categorize student responses. The lexical analysis models showed high agreement with human scoring, demonstrating that this approach can be successfully used to analyze large numbers of student written responses. The results of this analysis show that students’ ideas about one process in the central dogma can affect their thinking about subsequent and previous processes, leading to mixed models of conceptual understanding. PMID:27909016

Integrating molecular dynamics and co-evolutionary analysis for reliable target prediction and deregulation of the allosteric inhibition of aspartokinase for amino acid production.

PubMed

Chen, Zhen; Rappert, Sugima; Sun, Jibin; Zeng, An-Ping

2011-07-20

Deregulation of allosteric inhibition of enzymes is a challenge for strain engineering and has been achieved so far primarily by random mutation and trial-and-error. In this work, we used aspartokinase, an important allosteric enzyme for industrial amino acids production, to demonstrate a predictive approach that combines protein dynamics and evolution for a rational reengineering of enzyme allostery. Molecular dynamic simulation of aspartokinase III (AK3) from Escherichia coli and statistical coupling analysis of protein sequences of the aspartokinase family allowed to identify a cluster of residues which are correlated during protein motion and coupled during the evolution. This cluster of residues forms an interconnected network mediating the allosteric regulation, including most of the previously reported positions mutated in feedback insensitive AK3 mutants. Beyond these mutation positions, we have successfully constructed another twelve targeted mutations of AK3 desensitized toward lysine inhibition. Six threonine-insensitive mutants of aspartokinase I-homoserine dehydrogenase I (AK1-HD1) were also created based on the predictions. The proposed approach can be widely applied for the deregulation of other allosteric enzymes. Copyright © 2011 Elsevier B.V. All rights reserved.

Molecular analysis of abnormal hemoglobins in beta chain in Aegean region of Turkey and first reports of hemoglobin Andrew-Minneapolis and Hb Hinsdale from Turkey.

PubMed

Aykut, Ayça; Onay, Hüseyin; Durmaz, Asude; Karaca, Emin; Vergin, Canan; Aydınok, Yeşim; Özkınay, Ferda

2015-07-01

The Agean is one of the regions in Turkey where thalassemias and abnormal hemoglobins (Hbs) are prevalent. Combined heterozygosity of thalassemia mutations with a variety of structural Hb variants lead to an extremely wide spectrum of clinical and hematological phenotypes which is of importance for prenatal diagnosis. One hundred and seventeen patients and carriers diagnosed by hemoglobin electrophoresis (HPLC), at risk for abnormal hemoglobinopathies were screened for mutational analysis of the beta-globin gene. The full coding the 5' UTR, and the 3' UTR sequences of beta-globin gene (GenBank accession no. U01317) were amplified and sequenced. In this study, a total of 118 (12.24%) structural Hb variant alleles were identified in 1341 mutated beta-chain alleles in Medical Genetics Department of Ege University between January 2006 and November 2013. Here, we report the mutation spectrum of abnormal Hbs associated with the beta-globin gene in Aegean region of Turkey. In the present study, the Hb Hinsdale and Hb Andrew-Minneapolis variants are demonstrated for the first time in the Turkish population.

Single-Cell Analysis of Human Pancreas Reveals Transcriptional Signatures of Aging and Somatic Mutation Patterns.

PubMed

Enge, Martin; Arda, H Efsun; Mignardi, Marco; Beausang, John; Bottino, Rita; Kim, Seung K; Quake, Stephen R

2017-10-05

As organisms age, cells accumulate genetic and epigenetic errors that eventually lead to impaired organ function or catastrophic transformation such as cancer. Because aging reflects a stochastic process of increasing disorder, cells in an organ will be individually affected in different ways, thus rendering bulk analyses of postmitotic adult cells difficult to interpret. Here, we directly measure the effects of aging in human tissue by performing single-cell transcriptome analysis of 2,544 human pancreas cells from eight donors spanning six decades of life. We find that islet endocrine cells from older donors display increased levels of transcriptional noise and potential fate drift. By determining the mutational history of individual cells, we uncover a novel mutational signature in healthy aging endocrine cells. Our results demonstrate the feasibility of using single-cell RNA sequencing (RNA-seq) data from primary cells to derive insights into genetic and transcriptional processes that operate on aging human tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

Juvenile-onset Sporadic Amyotrophic Lateral Sclerosis with a Frameshift FUS Gene Mutation Presenting Unique Neuroradiological Findings and Cognitive Impairment.

PubMed

Hirayanagi, Kimitoshi; Sato, Masayuki; Furuta, Natsumi; Makioka, Kouki; Ikeda, Yoshio

2016-01-01

A 24-year-old Japanese woman developed anterocollis, weakness of the proximal arms, and subsequent cognitive impairment. A neurological examination revealed amyotrophic lateral sclerosis (ALS) without a family history. Systemic muscle atrophy progressed rapidly. Cerebral MRI clearly exhibited high signal intensities along the bilateral pyramidal tracts. An analysis of the FUS gene revealed a heterozygous two-base pair deletion, c.1507-1508delAG (p.G504WfsX515). A subset of juvenile-onset familial/sporadic ALS cases with FUS gene mutations reportedly demonstrates mental retardation or learning difficulty. Our study emphasizes the importance of conducting a FUS gene analysis in juvenile-onset ALS cases, even when no family occurrence is confirmed.

JAK2 mutation in a patient with CLL with coexistent myeloproliferative neoplasm (MPN).

PubMed

Kodali, Srinivas; Chen, Chi; Rathnasabapathy, Chenthilmurugan; Wang, Jen Chin

2009-12-01

JAK2 mutation has not been described in patients with chronic lymphocytic leukemia (CLL). We found JAK2 mutation in a patient with CLL and coexisting myeloproliferative neoplasm (MPN). In this patient, we demonstrated the presence of the JAK2 mutation in CD34(+) progenitor cells, myeloid lineage cells, megakaryocytes, B lymphocytes but not in T lymphocytes. This case represents the first case report of JAK2 mutation in CLL and may also suggest that, JAK2 mutation most likely represents a secondary event from primary gene mutations involving the primitive stem cells which give rise to MPN and CLL. Furthermore, in this case, we believe that we are the first to demonstrate that JAK2 mutation in myeloid and B lymphoid cells but not T lymphocytes in a case of coexisting CLL and MPN.

Biallelic Mutations in GNB3 Cause a Unique Form of Autosomal-Recessive Congenital Stationary Night Blindness

PubMed Central

Vincent, Ajoy; Audo, Isabelle; Tavares, Erika; Maynes, Jason T.; Tumber, Anupreet; Wright, Thomas; Li, Shuning; Michiels, Christelle; Banin, Eyal; Bocquet, Beatrice; De Baere, Elfride; Casteels, Ingele; Defoort-Dhellemmes, Sabine; Drumare, Isabelle; Friedburg, Christoph; Gottlob, Irene; Jacobson, Samuel G.; Kellner, Ulrich; Koenekoop, Robert; Kohl, Susanne; Leroy, Bart P.; Lorenz, Birgit; McLean, Rebecca; Meire, Francoise; Meunier, Isabelle; Munier, Francis; de Ravel, Thomy; Reiff, Charlotte M.; Mohand-Saïd, Saddek; Sharon, Dror; Schorderet, Daniel; Schwartz, Sharon; Zanlonghi, Xavier; Condroyer, Christel; MacDonald, Heather; Verdet, Robert; Sahel, José-Alain; Hamel, Christian P.; Zeitz, Christina; Héon, Elise

2016-01-01

Congenital stationary night blindness (CSNB) is a heterogeneous group of non-progressive inherited retinal disorders with characteristic electroretinogram (ERG) abnormalities. Riggs and Schubert-Bornschein are subtypes of CSNB and demonstrate distinct ERG features. Riggs CSNB demonstrates selective rod photoreceptor dysfunction and occurs due to mutations in genes encoding proteins involved in rod phototransduction cascade; night blindness is the only symptom and eye examination is otherwise normal. Schubert-Bornschein CSNB is a consequence of impaired signal transmission between the photoreceptors and bipolar cells. Schubert-Bornschein CSNB is subdivided into complete CSNB with an ON bipolar signaling defect and incomplete CSNB with both ON and OFF pathway involvement. Both subtypes are associated with variable degrees of night blindness or photophobia, reduced visual acuity, high myopia, and nystagmus. Whole-exome sequencing of a family screened negative for mutations in genes associated with CSNB identified biallelic mutations in the guanine nucleotide-binding protein subunit beta-3 gene (GNB3). Two siblings were compound heterozygous for a deletion (c.170_172delAGA [p.Lys57del]) and a nonsense mutation (c.1017G>A [p.Trp339∗]). The maternal aunt was homozygous for the nonsense mutation (c.1017G>A [p.Trp339∗]). Mutational analysis of GNB3 in a cohort of 58 subjects with CSNB identified a sporadic case individual with a homozygous GNB3 mutation (c.200C>T [p.Ser67Phe]). GNB3 encodes the β subunit of G protein heterotrimer (Gαβγ) and is known to modulate ON bipolar cell signaling and cone transducin function in mice. Affected human subjects showed an unusual CSNB phenotype with variable degrees of ON bipolar dysfunction and reduced cone sensitivity. This unique retinal disorder with dual anomaly in visual processing expands our knowledge about retinal signaling. PMID:27063057

A novel AVPR2 splice site mutation leads to partial X-linked nephrogenic diabetes insipidus in two brothers.

PubMed

Schernthaner-Reiter, Marie Helene; Adams, David; Trivellin, Giampaolo; Ramnitz, Mary Scott; Raygada, Margarita; Golas, Gretchen; Faucz, Fabio R; Nilsson, Ola; Nella, Aikaterini A; Dileepan, Kavitha; Lodish, Maya; Lee, Paul; Tifft, Cynthia; Markello, Thomas; Gahl, William; Stratakis, Constantine A

2016-05-01

X-linked nephrogenic diabetes insipidus (NDI, OMIM#304800) is caused by mutations in the arginine vasopressin (AVP, OMIM*192340) receptor type 2 (AVPR2, OMIM*300538) gene. A 20-month-old boy and his 8-year-old brother presented with polyuria, polydipsia, and failure to thrive. Both boys demonstrated partial DDAVP (1-desamino-8-D AVP or desmopressin) responses; thus, NDI diagnosis was delayed. While routine sequencing of AVPR2 showed a potential splice site variant, it was not until exome sequencing confirmed the AVPR2 splice site variant and did not reveal any more likely candidates that the patients' diagnosis was made and proper treatment was instituted. Both patients were hemizygous for two AVPR2 variants predicted in silico to affect AVPR2 messenger RNA (mRNA) splicing. A minigene assay revealed that the novel AVPR2 c.276A>G mutation creates a novel splice acceptor site leading to 5' truncation of AVPR2 exon 2 in HEK293 human kidney cells. Both patients have been treated with high-dose DDAVP with a remarkable improvement of their symptoms and accelerated linear growth and weight gain. We present here a unique case of partial X-linked NDI due to an AVPR2 splice site mutation; patients with diabetes insipidus of unknown etiology may harbor splice site mutations that are initially underestimated in their pathogenicity on sequence analysis. • X-linked nephrogenic diabetes insipidus is caused by AVPR2 mutations, and disease severity can vary depending on the functional effect of the mutation. What is New: • We demonstrate here that a splice site mutation in AVPR2 leads to partial X-linked NDI in two brothers. • Treatment with high-dose DDAVP led to improvement of polyuria and polydipsia, weight gain, and growth.

Selected exonic sequencing of the AGXT gene provides a genetic diagnosis in 50% of patients with primary hyperoxaluria type 1.

PubMed

Williams, Emma; Rumsby, Gill

2007-07-01

Definitive diagnosis of primary hyperoxaluria type 1 (PH1) requires analysis of alanine:glyoxylate aminotransferase (AGT) activity in the liver. We have previously shown that targeted screening for the 3 most common mutations in the AGXT gene (c.33_34insC, c.508G>A, and c.731T>C) can provide a molecular diagnosis in 34.5% of PH1 patients, eliminating the need for a liver biopsy. Having reviewed the distribution of all AGXT mutations, we have evaluated a diagnostic strategy that uses selected exon sequencing for the molecular diagnosis of PH1. We sequenced exons 1, 4, and 7 for 300 biopsy-confirmed PH1 patients and expressed the identified missense mutations in vitro. Our identification of at least 1 mutation in 224 patients (75%) and 2 mutations in 149 patients increased the diagnostic sensitivity to 50%. We detected 29 kinds of sequence changes, 15 of which were novel. Four of these mutations were in exon 1 (c.2_3delinsAT, c.30_32delCC, c.122G>A, c.126delG), 7 were in exon 4 (c.447_454delGCTGCTGT, c.449T>C, c.473C>T, c.481G>A, c.481G>T, c.497T>C, c.424-2A>G), and 4 were in exon 7 (c.725insT, c.737G>A, c.757T>C, c.776 + 1G>A). The missense changes were associated with severely decreased AGT catalytic activity and negative immunoreactivity when expressed in vitro. Missense mutation c.26C>A, previously described as a pathological mutation, had activity similar to that of the wild-type enzyme. Selective exon sequencing can allow a definitive diagnosis in 50% of PH1 patients. The test offers a rapid turnaround time (15 days) with minimal risk to the patient. Demonstration of the expression of missense changes is essential to demonstrate pathogenicity.

Recessive HYDIN Mutations Cause Primary Ciliary Dyskinesia without Randomization of Left-Right Body Asymmetry

PubMed Central

Olbrich, Heike; Schmidts, Miriam; Werner, Claudius; Onoufriadis, Alexandros; Loges, Niki T.; Raidt, Johanna; Banki, Nora Fanni; Shoemark, Amelia; Burgoyne, Tom; Al Turki, Saeed; Hurles, Matthew E.; Köhler, Gabriele; Schroeder, Josef; Nürnberg, Gudrun; Nürnberg, Peter; Chung, Eddie M.K.; Reinhardt, Richard; Marthin, June K.; Nielsen, Kim G.; Mitchison, Hannah M.; Omran, Heymut

2012-01-01

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder characterized by defective cilia and flagella motility. Chronic destructive-airway disease is caused by abnormal respiratory-tract mucociliary clearance. Abnormal propulsion of sperm flagella contributes to male infertility. Genetic defects in most individuals affected by PCD cause randomization of left-right body asymmetry; approximately half show situs inversus or situs ambiguous. Almost 70 years after the hy3 mouse possessing Hydin mutations was described as a recessive hydrocephalus model, we report HYDIN mutations in PCD-affected persons without hydrocephalus. By homozygosity mapping, we identified a PCD-associated locus, chromosomal region 16q21-q23, which contains HYDIN. However, a nearly identical 360 kb paralogous segment (HYDIN2) in chromosomal region 1q21.1 complicated mutational analysis. In three affected German siblings linked to HYDIN, we identified homozygous c.3985G>T mutations that affect an evolutionary conserved splice acceptor site and that subsequently cause aberrantly spliced transcripts predicting premature protein termination in respiratory cells. Parallel whole-exome sequencing identified a homozygous nonsense HYDIN mutation, c.922A>T (p.Lys307∗), in six individuals from three Faroe Island PCD-affected families that all carried an 8.8 Mb shared haplotype across HYDIN, indicating an ancestral founder mutation in this isolated population. We demonstrate by electron microscopy tomography that, consistent with the effects of loss-of-function mutations, HYDIN mutant respiratory cilia lack the C2b projection of the central pair (CP) apparatus; similar findings were reported in Hydin-deficient Chlamydomonas and mice. High-speed videomicroscopy demonstrated markedly reduced beating amplitudes of respiratory cilia and stiff sperm flagella. Like the hy3 mouse model, all nine PCD-affected persons had normal body composition because nodal cilia function is apparently not dependent on the function of the CP apparatus. PMID:23022101

TGM5 mutations impact epidermal differentiation in acral peeling skin syndrome.

PubMed

Pigors, Manuela; Kiritsi, Dimitra; Cobzaru, Cristina; Schwieger-Briel, Agnes; Suárez, Jose; Faletra, Flavio; Aho, Heikki; Mäkelä, Leeni; Kern, Johannes S; Bruckner-Tuderman, Leena; Has, Cristina

2012-10-01

Acral peeling skin syndrome (APSS) is an autosomal recessive skin disorder characterized by acral blistering and peeling of the outermost layers of the epidermis. It is caused by mutations in the gene for transglutaminase 5, TGM5. Here, we report on clinical and molecular findings in 11 patients and extend the TGM5 mutation database by four, to our knowledge, previously unreported mutations: p.M1T, p.L41P, p.L214CfsX15, and p.S604IfsX9. The recurrent mutation p.G113C was found in 9 patients, but also in 3 of 100 control individuals in a heterozygous state, indicating that APSS might be more widespread than hitherto expected. Using quantitative real-time PCR, immunoblotting, and immunofluorescence analysis, we demonstrate that expression and distribution of several epidermal differentiation markers and corneodesmosin (CDSN) is altered in APSS keratinocytes and skin. Although the expression of transglutaminases 1 and 3 was not changed, we found an upregulation of keratin 1, keratin 10, involucrin, loricrin, and CDSN, probably as compensatory mechanisms for stabilization of the epidermal barrier. Our results give insights into the consequences of TGM5 mutations on terminal epidermal differentiation.

Two co-existing germline mutations P53 V157D and PMS2 R20Q promote tumorigenesis in a familial cancer syndrome.

PubMed

Wang, Zuoyun; Sun, Yihua; Gao, Bin; Lu, Yi; Fang, Rong; Gao, Yijun; Xiao, Tian; Liu, Xin-Yuan; Pao, William; Zhao, Yun; Chen, Haiquan; Ji, Hongbin

2014-01-01

Germline mutations are responsible for familial cancer syndromes which account for approximately 5-10% of all types of cancers. These mutations mainly occur at tumor suppressor genes or genome stability genes, such as DNA repair genes. Here we have identified a cancer predisposition family, in which eight members were inflicted with a wide spectrum of cancer including one diagnosed with lung cancer at 22years old. Sequencing analysis of tumor samples as well as histologically normal specimens identified two germline mutations co-existing in the familial cancer syndrome, the mutation of tumor suppressor gene P53 V157D and mismatch repair gene PMS2 R20Q. We further demonstrate that P53 V157D and/or PMS2 R20Q mutant promotes lung cancer cell proliferation. These two mutants are capable of promoting colony formation in soft agar as well as tumor formation in transgenic drosophila system. Collectively, these data have uncovered the important role of co-existing germline P53 and PMS2 mutations in the familial cancer syndrome development. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Two co-existing germline mutations P53 V157D and PMS2 R20Q promote tumorigenesis in a familial cancer syndrome

PubMed Central

Wang, Zuoyun; Sun, Yihua; Gao, Bin; Lu, Yi; Fang, Rong; Gao, Yijun; Xiao, Tian; Liu, Xin-Yuan; Pao, William; Zhao, Yun; Chen, Haiquan; Ji, Hongbin

2014-01-01

Germline mutations are responsible for familial cancer syndromes which account for approximately 5–10% of all types of cancers. These mutations mainly occur at tumor suppressor genes or genome stability genes, such as DNA repair genes. Here we have identified a cancer predisposition family, in which eight members were inflicted with a wide spectrum of cancer including one diagnosed with lung cancer at 22 years old. Sequencing analysis of tumor samples as well as histologically normal specimens identified two germline mutations co-existing in the familial cancer syndrome, the mutation of tumor suppressor gene P53 V157D and mismatch repair gene PMS2 R20Q. We further demonstrate that P53 V157D and/or PMS2 R20Q mutant promotes lung cancer cell proliferation. These two mutants are capable of promoting colony formation in soft agar as well as tumor formation in transgenic drosophila system. Collectively, these data have uncovered the important role of co-existing germline P53 and PMS2 mutations in the familial cancer syndrome development. PMID:23981578

R124C mutation of the betaIGH3 gene leads to remarkable phenotypic variability in a Greek four-generation family with lattice corneal dystrophy type 1.

PubMed

Hellenbroich, Y; Tzivras, G; Neppert, B; Schwinger, E; Zühlke, C

2001-01-01

Five autosomal dominantly inherited corneal dystrophies are caused by missense mutations in the betaIGH3 gene on chromosome 5q31. Here we describe the clinical features and the analysis of the betaIGH3 gene in a Greek four-generation family with lattice corneal dystrophy type 1 (CDL1). Sequencing of the betaIGH3 cDNA from an affected family member revealed the R124C mutation. More recent data indicate that this is probably a mutation hot spot in CDL1. We could not find a common haplotype with another CDL1 family with the R124C mutation demonstrating that this mutation occurs independently in different families. The clinical course of the disease showed a remarkable variability between the affected family members. To investigate a possible role between the phenotypic variability and apolipoprotein E (ApoE), which co-localises with amyloid deposits in CDL1, we determined the ApoE genotype of all family members. The resulting data revealed no association with the variable clinical course. Copyright 2001 S. Karger AG, Basel

Identification and Functional Characterisation of Novel Glucokinase Mutations Causing Maturity-Onset Diabetes of the Young in Slovakia

PubMed Central

Valentínová, Lucia; Beer, Nicola L.; Staník, Juraj; Tribble, Nicholas D.; van de Bunt, Martijn; Hučková, Miroslava; Barrett, Amy; Klimeš, Iwar; Gašperíková, Daniela; Gloyn, Anna L.

2012-01-01

Heterozygous glucokinase (GCK) mutations cause a subtype of maturity-onset diabetes of the young (GCK-MODY). Over 600 GCK mutations have been reported of which ∼65% are missense. In many cases co-segregation has not been established and despite the importance of functional studies in ascribing pathogenicity for missense variants these have only been performed for <10% of mutations. The aim of this study was to determine the minimum prevalence of GCK-MODY amongst diabetic subjects in Slovakia by sequencing GCK in 100 Slovakian probands with a phenotype consistent with GCK-MODY and to explore the pathogenicity of identified variants through family and functional studies. Twenty-two mutations were identified in 36 families (17 missense) of which 7 (I110N, V200A, N204D, G258R, F419S, c.580-2A>C, c.1113–1114delGC) were novel. Parental DNA was available for 22 probands (covering 14/22 mutations) and co-segregation established in all cases. Bioinformatic analysis predicted all missense mutations to be damaging. Nine (I110N, V200A, N204D, G223S, G258R, F419S, V244G, L315H, I436N) mutations were functionally evaluated. Basic kinetic analysis explained pathogenicity for 7 mutants which showed reduced glucokinase activity with relative activity indices (RAI) between 0.6 to <0.001 compared to wild-type GCK (1.0). For the remaining 2 mutants additional molecular mechanisms were investigated. Differences in glucokinase regulatory protein (GKRP) –mediated-inhibition of GCK were observed for both L315H & I436N when compared to wild type (IC50 14.6±0.1 mM & 20.3±1.6 mM vs.13.3±0.1 mM respectively [p<0.03]). Protein instability as assessed by thermal lability studies demonstrated that both L315H and I436N show marked thermal instability compared to wild-type GCK (RAI at 55°C 8.8±0.8% & 3.1±0.4% vs. 42.5±3.9% respectively [p<0.001]). The minimum prevalence of GCK-MODY amongst Slovakian patients with diabetes was 0.03%. In conclusion, we have identified 22 GCK mutations in 36 Slovakian probands and demonstrate that combining family, bioinformatic and functional studies can aid the interpretation of variants identified by molecular diagnostic screening. PMID:22493702

Novel nonsense mutation of the endothelin-B receptor gene in a family with Waardenburg-Hirschsprung disease.

PubMed

Syrris, P; Carter, N D; Patton, M A

1999-11-05

Waardenburg syndrome (WS) comprises sensorineural hearing loss, hypopigmentation of skin and hair, and pigmentary disturbances of the irides. Four types of WS have been classified to date; in WS type IV (WS4), patients additionally have colonic aganglionosis (Hirschsprung disease, HSCR). Mutations in the endothelin-3 (EDN3), endothelin-B receptor (EDNRB), and Sox10 genes have been identified as causative for WS type IV. We screened a family with a combined WS-HSCR phenotype for mutations in the EDNRB locus using standard DNA mutation analysis and sequencing techniques. We have identified a novel nonsense mutation at codon 253 (CGA-->TGA, Arg-->STOP). This mutation leads to a premature end of the translation of EDNRB at exon 3, and it is predicted to produce a truncated and nonfunctional endothelin-B receptor. All affected relatives were heterozygous for the Arg(253)-->STOP mutation, whereas it was not observed in over 50 unrelated individuals used as controls. These data confirm the role of EDNRB in the cause of the Waardenburg-Hirschsprung syndrome and demonstrate that in WS-HSCR there is a lack of correlation between phenotype and genotype and a variable expression of disease even within the same family. Copyright 1999 Wiley-Liss, Inc.

In Vivo Rat T-Lymphocyte Pig-a Assay: Detection and Expansion of Cells Deficient in the GPI-Anchored CD48 Surface Marker for Analysis of Mutation in the Endogenous Pig-a Gene.

PubMed

Dobrovolsky, Vasily N; Revollo, Javier; Petibone, Dayton M; Heflich, Robert H

2017-01-01

The Pig-a assay is being developed as an in vivo gene mutation assay for regulatory safety assessments. The assay is based on detecting mutation in the endogenous Pig-a gene of treated rats by using flow cytometry to measure changes in cell surface markers of peripheral blood cells. Here we present a methodology for demonstrating that phenotypically mutant rat T-cells identified by flow cytometry contain mutations in the Pig-a gene, an important step for validating the assay. In our approach, the mutant phenotype T-cells are sorted into individual wells of 96-well plates and expanded into clones. Subsequent sequencing of genomic DNA from the expanded clones confirms that the Pig-a assay detects exactly what it claims to detect-cells with mutations in the endogenous Pig-a gene. In addition, determining the spectra of Pig-a mutations provides information for better understanding the mutational mechanism of compounds of interest. Our methodology of combining phenotypic antibody labeling, magnetic enrichment, sorting, and single-cell clonal expansion can be used in genotoxicity/mutagenicity studies and in other general immunotoxicology research requiring identification, isolation, and expansion of extremely rare subpopulations of T-cells.

Phenotype/genotype correlation in a case series of Stargardt's patients identifies novel mutations in the ABCA4 gene.

PubMed

Gemenetzi, M; Lotery, A J

2013-11-01

To investigate phenotypic variability in terms of best-corrected visual acuity (BCVA) in patients with Stargardt disease (STGD) and confirmed ABCA4 mutations. Entire coding region analysis of the ABCA4 gene by direct sequencing of seven patients with clinical findings of STGD seen in the Retina Clinics of Southampton Eye Unit between 2002 and 2011.Phenotypic variables recorded were BCVA, fluorescein angiographic appearance, electrophysiology, and visual fields. All patients had heterozygous amino acid-changing variants (missense mutations) in the ABCA4 gene. A splice sequence change was found in a 30-year-old patient with severly affected vision. Two novel sequence changes were identified: a missense mutation in a mildly affected 44-year-old patient and a frameshift mutation in a severly affected 34-year-old patient. The identified ABCA4 mutations were compatible with the resulting phenotypes in terms of BCVA. Higher BCVAs were recorded in patients with missense mutations. Sequence changes, predicted to have more deleterious effect on protein function, resulted in a more severe phenotype. This case series of STGD patients demonstrates novel genotype/phenotype correlations, which may be useful to counselling of patients. This information may prove useful in selection of candidates for clinical trials in ABCA4 disease.

The Sequences of 1504 Mutants in the Model Rice Variety Kitaake Facilitate Rapid Functional Genomic Studies

PubMed Central

Pham, Nikki T.; Wei, Tong; Schackwitz, Wendy S.; Lipzen, Anna M.; Duong, Phat Q.; Jones, Kyle C.; Ruan, Deling; Bauer, Diane; Peng, Yi; Schmutz, Jeremy

2017-01-01

The availability of a whole-genome sequenced mutant population and the cataloging of mutations of each line at a single-nucleotide resolution facilitate functional genomic analysis. To this end, we generated and sequenced a fast-neutron-induced mutant population in the model rice cultivar Kitaake (Oryza sativa ssp japonica), which completes its life cycle in 9 weeks. We sequenced 1504 mutant lines at 45-fold coverage and identified 91,513 mutations affecting 32,307 genes, i.e., 58% of all rice genes. We detected an average of 61 mutations per line. Mutation types include single-base substitutions, deletions, insertions, inversions, translocations, and tandem duplications. We observed a high proportion of loss-of-function mutations. We identified an inversion affecting a single gene as the causative mutation for the short-grain phenotype in one mutant line. This result reveals the usefulness of the resource for efficient, cost-effective identification of genes conferring specific phenotypes. To facilitate public access to this genetic resource, we established an open access database called KitBase that provides access to sequence data and seed stocks. This population complements other available mutant collections and gene-editing technologies. This work demonstrates how inexpensive next-generation sequencing can be applied to generate a high-density catalog of mutations. PMID:28576844

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.

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

Li, Guotian; Jain, Rashmi; Chern, Mawsheng

The availability of a whole-genome sequenced mutant population and the cataloging of mutations of each line at a single-nucleotide resolution facilitate functional genomic analysis. To this end, we generated and sequenced a fast-neutron-induced mutant population in the model rice cultivar Kitaake (Oryza sativa ssp japonica), which completes its life cycle in 9 weeks. We sequenced 1504 mutant lines at 45-fold coverage and identified 91,513 mutations affecting 32,307 genes, i.e., 58% of all rice genes. We detected an average of 61 mutations per line. Mutation types include single-base substitutions, deletions, insertions, inversions, translocations, and tandem duplications. We observed a high proportionmore » of loss-of-function mutations. We identified an inversion affecting a single gene as the causative mutation for the short-grain phenotype in one mutant line. This result reveals the usefulness of the resource for efficient, cost-effective identification of genes conferring specific phenotypes. To facilitate public access to this genetic resource, we established an open access database called KitBase that provides access to sequence data and seed stocks. This population complements other available mutant collections and gene-editing technologies. In conclusion, this work demonstrates how inexpensive next-generation sequencing can be applied to generate a high-density catalog of mutations.« less

Improving diagnosis and broadening the phenotypes in early-onset seizure and severe developmental delay disorders through gene panel analysis.

PubMed

Trump, Natalie; McTague, Amy; Brittain, Helen; Papandreou, Apostolos; Meyer, Esther; Ngoh, Adeline; Palmer, Rodger; Morrogh, Deborah; Boustred, Christopher; Hurst, Jane A; Jenkins, Lucy; Kurian, Manju A; Scott, Richard H

2016-05-01

We sought to investigate the diagnostic yield and mutation spectrum in previously reported genes for early-onset epilepsy and disorders of severe developmental delay. In 400 patients with these disorders with no known underlying aetiology and no major structural brain anomaly, we analysed 46 genes using a combination of targeted sequencing on an Illumina MiSeq platform and targeted, exon-level microarray copy number analysis. We identified causative mutations in 71/400 patients (18%). The diagnostic rate was highest among those with seizure onset within the first two months of life (39%), although overall it was similar in those with and without seizures. The most frequently mutated gene was SCN2A (11 patients, 3%). Other recurrently mutated genes included CDKL5, KCNQ2, SCN8A (six patients each), FOXG1, MECP2, SCN1A, STXBP1 (five patients each), KCNT1, PCDH19, TCF4 (three patients each) and ATP1A3, PRRT2 and SLC9A6 (two patients each). Mutations in EHMT1, GABRB3, LGI1, MBD5, PIGA, UBE3A and ZEB2 were each found in single patients. We found mutations in a number of genes in patients where either the electroclinical features or dysmorphic phenotypes were atypical for the identified gene. In only 11 cases (15%) had the clinician sufficient certainty to specify the mutated gene as the likely cause before testing. Our data demonstrate the considerable utility of a gene panel approach in the diagnosis of patients with early-onset epilepsy and severe developmental delay disorders., They provide further insights into the phenotypic spectrum and genotype-phenotype correlations for a number of the causative genes and emphasise the value of exon-level copy number testing in their analysis. 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/

Primary tumor sidedness is an independent prognostic marker for survival in metastatic colorectal cancer: Results from a large retrospective cohort with mutational analysis.

PubMed

Kamran, Sophia C; Clark, Jeffrey W; Zheng, Hui; Borger, Darrell R; Blaszkowsky, Lawrence S; Allen, Jill N; Kwak, Eunice L; Wo, Jennifer Y; Parikh, Aparna R; Nipp, Ryan D; Murphy, Janet E; Goyal, Lipika; Zhu, Andrew X; Iafrate, A John; Corcoran, Ryan B; Ryan, David P; Hong, Theodore S

2018-05-17

Recent reports demonstrate inferior outcomes associated with primary right-sided vs left-sided colorectal tumors in patients with metastatic colorectal cancer (mCRC). We sought to describe our experience with mCRC patients on whom we have molecular data to determine whether primary tumor sidedness was an independent prognostic marker for overall survival (OS). mCRC patients with documented primary tumor sidedness who received mutational profiling between 2009 and 2014 were identified (n = 367, median follow-up 30.4 months). Mutational profiling for >150 mutations across commonly mutated cancer genes including RAS, PIK3CA, BRAF, and PTEN as well as treatment data, including receipt of a biologic agent, were collected. Univariable/multivariable models were used to analyze relationships between collected data and OS. Among 367 patients, sidedness breakdown was as follows: 234 left (64%), 133 right (36%). 56% were male, with a median age at diagnosis of 57 (range 24-89). A total of 143 patients had RAS mutations. Five-year OS was 41%, median OS was 54 months (range 1-149). Five-year OS for left- vs right-sided tumors was 46% vs 24% (P < .0001). On univariable analysis, among both RAS wildtype and mutant tumors, left-sided tumors continued to have improved OS vs right-sided tumors (HR: 0.49, 95% CI: 0.34-0.69 RAS wildtype; HR: 0.61, 95% CI: 0.40-0.95 RAS mutant). Left-sidedness was an important prognostic factor for OS among RAS wildtype patients despite treatment with or without a biologic agent (P < .05). Left-sidedness remained significant for improved OS on multivariable analysis (P < .0001). Left-sided primary tumor remained most important prognostic factor for OS, even when adjusting for mutational status and receipt of biologic agent. © 2018 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

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

Single-Step qPCR and dPCR Detection of Diverse CRISPR-Cas9 Gene Editing Events In Vivo.

PubMed

Falabella, Micol; Sun, Linqing; Barr, Justin; Pena, Andressa Z; Kershaw, Erin E; Gingras, Sebastien; Goncharova, Elena A; Kaufman, Brett A

2017-10-05

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based technology is currently the most flexible means to create targeted mutations by recombination or indel mutations by nonhomologous end joining. During mouse transgenesis, recombinant and indel alleles are often pursued simultaneously. Multiple alleles can be formed in each animal to create significant genetic complexity that complicates the CRISPR-Cas9 approach and analysis. Currently, there are no rapid methods to measure the extent of on-site editing with broad mutation sensitivity. In this study, we demonstrate the allelic diversity arising from targeted CRISPR editing in founder mice. Using this DNA sample collection, we validated specific quantitative and digital PCR methods (qPCR and dPCR, respectively) for measuring the frequency of on-target editing in founder mice. We found that locked nucleic acid (LNA) probes combined with an internal reference probe (Drop-Off Assay) provide accurate measurements of editing rates. The Drop-Off LNA Assay also detected on-target CRISPR-Cas9 gene editing in blastocysts with a sensitivity comparable to PCR-clone sequencing. Lastly, we demonstrate that the allele-specific LNA probes used in qPCR competitor assays can accurately detect recombinant mutations in founder mice. In summary, we show that LNA-based qPCR and dPCR assays provide a rapid method for quantifying the extent of on-target genome editing in vivo , testing RNA guides, and detecting recombinant mutations. Copyright © 2017 Falabella et al.

First molecular genotyping of insensitive acetylcholinesterase associated with malathion resistance in Culex quinquefasciatus Say populations in Malaysia.

PubMed

Low, Van Lun; Chen, Chee Dhang; Lim, Phaik Eem; Lee, Han Lim; Lim, Yvonne Ai Lian; Tan, Tiong Kai; Sofian-Azirun, Mohd

2013-12-01

Given that there is limited available information on the insensitive acetylcholinesterase in insect species in Malaysia, the present study aims to detect the presence of G119S mutation in the acetylcholinesterase gene of Culex quinquefasciatus from 14 residential areas across 13 states and a federal territory in Malaysia. The ace-1 sequence and PCR-RFLP test revealed the presence of glycine-serine ace-1 mutation in the wild populations of Cx. quinquefasciatus. Both direct sequencing and PCR-RFLP methods demonstrated similar results and revealed the presence of a heterozygous genotype at a very low frequency (18 out of 140 individuals), while a homozygous resistant genotype was not detected across any study site in Malaysia. In addition, statistical analysis also revealed that malathion resistance is associated with the frequency of ace-1(R) in Cx. quinquefasciatus populations. This study has demonstrated the first field-evolved instance of G119S mutation in Malaysian populations. Molecular identification of insensitive acetylcholinesterase provides significant insights into the evolution and adaptation of the Malaysian Cx. quinquefasciatus populations. © 2013 Society of Chemical Industry.

Text mining facilitates database curation - extraction of mutation-disease associations from Bio-medical literature.

PubMed

Ravikumar, Komandur Elayavilli; Wagholikar, Kavishwar B; Li, Dingcheng; Kocher, Jean-Pierre; Liu, Hongfang

2015-06-06

Advances in the next generation sequencing technology has accelerated the pace of individualized medicine (IM), which aims to incorporate genetic/genomic information into medicine. One immediate need in interpreting sequencing data is the assembly of information about genetic variants and their corresponding associations with other entities (e.g., diseases or medications). Even with dedicated effort to capture such information in biological databases, much of this information remains 'locked' in the unstructured text of biomedical publications. There is a substantial lag between the publication and the subsequent abstraction of such information into databases. Multiple text mining systems have been developed, but most of them focus on the sentence level association extraction with performance evaluation based on gold standard text annotations specifically prepared for text mining systems. We developed and evaluated a text mining system, MutD, which extracts protein mutation-disease associations from MEDLINE abstracts by incorporating discourse level analysis, using a benchmark data set extracted from curated database records. MutD achieves an F-measure of 64.3% for reconstructing protein mutation disease associations in curated database records. Discourse level analysis component of MutD contributed to a gain of more than 10% in F-measure when compared against the sentence level association extraction. Our error analysis indicates that 23 of the 64 precision errors are true associations that were not captured by database curators and 68 of the 113 recall errors are caused by the absence of associated disease entities in the abstract. After adjusting for the defects in the curated database, the revised F-measure of MutD in association detection reaches 81.5%. Our quantitative analysis reveals that MutD can effectively extract protein mutation disease associations when benchmarking based on curated database records. The analysis also demonstrates that incorporating discourse level analysis significantly improved the performance of extracting the protein-mutation-disease association. Future work includes the extension of MutD for full text articles.

DNA methylation-based reclassification of olfactory neuroblastoma.

PubMed

Capper, David; Engel, Nils W; Stichel, Damian; Lechner, Matt; Glöss, Stefanie; Schmid, Simone; Koelsche, Christian; Schrimpf, Daniel; Niesen, Judith; Wefers, Annika K; Jones, David T W; Sill, Martin; Weigert, Oliver; Ligon, Keith L; Olar, Adriana; Koch, Arend; Forster, Martin; Moran, Sebastian; Tirado, Oscar M; Sáinz-Japeado, Miguel; Mora, Jaume; Esteller, Manel; Alonso, Javier; Del Muro, Xavier Garcia; Paulus, Werner; Felsberg, Jörg; Reifenberger, Guido; Glatzel, Markus; Frank, Stephan; Monoranu, Camelia M; Lund, Valerie J; von Deimling, Andreas; Pfister, Stefan; Buslei, Rolf; Ribbat-Idel, Julika; Perner, Sven; Gudziol, Volker; Meinhardt, Matthias; Schüller, Ulrich

2018-05-05

Olfactory neuroblastoma/esthesioneuroblastoma (ONB) is an uncommon neuroectodermal neoplasm thought to arise from the olfactory epithelium. Little is known about its molecular pathogenesis. For this study, a retrospective cohort of n = 66 tumor samples with the institutional diagnosis of ONB was analyzed by immunohistochemistry, genome-wide DNA methylation profiling, copy number analysis, and in a subset, next-generation panel sequencing of 560 tumor-associated genes. DNA methylation profiles were compared to those of relevant differential diagnoses of ONB. Unsupervised hierarchical clustering analysis of DNA methylation data revealed four subgroups among institutionally diagnosed ONB. The largest group (n = 42, 64%, Core ONB) presented with classical ONB histology and no overlap with other classes upon methylation profiling-based t-distributed stochastic neighbor embedding (t-SNE) analysis. A second DNA methylation group (n = 7, 11%) with CpG island methylator phenotype (CIMP) consisted of cases with strong expression of cytokeratin, no or scarce chromogranin A expression and IDH2 hotspot mutation in all cases. T-SNE analysis clustered these cases together with sinonasal carcinoma with IDH2 mutation. Four cases (6%) formed a small group characterized by an overall high level of DNA methylation, but without CIMP. The fourth group consisted of 13 cases that had heterogeneous DNA methylation profiles and strong cytokeratin expression in most cases. In t-SNE analysis, these cases mostly grouped among sinonasal adenocarcinoma, squamous cell carcinoma, and undifferentiated carcinoma. Copy number analysis indicated highly recurrent chromosomal changes among Core ONB with a high frequency of combined loss of chromosome 1-4, 8-10, and 12. NGS sequencing did not reveal highly recurrent mutations in ONB, with the only recurrently mutated genes being TP53 and DNMT3A. In conclusion, we demonstrate that institutionally diagnosed ONB are a heterogeneous group of tumors. Expression of cytokeratin, chromogranin A, the mutational status of IDH2 as well as DNA methylation patterns may greatly aid in the precise classification of ONB.

Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons

PubMed Central

Arber, Charles; Bartolome, Fernando; de Vicente, Macarena; Houlden, Henry

2017-01-01

Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP. Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies. PMID:28360103

A characteristic phenotypic retinal appearance in Norrie disease.

PubMed

Drenser, Kimberly A; Fecko, Alice; Dailey, Wendy; Trese, Michael T

2007-02-01

To describe a striking retinal finding that the authors have only seen in Norrie disease eyes and to determine if a particular genotype corresponds to this dramatic presentation. This is a retrospective, interventional case report of four patients seen in the clinic over a 1-year period. All patients had analysis of the Norrie gene by direct sequencing. All patients presented with a similar retinal appearance of dense stalk tissue, globular dystrophic retina, and peripheral avascular retina with pigmentary changes. Each patient was found to have a mutation in the Norrie gene affecting a cystine residue in the cystine knot domain. The mutations are predicted to disrupt the structure of the protein product, norrin, which is required for activation of the Wnt receptor:beta-catenin pathway. No other vitreoretinopathy that the authors have seen demonstrates this characteristic retinal presentation of severe retinal dysplasia. All four patients were found to have mutations in the Norrie gene which alter the cystine knot motif. Mutations affecting this domain appear to have devastating effects on retinal development and indicate phenotype correlates with mutations affecting the cystine knot domain.

Polyclonal secondary FGFR2 mutations drive acquired resistance to FGFR inhibition in patients with FGFR2 fusion-positive cholangiocarcinoma

PubMed Central

Goyal, Lipika; Saha, Supriya K.; Liu, Leah Y.; Siravegna, Giulia; Leshchiner, Ignaty; Ahronian, Leanne G.; Lennerz, Jochen K.; Vu, Phuong; Deshpande, Vikram; Kambadakone, Avinash; Mussolin, Benedetta; Reyes, Stephanie; Henderson, Laura; Sun, Jiaoyuan Elisabeth; Van Seventer, Emily E.; Gurski, Joseph M.; Baltschukat, Sabrina; Schacher-Engstler, Barbara; Barys, Louise; Stamm, Christelle; Furet, Pascal; Ryan, David P.; Stone, James R.; Iafrate, A. John; Getz, Gad; Porta, Diana Graus; Tiedt, Ralph; Bardelli, Alberto; Juric, Dejan; Corcoran, Ryan B.; Bardeesy, Nabeel; Zhu, Andrew X.

2017-01-01

Genetic alterations in the fibroblast growth factor receptor (FGFR) pathway are promising therapeutic targets in many cancers, including intrahepatic cholangiocarcinoma (ICC). The FGFR inhibitor BGJ398 displayed encouraging efficacy in patients with FGFR2 fusion-positive ICC in a phase II trial, but the durability of response was limited in some patients. Here, we report the molecular basis for acquired resistance to BGJ398 in three patients via integrative genomic characterization of cell-free circulating tumor DNA (cfDNA), primary tumors, and metastases. Serial analysis of cfDNA demonstrated multiple recurrent point mutations in the FGFR2 kinase domain at progression. Accordingly, biopsy of post-progression lesions and rapid autopsy revealed marked inter- and intra-lesional heterogeneity, with different FGFR2 mutations in individual resistant clones. Molecular modeling and in vitro studies indicated that each mutation lead to BGJ398 resistance and was surmountable by structurally distinct FGFR inhibitors. Thus, polyclonal secondary FGFR2 mutations represent an important clinical resistance mechanism that may guide development of future therapeutic strategies. PMID:28034880

A novel mutation of the NDUFS7 gene leads to activation of a cryptic exon and impaired assembly of mitochondrial complex I in a patient with Leigh syndrome.

PubMed

Lebon, Sophie; Minai, Limor; Chretien, Dominique; Corcos, Johanna; Serre, Valérie; Kadhom, Noman; Steffann, Julie; Pauchard, Jean-Yves; Munnich, Arnold; Bonnefont, Jean-Paul; Rötig, Agnès

2007-01-01

Complex I deficiency is a frequent cause of mitochondrial disease as it accounts for one third of these disorders. By genotyping several putative disease loci using microsatellite markers we were able to describe a new NDUFS7 mutation in a consanguineous family with Leigh syndrome and isolated complex I deficiency. This mutation lies in the first intron of the NDUFS7 gene (c.17-1167 C>G) and creates a strong donor splice site resulting in the generation of a cryptic exon. This mutation is predicted to result in a shortened mutant protein of 41 instead of 213 amino acids containing only the first five amino acids of the normal protein. Analysis of the assembly state of the respiratory chain complexes under native condition revealed a marked decrease of fully assembled complex I while the quantity of the other complexes was not altered. These results report the first intronic NDUFS7 gene mutation and demonstrate the crucial role of NDUFS7 in the biogenesis of complex I.

Mutation in Torenia fournieri Lind. UFO homolog confers loss of TfLFY interaction and results in a petal to sepal transformation.

PubMed

Sasaki, Katsutomo; Yamaguchi, Hiroyasu; Aida, Ryutaro; Shikata, Masahito; Abe, Tomoko; Ohtsubo, Norihiro

2012-09-01

We identified a Torenia fournieri Lind. mutant (no. 252) that exhibited a sepaloid phenotype in which the second whorls were changed to sepal-like organs. This mutant had no stamens, and the floral organs consisted of sepals and carpels. Although the expression of a torenia class B MADS-box gene, GLOBOSA (TfGLO), was abolished in the 252 mutant, no mutation of TfGLO was found. Among torenia homologs such as APETALA1 (AP1), LEAFY (LFY), and UNUSUAL FLORAL ORGANS (UFO), which regulate expression of class B genes in Arabidopsis, only accumulation of the TfUFO transcript was diminished in the 252 mutant. Furthermore, a missense mutation was found in the coding region of the mutant TfUFO. Intact TfUFO complemented the mutant phenotype whereas mutated TfUFO did not; in addition, the transgenic phenotype of TfUFO-knockdown torenias coincided with the mutant phenotype. Yeast two-hybrid analysis revealed that the mutated TfUFO lost its ability to interact with TfLFY protein. In situ hybridization analysis indicated that the transcripts of TfUFO and TfLFY were partially accumulated in the same region. These results clearly demonstrate that the defect in TfUFO caused the sepaloid phenotype in the 252 mutant due to the loss of interaction with TfLFY. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

Common mutation causes cystinosis in the majority of black South African patients.

PubMed

Owen, E Patricia; Nandhlal, Jenisha; Leisegang, Felicity; Van der Watt, George; Nourse, Peter; Gajjar, Priya

2015-04-01

The mutations responsible for cystinosis in South African patients are currently unknown. A pertinent question is whether they are similar to those described elsewhere in the world. Children who were being managed for cystinosis in the Western Cape Province of South Africa between 2002 and 2013 were studied. All underwent molecular analysis to detect sequence variations in the cystinosis gene. This cohort study included 20 patients, 13 of whom were Xhosa-speaking black South Africans and seven were Cape Coloureds (mixed race); none were Caucasian. All had nephropathic infantile-type cystinosis with evidence of proximal tubulopathy, with glycosuria and renal phosphate wasting. Diagnosis was confirmed in 19 cases by demonstrating an elevated cystine concentration in leukocytes. Molecular analysis of the cystinosin gene revealed that 19 patients had a G > A mutation in intron 11 (CTNS-c.971-12G > A p.D324AfsX44) which caused an out-of-frame 10-bp insertion. Of these 19 patients, 16 were homozygous for this mutation, which was the most frequent mutation identified in the alleles of the black South African and Cape Coloured patients (96 and 71 %, respectively). We recommend that black South African and Cape Coloured patients presenting with cystinosis be tested for CTNS-c.971-12G > A in the first instance, with the possibility of prenatal testing being offered to at-risk families.

Glucose-6-phosphate dehydrogenase deficiency in northern Mexico and description of a novel mutation.

PubMed

García-Magallanes, N; Luque-Ortega, F; Aguilar-Medina, E M; Ramos-Payán, R; Galaviz-Hernández, C; Romero-Quintana, J G; Del Pozo-Yauner, L; Rangel-Villalobos, H; Arámbula-Meraz, E

2014-08-01

Glucose-6-phosphate dehydrogenase deficiency (G6PD) is the most common enzyme pathology in humans; it is X-linked inherited and causes neonatal hyperbilirubinaemia, chronic nonspherocytic haemolytic anaemia and drug-induced acute haemolytic anaemia. G6PD deficiency has scarcely been studied in the northern region of Mexico, which is important because of the genetic heterogeneity described in Mexican population. Therefore, samples from the northern Mexico were biochemically screened for G6PD deficiency, and PCR-RFLPs, and DNA sequencing used to identify mutations in positive samples. The frequency of G6PD deficiency in the population was 0.95% (n = 1993); the mutations in 86% of these samples were G6PD A(-202A/376G), G6PDA(-376G/968C) and G6PD Santamaria(376G/542T). Contrary to previous reports, we demonstrated that G6PD deficiency distribution is relatively homogenous throughout the country (P = 0.48336), and the unique exception with high frequency of G6PD deficiency does not involve a coastal population (Chihuahua: 2.4%). Analysis of eight polymorphic sites showed only 10 haplotypes. In one individual we identified a new G6PD mutation named Mexico DF(193A>G) (rs199474830), which probably results in a damaging functional effect, according to PolyPhen analysis. Proteomic impact of the mutation is also described.

Somatic Mutations and Neoepitope Homology in Melanomas Treated with CTLA-4 Blockade.

PubMed

Nathanson, Tavi; Ahuja, Arun; Rubinsteyn, Alexander; Aksoy, Bulent Arman; Hellmann, Matthew D; Miao, Diana; Van Allen, Eliezer; Merghoub, Taha; Wolchok, Jedd D; Snyder, Alexandra; Hammerbacher, Jeff

2017-01-01

Immune checkpoint inhibitors are promising treatments for patients with a variety of malignancies. Toward understanding the determinants of response to immune checkpoint inhibitors, it was previously demonstrated that the presence of somatic mutations is associated with benefit from checkpoint inhibition. A hypothesis was posited that neoantigen homology to pathogens may in part explain the link between somatic mutations and response. To further examine this hypothesis, we reanalyzed cancer exome data obtained from our previously published study of 64 melanoma patients treated with CTLA-4 blockade and a new dataset of RNA-Seq data from 24 of these patients. We found that the ability to accurately predict patient benefit did not increase as the analysis narrowed from somatic mutation burden, to inclusion of only those mutations predicted to be MHC class I neoantigens, to only including those neoantigens that were expressed or that had homology to pathogens. The only association between somatic mutation burden and response was found when examining samples obtained prior to treatment. Neoantigen and expressed neoantigen burden were also associated with response, but neither was more predictive than somatic mutation burden. Neither the previously described tetrapeptide signature nor an updated method to evaluate neoepitope homology to pathogens was more predictive than mutation burden. Cancer Immunol Res; 5(1); 84-91. ©2016 AACR. ©2016 American Association for Cancer Research.

Development of a PCR/LDR/capillary electrophoresis assay with potential for the detection of a beta-thalassemia fetal mutation in maternal plasma.

PubMed

Yi, Ping; Chen, Zhuqin; Yu, Lili; Zheng, Yingru; Liu, Guodong; Xie, Haichang; Zhou, Yuanguo; Zheng, Xiuhui; Han, Jian; Li, Li

2010-08-01

Analysis of fetal DNA in maternal plasma has recently been introduced for non-invasive prenatal diagnosis. We have now investigated the feasibility of polymerase chain reaction (PCR)/ligase detection reaction (LDR)/capillary electrophoresis for the detection of fetal point mutations, such as the beta-thalassemia mutation, IVS2 654(C --> T), in maternal plasma DNA. The sensitivity of LDR/capillary electrophoresis was examined by quantifying the mutant PCR products in the presence of a vast excess of non-mutant competitor template, a situation that mimics the detection of rare fetal mutations in the presence of excess maternal DNA. PCR/LDR/capillary electrophoresis was applied to detect the mutation, IVS2 654(C --> T), in an experimental model at different sensitivity levels and from 10 maternal plasma samples. Our results demonstrated that this approach to detect a low abundance IVS2 654(C --> T) mutation achieved a sensitivity of approximately 1:10,000. The approach was applied to maternal plasma DNA to detect the paternally inherited fetal IVS2 654(C --> T) mutation, and the results were equivalent to those obtained by PCR/reverse dot blot of amniotic fluid cell DNA. PCR/LDR/capillary electrophoresis has a very high sensitivity that can distinguish low abundance single nucleotide differences and can detect paternally inherited fetal point mutations in maternal plasma.

Recessive myosin myopathy with external ophthalmoplegia associated with MYH2 mutations.

PubMed

Tajsharghi, Homa; Hammans, Simon; Lindberg, Christopher; Lossos, Alexander; Clarke, Nigel F; Mazanti, Ingrid; Waddell, Leigh B; Fellig, Yakov; Foulds, Nicola; Katifi, Haider; Webster, Richard; Raheem, Olayinka; Udd, Bjarne; Argov, Zohar; Oldfors, Anders

2014-06-01

Myosin myopathies comprise a group of inherited diseases caused by mutations in myosin heavy chain (MyHC) genes. Homozygous or compound heterozygous truncating MYH2 mutations have been demonstrated to cause recessive myopathy with ophthalmoplegia, mild-to-moderate muscle weakness and complete lack of type 2A muscle fibers. In this study, we describe for the first time the clinical and morphological characteristics of recessive myosin IIa myopathy associated with MYH2 missense mutations. Seven patients of five different families with a myopathy characterized by ophthalmoplegia and mild-to-moderate muscle weakness were investigated. Muscle biopsy was performed to study morphological changes and MyHC isoform expression. Five of the patients were homozygous for MYH2 missense mutations, one patient was compound heterozygous for a missense and a nonsense mutation and one patient was homozygous for a frame-shift MYH2 mutation. Muscle biopsy demonstrated small or absent type 2A muscle fibers and reduced or absent expression of the corresponding MyHC IIa transcript and protein. We conclude that mild muscle weakness and ophthalmoplegia in combination with muscle biopsy demonstrating small or absent type 2A muscle fibers are the hallmark of recessive myopathy associated with MYH2 mutations.

De novo and rare mutations in the HSPA1L heat shock gene associated with inflammatory bowel disease.

PubMed

Takahashi, Shinichi; Andreoletti, Gaia; Chen, Rui; Munehira, Yoichi; Batra, Akshay; Afzal, Nadeem A; Beattie, R Mark; Bernstein, Jonathan A; Ennis, Sarah; Snyder, Michael

2017-01-26

Inflammatory bowel disease (IBD) is a chronic, relapsing inflammatory disease of the gastrointestinal tract which includes ulcerative colitis and Crohn's disease. Genetic risk factors for IBD are not well understood. We performed a family-based whole exome sequencing (WES) analysis on a core family (Family A) to identify potential causal mutations and then analyzed exome data from a Caucasian pediatric cohort (136 patients and 106 controls) to validate the presence of mutations in the candidate gene, heat shock 70 kDa protein 1-like (HSPA1L). Biochemical assays of the de novo and rare (minor allele frequency, MAF < 0.01) mutation variant proteins further validated the predicted deleterious effects of the identified alleles. In the proband of Family A, we found a heterozygous de novo mutation (c.830C > T; p.Ser277Leu) in HSPA1L. Through analysis of WES data of 136 patients, we identified five additional rare HSPA1L mutations (p.Gly77Ser, p.Leu172del, p.Thr267Ile, p.Ala268Thr, p.Glu558Asp) in six patients. In contrast, rare HSPA1L mutations were not observed in controls, and were significantly enriched in patients (P = 0.02). Interestingly, we did not find non-synonymous rare mutations in the HSP70 isoforms HSPA1A and HSPA1B. Biochemical assays revealed that all six rare HSPA1L variant proteins showed decreased chaperone activity in vitro. Moreover, three variants demonstrated dominant negative effects on HSPA1L and HSPA1A protein activity. Our results indicate that de novo and rare mutations in HSPA1L are associated with IBD and provide insights into the pathogenesis of IBD, and also expand our understanding of the roles of HSP70s in human disease.

Wolfram Syndrome in the Japanese Population; Molecular Analysis of WFS1 Gene and Characterization of Clinical Features

PubMed Central

Inoue, Hiroshi; Okuya, Shigeru; Ohta, Yasuharu; Akiyama, Masaru; Taguchi, Akihiko; Kora, Yukari; Okayama, Naoko; Yamada, Yuichiro; Wada, Yasuhiko; Amemiya, Shin; Sugihara, Shigetaka; Nakao, Yuzo; Oka, Yoshitomo; Tanizawa, Yukio

2014-01-01

Background Wolfram syndrome (WFS) is a recessive neurologic and endocrinologic degenerative disorder, and is also known as DIDMOAD (Diabetes Insipidus, early-onset Diabetes Mellitus, progressive Optic Atrophy and Deafness) syndrome. Most affected individuals carry recessive mutations in the Wolfram syndrome 1 gene (WFS1). However, the phenotypic pleiomorphism, rarity and molecular complexity of this disease complicate our efforts to understand WFS. To address this limitation, we aimed to describe complications and to elucidate the contributions of WFS1 mutations to clinical manifestations in Japanese patients with WFS. Methodology The minimal ascertainment criterion for diagnosing WFS was having both early onset diabetes mellitus and bilateral optic atrophy. Genetic analysis for WFS1 was performed by direct sequencing. Principal Findings Sixty-seven patients were identified nationally for a prevalence of one per 710,000, with 33 patients (49%) having all 4 components of DIDMOAD. In 40 subjects who agreed to participate in this investigation from 30 unrelated families, the earliest manifestation was DM at a median age of 8.7 years, followed by OA at a median age of 15.8 years. However, either OA or DI was the first diagnosed feature in 6 subjects. In 10, features other than DM predated OA. Twenty-seven patients (67.5%) had a broad spectrum of recessive mutations in WFS1. Two patients had mutations in only one allele. Eleven patients (27.5%) had intact WFS1 alleles. Ages at onset of both DM and OA in patients with recessive WFS1 mutations were indistinguishable from those in patients without WFS1 mutations. In the patients with predicted complete loss-of-function mutations, ages at the onsets of both DM and OA were significantly earlier than those in patients with predicted partial-loss-of function mutations. Conclusion/Significance This study emphasizes the clinical and genetic heterogeneity in patients with WFS. Genotype-phenotype correlations may exist in patients with WFS1 mutations, as demonstrated by the disease onset. PMID:25211237

Loss of SPEF2 Function in Mice Results in Spermatogenesis Defects and Primary Ciliary Dyskinesia1

PubMed Central

Sironen, Anu; Kotaja, Noora; Mulhern, Howard; Wyatt, Todd A.; Sisson, Joseph H.; Pavlik, Jacqueline A.; Miiluniemi, Mari; Fleming, Mark D.; Lee, Lance

2011-01-01

Primary ciliary dyskinesia (PCD) results from defects in motile cilia function. Mice homozygous for the mutation big giant head (bgh) have several abnormalities commonly associated with PCD, including hydrocephalus, male infertility, and sinusitis. In the present study, we use a variety of histopathological and cell biological techniques to characterize the bgh phenotype, and we identify the bgh mutation using a positional cloning approach. Histopathological, immunofluorescence, and electron microscopic analyses demonstrate that the male infertility results from shortened flagella and disorganized axonemal and accessory structures in elongating spermatids and mature sperm. In addition, there is a reduced number of elongating spermatids during spermatogenesis and mature sperm in the epididymis. Histological analyses show that the hydrocephalus is characterized by severe dilatation of the lateral ventricles and that bgh sinuses have an accumulation of mucus infiltrated by neutrophils. In contrast to the sperm phenotype, electron microscopy demonstrates that mutant respiratory epithelial cilia are ultrastructurally normal, but video microscopic analysis shows that their beat frequency is lower than that of wild-type cilia. Through a positional cloning approach, we identified two sequence variants in the gene encoding sperm flagellar protein 2 (SPEF2), which has been postulated to play an important role in spermatogenesis and flagellar assembly. A causative nonsense mutation was validated by Western blot analysis, strongly suggesting that the bgh phenotype results from the loss of SPEF2 function. Taken together, the data in this study demonstrate that SPEF2 is required for cilia function and identify a new genetic cause of PCD in mice. PMID:21715716

Retracing Evolution of Red Fluorescence in GFP-Like Proteins from Faviina Corals

PubMed Central

Field, Steven F.; Matz, Mikhail V.

2010-01-01

Proteins of the green fluorescent protein family represent a convenient experimental model to study evolution of novelty at the molecular level. Here, we focus on the origin of Kaede-like red fluorescent proteins characteristic of the corals of the Faviina suborder. We demonstrate, using an original approach involving resurrection and analysis of the library of possible evolutionary intermediates, that it takes on the order of 12 mutations, some of which strongly interact epistatically, to fully recapitulate the evolution of a red fluorescent phenotype from the ancestral green. Five of the identified mutations would not have been found without the help of ancestral reconstruction, because the corresponding site states are shared between extant red and green proteins due to their recent descent from a dual-function common ancestor. Seven of the 12 mutations affect residues that are not in close contact with the chromophore and thus must exert their effect indirectly through adjustments of the overall protein fold; the relevance of these mutations could not have been anticipated from the purely theoretical analysis of the protein's structure. Our results introduce a powerful experimental approach for comparative analysis of functional specificity in protein families even in the cases of pronounced epistasis, provide foundation for the detailed studies of evolutionary trajectories leading to novelty and complexity, and will help rational modification of existing fluorescent labels. PMID:19793832

Reduced expression of APC-1B but not APC-1A by the deletion of promoter 1B is responsible for familial adenomatous polyposis.

PubMed

Yamaguchi, Kiyoshi; Nagayama, Satoshi; Shimizu, Eigo; Komura, Mitsuhiro; Yamaguchi, Rui; Shibuya, Tetsuo; Arai, Masami; Hatakeyama, Seira; Ikenoue, Tsuneo; Ueno, Masashi; Miyano, Satoru; Imoto, Seiya; Furukawa, Yoichi

2016-05-24

Germline mutations in the tumor suppressor gene APC are associated with familial adenomatous polyposis (FAP). Here we applied whole-genome sequencing (WGS) to the DNA of a sporadic FAP patient in which we did not find any pathological APC mutations by direct sequencing. WGS identified a promoter deletion of approximately 10 kb encompassing promoter 1B and exon1B of APC. Additional allele-specific expression analysis by deep cDNA sequencing revealed that the deletion reduced the expression of the mutated APC allele to as low as 11.2% in the total APC transcripts, suggesting that the residual mutant transcripts were driven by other promoter(s). Furthermore, cap analysis of gene expression (CAGE) demonstrated that the deleted promoter 1B region is responsible for the great majority of APC transcription in many tissues except the brain. The deletion decreased the transcripts of APC-1B to 39-45% in the patient compared to the healthy controls, but it did not decrease those of APC-1A. Different deletions including promoter 1B have been reported in FAP patients. Taken together, our results strengthen the evidence that analysis of structural variations in promoter 1B should be considered for the FAP patients whose pathological mutations are not identified by conventional direct sequencing.

Integrated whole-genome and transcriptome sequence analysis reveals the genetic characteristics of a riboflavin-overproducing Bacillus subtilis.

PubMed

Wang, Guanglu; Shi, Ting; Chen, Tao; Wang, Xiaoyue; Wang, Yongcheng; Liu, Dingyu; Guo, Jiaxin; Fu, Jing; Feng, Lili; Wang, Zhiwen; Zhao, Xueming

2018-06-02

Commercial riboflavin production with Bacillus subtilis has been developed by combining rational and classical strain development for almost two decades, but how an improved riboflavin producer can be created rationally is still not completely understood. In this study, we demonstrate the combined use of integrated genomic and transcriptomic analysis of the genetic basis for riboflavin over-production in B. subtilis. This methodology succeeded in discerning the positive mutations in the mutagenesis derived riboflavin producer B. subtilis 24/pMX45 through whole-genome sequencing and transcriptome sequencing. These included RibC (G199D), ribD + (G+39A), PurA (P242L), CcpN(A44S), YvrH (R222Q) and two nonsense mutations YhcF (R90*) and YwaA (Q68*). Reintroducing these specific mutations into the wild-type strain recovered the riboflavin overproduction phenotype and subsequent metabolic engineering greatly improved riboflavin production, achieving an up to 3.4-fold increase of the riboflavin titer over the sequenced producer. A novel mutation, YvrH (R222Q), involved in a typical two-component regulatory system deregulated the purine de novo synthesis pathway and increased the pool of intracellular purine metabolites, which in turn increased riboflavin production. Taken together, we present a case study of combining genome and transcriptome analysis to elucidate the genetic underpinnings of a complex cellular property, which enabled the transfer of beneficial mutations to engineer a reference strain into an overproducer. Copyright © 2018 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Exploring the effect of D61G mutation on SHP2 cause gain of function activity by a molecular dynamics study.

PubMed

Li, Hong-Lian; Ma, Ying; Zheng, Chang-Jie; Jin, Wen-Yan; Liu, Wen-Shan; Wang, Run-Ling

2017-11-24

Noonan syndrome (NS) is a common autosomal dominant congenital disorder which could cause the congenital cardiopathy and cancer predisposition. Previous studies reported that the knock-in mouse models of the mutant D61G of SHP2 exhibited the major features of NS, which demonstrated that the mutation D61G of SHP2 could cause NS. To explore the effect of D61G mutation on SHP2 and explain the high activity of the mutant, molecular dynamic simulations were performed on wild type (WT) of SHP2 and the mutated SHP2-D61G, respectively. The principal component analysis and dynamic cross-correlation mapping, associated with secondary structure, showed that the D61G mutation affected the motions of two regions (residues Asn 58-Thr 59 and Val 460-His 462) in SHP2 from β to turn. Moreover, the residue interaction networks analysis, the hydrogen bond occupancy analysis and the binding free energies were calculated to gain detailed insight into the influence of the mutant D61G on the two regions, revealing that the major differences between SHP2-WT and SHP2-D61G were the different interactions between Gly 61 and Gly 462, Gly 61 and Ala 461, Gln 506 and Ile 463, Gly 61 and Asn 58, Ile 463 and Thr 466, Gly 462 and Cys 459. Consequently, our findings here may provide knowledge to understand the increased activity of SHP2 caused by the mutant D61G.

Gitelman syndrome in a South African family presenting with hypokalaemia and unusual food cravings.

PubMed

van der Merwe, Pieter Du Toit; Rensburg, Megan A; Haylett, William L; Bardien, Soraya; Davids, M Razeen

2017-01-26

Gitelman syndrome (GS) is an autosomal recessive renal tubular disorder characterised by renal salt wasting with hypokalaemia, metabolic alkalosis, hypomagnesaemia and hypocalciuria. It is caused by mutations in SLC12A3 encoding the sodium-chloride cotransporter on the apical membrane of the distal convoluted tubule. We report a South African family with five affected individuals presenting with hypokalaemia and unusual food cravings. The affected individuals and two unaffected first degree relatives were enrolled into the study. Phenotypes were evaluated through history, physical examination and biochemical analysis of blood and urine. Mutation screening was performed by sequencing of SLC12A3, and determining the allele frequencies of the sequence variants found in this family in 117 ethnically matched controls. The index patient, her sister, father and two aunts had a history of severe salt cravings, fatigue and tetanic episodes, leading to consumption of large quantities of salt and vinegar. All affected individuals demonstrated hypokalaemia with renal potassium wasting. Genetic analysis revealed that the pseudo-dominant pattern of inheritance was due to compound heterozygosity with two novel mutations: a S546G substitution in exon 13, and insertion of AGCCCC at c.1930 in exon 16. These variants were present in the five affected individuals, but only one variant each in the unaffected family members. Neither variant was found in any of the controls. The diagnosis of GS was established in five members of a South African family through clinical assessment, biochemical analysis and mutation screening of the SLC12A3 gene, which identified two novel putative pathogenic mutations.

Digital PCR analysis of plasma cell-free DNA for non-invasive detection of drug resistance mechanisms in EGFR mutant NSCLC: Correlation with paired tumor samples

PubMed Central

Ishii, Hidenobu; Azuma, Koichi; Sakai, Kazuko; Kawahara, Akihiko; Yamada, Kazuhiko; Tokito, Takaaki; Okamoto, Isamu; Nishio, Kazuto; Hoshino, Tomoaki

2015-01-01

As the development of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has become an issue of concern, identification of the mechanisms responsible has become an urgent priority. However, for research purposes, it is not easy to obtain tumor samples from patients with EGFR mutation-positive non-small-cell lung cancer (NSCLC) that has relapsed after treatment with EGFR-TKIs. Here, using digital PCR assay as an alternative and noninvasive method, we examined plasma and tumor samples from patients with relapsed NSCLC to establish the inter-relationships existing among T790M mutation, activating EGFR mutations, HER2 amplification, and MET amplification. Paired samples of tumor and blood were obtained from a total of 18 patients with NSCLC after they had developed resistance to EGFR-TKI treatment, and the mechanisms of resistance were analyzed by digital PCR. Digital PCR analysis of T790M mutation in plasma had a sensitivity of 81.8% and specificity of 85.7%, the overall concordance between plasma and tissue samples being 83.3%. MET gene copy number gain in tumor DNA was observed by digital PCR in three patients, of whom one exhibited positivity for MET amplification by FISH, whereas no patient demonstrated MET and HER2 copy number gain in plasma DNA. Digital PCR analysis of plasma is feasible and accurate for detection of T790M mutation in NSCLC that becomes resistant to treatment with EGFR-TKIs. PMID:26334838

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

Koivisto, U.M.; Viikari, J.S.; Kontula, K.

Two deletions of the low-density lipoprotein (LDL) receptor gene were previously shown to account for about two thirds of all mutations causing familial hypercholesterolemia (FH) in Finland. We screened the DNA samples from a cohort representing the remaining 30% of Finnish heterozygous FH patients by amplifying all the 18 exons of the receptor gene by PCR and searching for DNA variations with the SSCP technique. Ten novel mutations were identified, comprising two nonsense and seven missense mutations as well as one frameshift mutation caused by a 13-bp deletion. A single nucleotide change, substituting adenine for guanidine at position 2533 andmore » resulting in an amino acid change of glycine to aspartic acid at codon 823, was found in DNA samples from 14 unrelated FH probands. This mutation (FH-Turku) affects the sequence encoding the putative basolateral sorting signal of the LDL receptor protein; however, the exact functional consequences of this mutation are yet to be examined. The FH-Turku gene and another point mutation (Leu380{r_arrow}His or FH-Pori) together account for {approximately}8% of the FH-causing genes in Finland and are particularly common among FH patients from the southwestern part of the country (combined, 30%). Primer-introduced restriction analysis was applied for convenient assay of the FH-Turku and FH-Pori point mutations. In conclusion, this paper demonstrates the unique genetic background of FH in Finland and describes a commonly occurring FH gene with a missense mutation closest to the C terminus thus far reported. 32 refs., 5 figs., 2 tabs.« less

Novel Compound Heterozygous CLCNKB Gene Mutations (c.1755A>G/ c.848_850delTCT) Cause Classic Bartter Syndrome.

PubMed

Wang, Chunli; Chen, Ying; Zheng, Bixia; Zhu, Mengshu; Fan, Jia; Wang, Juejin; Jia, Zhanjun; Huang, Songming; Zhang, Aihua

2018-02-14

Inactivated variants in CLCNKB gene encoding the basolateral chloride channel ClC-Kb cause classic Bartter syndrome characterized by hypokalemic metabolic alkalosis and hyperreninemic hyperaldosteronism. Here we identified two cBS siblings presenting hypokalemia in a Chinese family due to novel compound heterozygous CLCNKB mutations (c.848_850delTCT/c.1755A>G). Compound heterozygosity was confirmed by amplifying and sequencing the patient's genomic DNA. The synonymous mutation c.1755A>G (Thr585Thr) was located at +2bp from the 5' splice donor site in exon 15, further transcript analysis demonstrated that this single nucleotide mutation causes exclusion of exon 15 in the cDNA from the proband and his mother. Furthermore, we investigated the expression and protein trafficking change of c.848_850delTCT (TCT) and exon 15 deletion（E15）mutation in vitro. The E15 mutation markedly decreased the expression of ClC-Kb and resulted in a low-molecular-weight band (~55kD) trapping in the endoplasmic reticulum, while the TCT mutant only decreased the total and plasma membrane ClC-Kb protein expression but did not affect the subcellular localization. Finally, we studied the physiological functions of mutations by using whole-cell patch clamp and found that E15 or TCT mutation decreased the current of ClC-Kb/barttin channel. These results suggested that the compound defective mutations of CLCNKB gene are the molecular mechanism of the two cBS siblings.

Muscular dystrophies due to defective glycosylation of dystroglycan

PubMed Central

Muntoni, F; Brockington, M; Godfrey, C; Ackroyd, M; Robb, S.; Manzur, A; Kinali, M; Mercuri, E; Kaluarachchi, M; Feng, L; Jimenez-Mallebrera, C.; Clement, E; Torelli, S; Sewry, CA; Brown, SC

2007-01-01

Summary Muscular dystrophies are a clinically and genetically heterogeneous group of disorders. Until recently most of the proteins associated with muscular dystrophies were believed to be proteins of the sarcolemma associated with reinforcing the plasma membrane or in facilitating its re-sealing following injury. In the last few years a novel and frequent pathogenic mechanism has been identified that involves the abnormal glycosylation of alpha-dystroglycan (ADG). This peripheral membrane protein undergoes complex and crucial glycosylation steps that enable it to interact with LG domain containing extracellular matrix proteins such as laminins, agrin and perlecan. Mutations in six genes (POMT1, POMT2, POMGnT1, fukutin, FKRP and LARGE) have been identified in patients with reduced glycosylation of ADG. While initially a clear correlation between gene defect and phenotype was observed for each of these 6 genes (for example, Walker Warburg syndrome was associated with mutations in POMT1 and POMT2, Fukuyama congenital muscular dystrophy associated with fukutin mutations, and Muscle Eye Brain disease associated with POMGnT1 mutations), we have recently demonstrated that allelic mutations in each of these 6 genes can result in a much wider spectrum of clinical conditions. Thus, the crucial aspect in determining the phenotypic severity is not which gene is primarily mutated, but how severely the mutation affects the glycosylation of ADG. Systematic mutation analysis of these 6 glycosyltransferases in patients with a dystroglycan glycosylation disorder identifies mutations in approximately 65% suggesting that more genes have yet to be identified. PMID:18646561

Identification of HNF4A Mutation p.T130I and HNF1A Mutations p.I27L and p.S487N in a Han Chinese Family with Early-Onset Maternally Inherited Type 2 Diabetes.

PubMed

Yang, Ying; Zhou, Tai-Cheng; Liu, Yong-Ying; Li, Xiao; Wang, Wen-Xue; Irwin, David M; Zhang, Ya-Ping

2016-01-01

Maturity-onset diabetes of the young (MODY) is characterized by the onset of diabetes before the age of 25 years, positive family history, high genetic predisposition, monogenic mutations, and an autosomal dominant mode of inheritance. Here, we aimed to investigate the mutations and to characterize the phenotypes of a Han Chinese family with early-onset maternally inherited type 2 diabetes. Detailed clinical assessments and genetic screening for mutations in the HNF4α, GCK, HNF-1α, IPF-1, HNF1β, and NEUROD1 genes were carried out in this family. One HNF4A mutation (p.T130I) and two HNF1A polymorphisms (p.I27L and p.S487N) were identified. Mutation p.T130I was associated with both early-onset and late-onset diabetes and caused downregulated HNF4A expression, whereas HNF1A polymorphisms p.I27L and p.S487N were associated with the age of diagnosis of diabetes. We demonstrated that mutation p.T130I in HNF4A was pathogenic as were the predicted polymorphisms p.I27L and p.S487N in HNF1A by genetic and functional analysis. Our results show that mutations in HNF4A and HNF1A genes might account for this early-onset inherited type 2 diabetes.

High MACC1 expression in combination with mutated KRAS G13 indicates poor survival of colorectal cancer patients.

PubMed

Ilm, Katharina; Kemmner, Wolfgang; Osterland, Marc; Burock, Susen; Koch, Gudrun; Herrmann, Pia; Schlag, Peter M; Stein, Ulrike

2015-02-14

The metastasis-associated in colon cancer 1 (MACC1) gene has been identified as prognostic biomarker for colorectal cancer (CRC). Here, we aimed at the refinement of risk assessment by separate and combined survival analyses of MACC1 expression with any of the markers KRAS mutated in codon 12 (KRAS G12) or codon 13 (KRAS G13), BRAF V600 mutation and MSI status in a retrospective study of 99 CRC patients with tumors UICC staged I, II and III. We showed that only high MACC1 expression (HR: 6.09, 95% CI: 2.50-14.85, P < 0.001) and KRAS G13 mutation (HR: 5.19, 95% CI: 1.06-25.45, P = 0.042) were independent prognostic markers for shorter metastasis-free survival (MFS). Accordingly, Cox regression analysis revealed that patients with high MACC1 expression and KRAS G13 mutation exhibited the worst prognosis (HR: 14.48, 95% CI: 3.37-62.18, P < 0.001). Patients were classified based on their molecular characteristics into four clusters with significant differences in MFS (P = 0.003) by using the SPSS 2-step cluster function and Kaplan-Meier survival analysis. According to our results, patients with high MACC1 expression and mutated KRAS G13 exhibited the highest risk for metachronous metastases formation. Moreover, we demonstrated that the "Traditional pathway" with an intermediate risk for metastasis formation can be further subdivided by assessing MACC1 expression into a low and high risk group with regard to MFS prognosis. This is the first report showing that identification of CRC patients at high risk for metastasis is possible by assessing MACC1 expression in combination with KRAS G13 mutation.

Promoter mutation is a common variant in GJC2-associated Pelizaeus-Merzbacher-like disease.

PubMed

Meyer, E; Kurian, M A; Morgan, N V; McNeill, A; Pasha, S; Tee, L; Younis, R; Norman, A; van der Knaap, M S; Wassmer, E; Trembath, R C; Brueton, L; Maher, E R

2011-12-01

Pelizaeus-Merzbacher-like disease (PMLD) is a clinically and genetically heterogeneous neurological disorder of cerebral hypomyelination. It is clinically characterised by early onset (usually infantile) nystagmus, impaired motor development, ataxia, choreoathetoid movements, dysarthria and progressive limb spasticity. We undertook autozygosity mapping studies in a large consanguineous family of Pakistani origin in which affected children had progressive lower limb spasticity and features of cerebral hypomyelination on MR brain imaging. SNP microarray and microsatellite marker analysis demonstrated linkage to chromosome 1q42.13-1q42.2. Direct sequencing of the gap junction protein gamma-2 gene, GJC2, identified a promoter region mutation (c.-167A>G) in the non-coding exon 1. The c.-167A>G promoter mutation was identified in a further 4 individuals from two families (who were also of Pakistani origin) with clinical and radiological features of PMLD in whom previous routine diagnostic screening of GJC2 had been reported as negative. A common haplotype was identified at the GJC2 locus in the three mutation-positive families, consistent with a common origin for the mutation and likely founder effect. This promoter mutation has only recently been reported in GJC2-PMLD but it has been postulated to affect the binding of the transcription factor SOX10 and appears to be a prevalent mutation, accounting for ~29% of reported patients with GJC2-PMLD. We propose that diagnostic screening of GJC2 should include sequence analysis of the non-coding exon 1, as well as the coding regions to avoid misdiagnosis or diagnostic delay in suspected PMLD. Copyright © 2011 Elsevier Inc. All rights reserved.

Effect of HFE gene polymorphism on sustained virological response in patients with chronic hepatitis C and elevated serum ferritin.

PubMed

Coelho-Borges, Silvia; Cheinquer, Hugo; Wolff, Fernando Herz; Cheinquer, Nelson; Krug, Luciano; Ashton-Prolla, Patricia

2012-01-01

Abnormal serum ferritin levels are found in approximately 20%-30% of the patients with chronic hepatitis C and are associated with a lower response rate to interferon therapy. To determine if the presence of HFE gene mutations had any effect on the sustained virological response rate to interferon based therapy in chronic hepatitis C patients with elevated serum ferritin. A total of 44 treatment naÏve patients with histologically demonstrated chronic hepatitis C, all infected with hepatitis C virus genotype non-1 (38 genotype 3; 6 genotype 2) and serum ferritin above 500 ng/mL were treated with interferon (3 MU, 3 times a week) and ribavirin (1.000 mg, daily) for 24 weeks. Sustained virological response was defined as negative qualitative HCV-RNA more than 24 weeks after the end of treatment. Serum HCV-RNA was measured by qualitative in house polymerase chain reaction with a limit of detection of 200 IU/mL. HFE gene mutation was detected using restriction-enzyme digestion with RsaI (C282Y mutation analysis) and BclI (H63D mutation analysis) in 16 (37%) patients, all heterozygous (11 H63D, 2 C282Y and 3 both). Sustained virological response was achieved in 0 of 16 patients with HFE gene mutations and 11 (41%) of 27 patients without HFE gene mutations (P = 0.002; exact Fisher test). Heterozigozity for H63D and/or C282Y HFE gene mutation predicts absence of sustained virological response to combination treatment with interferon and ribavirin in patients with chronic hepatitis C, non-1 genotype and serum ferritin levels above 500 ng/mL.

A novel loss-of-function mutation in GPR54/KISS1R leads to hypogonadotropic hypogonadism in a highly consanguineous family.

PubMed

Nimri, Revital; Lebenthal, Yael; Lazar, Liora; Chevrier, Lucie; Phillip, Moshe; Bar, Meytal; Hernandez-Mora, Eva; de Roux, Nicolas; Gat-Yablonski, Galia

2011-03-01

The G protein-coupled receptor 54 (GPR54), the kisspeptin receptor, is essential for stimulation of GnRH secretion and induction of puberty. Recently loss-of-function mutations of the GPR54 have been implicated as a cause of isolated idiopathic hypogonadotropic hypogonadism (IHH). The objective of the study was to identify the genetic cause of IHH in a consanguineous pedigree and to characterize the phenotypic features from infancy through early adulthood. In six patients with normosmic IHH belonging to two families of Israeli Muslim-Arab origin highly related to one another, DNA was analyzed for mutations in the GnRHR and GPR54 genes, with functional analysis of the mutation found. The five males underwent comprehensive endocrine evaluation and were under longitudinal follow-up; the one female presented in early adulthood. A new homozygous mutation (c.T815C) in GPR54 leading to a phenylalanine substitution by serine (p.F272S) was detected in all patients. Functional analysis showed an almost complete inhibition of kisspeptin-induced GPR54 signaling and a dramatic decrease of the mutated receptor expression at the cell surface. The males exhibited the same clinical features from infancy to adulthood, characterized by cryptorchidism, a relatively short penis, and no spontaneous pubertal development. The female patient presented at 18 yr with impuberism and primary amenorrhea. Repeated stimulation tests demonstrated complete gonadotropin deficiency throughout follow-up. A novel loss-of-function mutation (p.F272S) in the GPR54 gene is associated with familial normosmic IHH. Underdeveloped external genitalia and impuberism point to the major role of GPR54 in the activation of the gonadotropic axis from intrauterine life to adulthood.

Prognostic relevance of autophagy-related markers LC3, p62/sequestosome 1, Beclin-1 and ULK1 in colorectal cancer patients with respect to KRAS mutational status.

PubMed

Schmitz, Klaus Juergen; Ademi, Ceflije; Bertram, Stefanie; Schmid, Kurt Werner; Baba, Hideo Andreas

2016-07-22

Autophagy is a cellular pathway that regulates transportation of cytoplasmic macromolecules and organelles to lysosomes for degradation. Autophagy is involved in both tumorigenesis and tumour suppression. Here we investigated the potential prognostic value of the autophagy-related proteins Beclin-1, p62, LC3 and uncoordinated (UNC) 51-like kinase 1 (ULK1) in a cohort of colorectal cancer (CRC) specimens. In this study, we analysed the immunoexpression of the autophagy-related proteins p62, LC3, Beclin-1 and ULK1 in 127 CRC patients with known KRAS mutational status and detailed clinical follow-up. Survival analysis of p62 staining showed a significant correlation of cytoplasmic (not nuclear) p62 expression with a favourable tumour-specific overall survival (OS). The prognostic power of cytoplasmic p62 was found in the KRAS-mutated subgroup but was lost in the KRAS wildtype subgroup. Survival analysis of Beclin-1 staining did not show an association with OS in the complete cohort. LC3 overexpression demonstrated a slight, though not significant, association with decreased OS. Upon stratifying cases by KRAS mutational status, nuclear (not cytoplasmic) Beclin-1 staining was associated with a significantly decreased OS in the KRAS-mutated subgroup but not in the KRAS wildtype CRCs. In addition, LC3 overexpression was significantly associated with decreased OS in the KRAS-mutated CRC subgroup. ULK1 expression was not correlated to survival. Immunohistochemical analyses of LC3, p62 and Beclin-1 may constitute promising novel prognostic markers in CRC, especially in KRAS-mutated CRCs. This strategy might help in identifying high-risk patients who would benefit from autophagy-related anticancer drugs.

Insight into resistance mechanism of anaplastic lymphoma kinase to alectinib and JH-VIII-157-02 caused by G1202R solvent front mutation.

PubMed

Wang, Han; Wang, Yao; Guo, Wentao; Du, Bin; Huang, Xiaobing; Wu, Riping; Yang, Baoyu; Lin, Xiaoyan; Wu, Yilan

2018-01-01

Mutated anaplastic lymphoma kinase (ALK) drives the development of advanced non-small cell lung cancer (NSCLC). Most reported small-molecule inhibitors targeting the ALK domain do not display good inhibition of the G1202R solvent front mutation. The solvent front mutation was assumed to hinder drug binding. However, a different fact could be uncovered by the simulations reported in this study through a structural analog of alectinib (JH-VIII-157-02), which demonstrated potent effects against the G1202R mutation. Molecular docking, conventional molecular dynamics (MD) simulations, free energy calculations, and umbrella sampling (US) simulations were carried out to make clear the principles of the binding preferences of alectinib and JH-VIII-157-02 toward ALK WT and the ALK G1202R (ALK G1202R ) mutation. JH-VIII-157-02 has similar binding affinities to both ALK WT and ALK G1202R whereas it has has a much lower binding affinity for alectinib to ALK G1202R . Analysis of individual energy terms indicate the major variation involves the van der Waals and entropy terms. Structural analysis reveals that the conformational change of the ATP-binding glycine-rich loop was primarily responsible for the alectinib resistance, not JH-VIII-157-02. In addition, US simulations prove JH-VIII-157-02 has similar dissociative processes from both ALK WT and ALK G1202R , while alectinib is more easily dissociated from ALK G1202R than from ALK WT , thus indicating lesser residence time. Both the binding affinity and the drug residence time should be emphasized in rational drug design to overcome the G1202R solvent front mutation in ALK resistance.

Insight into resistance mechanism of anaplastic lymphoma kinase to alectinib and JH-VIII-157-02 caused by G1202R solvent front mutation

PubMed Central

Wang, Han; Wang, Yao; Guo, Wentao; Du, Bin; Huang, Xiaobing; Wu, Riping; Yang, Baoyu; Lin, Xiaoyan; Wu, Yilan

2018-01-01

Background Mutated anaplastic lymphoma kinase (ALK) drives the development of advanced non-small cell lung cancer (NSCLC). Most reported small-molecule inhibitors targeting the ALK domain do not display good inhibition of the G1202R solvent front mutation. The solvent front mutation was assumed to hinder drug binding. However, a different fact could be uncovered by the simulations reported in this study through a structural analog of alectinib (JH-VIII-157-02), which demonstrated potent effects against the G1202R mutation. Methods Molecular docking, conventional molecular dynamics (MD) simulations, free energy calculations, and umbrella sampling (US) simulations were carried out to make clear the principles of the binding preferences of alectinib and JH-VIII-157-02 toward ALKWT and the ALK G1202R (ALKG1202R) mutation. Results JH-VIII-157-02 has similar binding affinities to both ALKWT and ALKG1202R whereas it has has a much lower binding affinity for alectinib to ALKG1202R. Analysis of individual energy terms indicate the major variation involves the van der Waals and entropy terms. Structural analysis reveals that the conformational change of the ATP-binding glycine-rich loop was primarily responsible for the alectinib resistance, not JH-VIII-157-02. In addition, US simulations prove JH-VIII-157-02 has similar dissociative processes from both ALKWT and ALKG1202R, while alectinib is more easily dissociated from ALKG1202R than from ALKWT, thus indicating lesser residence time. Conclusion Both the binding affinity and the drug residence time should be emphasized in rational drug design to overcome the G1202R solvent front mutation in ALK resistance. PMID:29785088

Alternating Hemiplegia of Childhood: Retrospective Genetic Study and Genotype-Phenotype Correlations in 187 Subjects from the US AHCF Registry

PubMed Central

Viollet, Louis; Glusman, Gustavo; Murphy, Kelley J.; Newcomb, Tara M.; Reyna, Sandra P.; Sweney, Matthew; Nelson, Benjamin; Andermann, Frederick; Andermann, Eva; Acsadi, Gyula; Barbano, Richard L.; Brown, Candida; Brunkow, Mary E.; Chugani, Harry T.; Cheyette, Sarah R.; Collins, Abigail; DeBrosse, Suzanne D.; Galas, David; Friedman, Jennifer; Hood, Lee; Huff, Chad; Jorde, Lynn B.; King, Mary D.; LaSalle, Bernie; Leventer, Richard J.; Lewelt, Aga J.; Massart, Mylynda B.; Mérida, Mario R.; Ptáček, Louis J.; Roach, Jared C.; Rust, Robert S.; Renault, Francis; Sanger, Terry D.; Sotero de Menezes, Marcio A.; Tennyson, Rachel; Uldall, Peter; Zhang, Yue; Zupanc, Mary; Xin, Winnie; Silver, Kenneth; Swoboda, Kathryn J.

2015-01-01

Mutations in ATP1A3 cause Alternating Hemiplegia of Childhood (AHC) by disrupting function of the neuronal Na+/K+ ATPase. Published studies to date indicate 2 recurrent mutations, D801N and E815K, and a more severe phenotype in the E815K cohort. We performed mutation analysis and retrospective genotype-phenotype correlations in all eligible patients with AHC enrolled in the US AHC Foundation registry from 1997-2012. Clinical data were abstracted from standardized caregivers’ questionnaires and medical records and confirmed by expert clinicians. We identified ATP1A3 mutations by Sanger and whole genome sequencing, and compared phenotypes within and between 4 groups of subjects, those with D801N, E815K, other ATP1A3 or no ATP1A3 mutations. We identified heterozygous ATP1A3 mutations in 154 of 187 (82%) AHC patients. Of 34 unique mutations, 31 (91%) are missense, and 16 (47%) had not been previously reported. Concordant with prior studies, more than 2/3 of all mutations are clustered in exons 17 and 18. Of 143 simplex occurrences, 58 had D801N (40%), 38 had E815K (26%) and 11 had G937R (8%) mutations. Patients with an E815K mutation demonstrate an earlier age of onset, more severe motor impairment and a higher prevalence of status epilepticus. This study further expands the number and spectrum of ATP1A3 mutations associated with AHC and confirms a more deleterious effect of the E815K mutation on selected neurologic outcomes. However, the complexity of the disorder and the extensive phenotypic variability among subgroups merits caution and emphasizes the need for further studies. PMID:25996915

BAG3-related myopathy, polyneuropathy and cardiomyopathy with long QT syndrome.

PubMed

Kostera-Pruszczyk, Anna; Suszek, Małgorzata; Płoski, Rafał; Franaszczyk, Maria; Potulska-Chromik, Anna; Pruszczyk, Piotr; Sadurska, Elżbieta; Karolczak, Justyna; Kamińska, Anna M; Rędowicz, Maria Jolanta

2015-12-01

BAG3 belongs to BAG family of molecular chaperone regulators interacting with HSP70 and anti-apoptotic protein Bcl-2. It is ubiquitously expressed with strong expression in skeletal and cardiac muscle, and is involved in a panoply of cellular processes. Mutations in BAG3 and aberrations in its expression cause fulminant myopathies, presenting with progressive limb and axial muscle weakness, and respiratory insufficiency and neuropathy. Herein, we report a sporadic case of a 15-years old girl with symptoms of myopathy, demyelinating polyneuropathy and asymptomatic long QT syndrome. Genetic testing demonstrated heterozygous mutation Pro209Leu (c.626C > T) in exon 3 of BAG3 gene causing severe myopathy and neuropathy, often associated with restrictive cardiomyopathy. We did not find a mutation in any known LQT syndrome genes. Analysis of muscle biopsy revealed profound disintegration of Z-discs with extensive accumulation of granular debris and large inclusions within fibers. We demonstrated profound alterations in BAG3 distribution as the protein localized to long filamentous structures present across the fibers that were positively stained not only for α-actinin but also for desmin and filamin indicating that those disintegrated Z-disc regions contained also other sarcomeric proteins. The mutation caused a decrease in the content of BAG3 and HSP70, and also of α-actinin desmin, filamin and fast myosin heavy chain, confirming its severe effect on the muscle fiber morphology and thus function. We provide further evidence that BAG3 is associated with Z-disc maintenance, and the Pro209Leu mutation may occur worldwide. We also provide a summary of cases associated with this mutation reported so far.

Distinct biological subtypes and patterns of genome evolution in lymphoma revealed by circulating tumor DNA.

PubMed

Scherer, Florian; Kurtz, David M; Newman, Aaron M; Stehr, Henning; Craig, Alexander F M; Esfahani, Mohammad Shahrokh; Lovejoy, Alexander F; Chabon, Jacob J; Klass, Daniel M; Liu, Chih Long; Zhou, Li; Glover, Cynthia; Visser, Brendan C; Poultsides, George A; Advani, Ranjana H; Maeda, Lauren S; Gupta, Neel K; Levy, Ronald; Ohgami, Robert S; Kunder, Christian A; Diehn, Maximilian; Alizadeh, Ash A

2016-11-09

Patients with diffuse large B cell lymphoma (DLBCL) exhibit marked diversity in tumor behavior and outcomes, yet the identification of poor-risk groups remains challenging. In addition, the biology underlying these differences is incompletely understood. We hypothesized that characterization of mutational heterogeneity and genomic evolution using circulating tumor DNA (ctDNA) profiling could reveal molecular determinants of adverse outcomes. To address this hypothesis, we applied cancer personalized profiling by deep sequencing (CAPP-Seq) analysis to tumor biopsies and cell-free DNA samples from 92 lymphoma patients and 24 healthy subjects. At diagnosis, the amount of ctDNA was found to strongly correlate with clinical indices and was independently predictive of patient outcomes. We demonstrate that ctDNA genotyping can classify transcriptionally defined tumor subtypes, including DLBCL cell of origin, directly from plasma. By simultaneously tracking multiple somatic mutations in ctDNA, our approach outperformed immunoglobulin sequencing and radiographic imaging for the detection of minimal residual disease and facilitated noninvasive identification of emergent resistance mutations to targeted therapies. In addition, we identified distinct patterns of clonal evolution distinguishing indolent follicular lymphomas from those that transformed into DLBCL, allowing for potential noninvasive prediction of histological transformation. Collectively, our results demonstrate that ctDNA analysis reveals biological factors that underlie lymphoma clinical outcomes and could facilitate individualized therapy. Copyright © 2016, American Association for the Advancement of Science.

Clustered Regularly Interspaced Short Palindromic Repeats-Based Genome Surgery for the Treatment of Autosomal Dominant Retinitis Pigmentosa.

PubMed

Tsai, Yi-Ting; Wu, Wen-Hsuan; Lee, Ting-Ting; Wu, Wei-Pu; Xu, Christine L; Park, Karen S; Cui, Xuan; Justus, Sally; Lin, Chyuan-Sheng; Jauregui, Ruben; Su, Pei-Yin; Tsang, Stephen H

2018-05-05

To develop a universal gene therapy to overcome the genetic heterogeneity in retinitis pigmentosa (RP) resulting from mutations in rhodopsin (RHO). Experimental study for a combination gene therapy that uses both gene ablation and gene replacement. This study included 2 kinds of human RHO mutation knock-in mouse models: Rho P23H and Rho D190N . In total, 23 Rho P23H/P23H , 43 Rho P23H/+ , and 31 Rho D190N/+ mice were used for analysis. This study involved gene therapy using dual adeno-associated viruses (AAVs) that (1) destroy expression of the endogenous Rho gene in a mutation-independent manner via an improved clustered regularly interspaced short palindromic repeats-based gene deletion and (2) enable expression of wild-type protein via exogenous cDNA. Electroretinographic and histologic analysis. The thickness of the outer nuclear layer (ONL) after the subretinal injection of combination ablate-and-replace gene therapy was approximately 17% to 36% more than the ONL thickness resulting from gene replacement-only therapy at 3 months after AAV injection. Furthermore, electroretinography results demonstrated that the a and b waves of both Rho P23H and Rho D190N disease models were preserved more significantly using ablate-and-replace gene therapy (P < 0.001), but not by gene replacement monotherapy. As a proof of concept, our results suggest that the ablate-and-replace strategy can ameliorate disease progression as measured by photoreceptor structure and function for both of the human mutation knock-in models. These results demonstrate the potency of the ablate-and-replace strategy to treat RP caused by different Rho mutations. Furthermore, because ablate-and-replace treatment is mutation independent, this strategy may be used to treat a wide array of dominant diseases in ophthalmology and other fields. Clinical trials using ablate-and-replace gene therapy would allow researchers to determine if this strategy provides any benefits for patients with diseases of interest. Copyright © 2018 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

Mutation analysis in a German family identified a new cataract-causing allele in the CRYBB2 gene

PubMed Central

Pauli, Silke; Söker, Torben; Klopp, Norman; Illig, Thomas; Engel, Wolfgang

2007-01-01

Purpose The study demonstrates the functional candidate gene analysis in a cataract family of German descent. Methods We screened a German family, clinically documented to have congenital cataracts, for mutation in the candidate genes CRYG (A to D) and CRYBB2 through polymerase chain reaction analyses and sequencing. Results Congenital cataract was first observed in a daughter of healthy parents. Her two children (a boy and a girl) also suffer from congenital cataracts and have been operated within the first weeks of birth. Morphologically, the cataract is characterized as nuclear with an additional ring-shaped cortical opacity. Molecular analysis revealed no causative mutation in any of the CRYG genes. However, sequencing of the exons of the CRYBB2 gene identified a sequence variation in exon 5 (383 A>T) with a substitution of Asp to Val at position 128. All three affected family members revealed this change but it was not observed in any of the unaffected persons of the family. The putative mutation creates a restriction site for the enzyme TaiI. This mutation was checked for in controls of randomly selected DNA samples from ophthalmologically normal individuals from the population-based KORA S4 study (n=96) and no mutation was observed. Moreover, the Asp at position 128 is within a stretch of 12 amino acids, which are highly conserved throughout the animal kingdom. For the mutant protein, the isoelectric point is raised from pH 6.50 to 6.75. Additionally, the random coil structure of the protein between the amino acids 126-139 is interrupted by a short extended strand structure. In addition, this region becomes hydrophobic (from neutral to +1) and the electrostatic potential in the region surrounding the exchanged amino acid alters from a mainly negative potential to an enlarged positive potential. Conclusions The D128V mutation segregates only in affected family members and is not seen in representative controls. It represents the first mutation outside exon 6 of the human CRYBB2 gene. PMID:17653036

Validation and comparison of two NGS assays for the detection of EGFR T790M resistance mutation in liquid biopsies of NSCLC patients.

PubMed

Vollbrecht, Claudia; Lehmann, Annika; Lenze, Dido; Hummel, Michael

2018-04-06

Analysis of circulating cell-free DNA (cfDNA) derived from peripheral blood ("liquid biopsy") is an attractive alternative to identify non-small cell lung cancer (NSCLC) patients with the EGFR T790M mutation eligible for 3rd generation tyrosine kinase inhibitor therapy. We evaluated two PCR-based next generation sequencing (NGS) approaches, one including unique molecular identifiers (UMI), with focus on highly sensitive EGFR T790M mutation detection. Therefore, we extracted and sequenced cfDNA from synthetic plasma samples spiked with mutated DNA at decreasing allele frequencies and from 21 diagnostic NSCLC patients. Data evaluation was performed to determine the limit of detection (LoD), accuracy, specificity and sensitivity of both assays. Considering all tested reference dilutions and mutations the UMI assay performed best in terms of LoD (1% vs. 5%), sensitivity (95.8% vs. 81.3%), specificity (100% vs. 93.8%) and accuracy (96.9% vs. 84.4%). Comparing mutation status of diagnostic samples with both assays showed 81.3% concordance with primary mutation verifiable in 52% of cases. EGFR T790M was detected concordantly in 6/7 patients with allele frequencies from 0.1% to 27%. In one patient, the T790M mutation was exclusively detectable with the UMI assay. Our data demonstrate that both assays are applicable as multi-biomarker NGS tools enabling the simultaneous detection of primary EGFR driver and resistance mutations. However, for mutations with low allelic frequencies the use of NGS panels with UMI facilitates a more sensitive and reliable detection.

Validation and comparison of two NGS assays for the detection of EGFR T790M resistance mutation in liquid biopsies of NSCLC patients

PubMed Central

Vollbrecht, Claudia; Lehmann, Annika; Lenze, Dido; Hummel, Michael

2018-01-01

Analysis of circulating cell-free DNA (cfDNA) derived from peripheral blood (“liquid biopsy”) is an attractive alternative to identify non-small cell lung cancer (NSCLC) patients with the EGFR T790M mutation eligible for 3rd generation tyrosine kinase inhibitor therapy. We evaluated two PCR-based next generation sequencing (NGS) approaches, one including unique molecular identifiers (UMI), with focus on highly sensitive EGFR T790M mutation detection. Therefore, we extracted and sequenced cfDNA from synthetic plasma samples spiked with mutated DNA at decreasing allele frequencies and from 21 diagnostic NSCLC patients. Data evaluation was performed to determine the limit of detection (LoD), accuracy, specificity and sensitivity of both assays. Considering all tested reference dilutions and mutations the UMI assay performed best in terms of LoD (1% vs. 5%), sensitivity (95.8% vs. 81.3%), specificity (100% vs. 93.8%) and accuracy (96.9% vs. 84.4%). Comparing mutation status of diagnostic samples with both assays showed 81.3% concordance with primary mutation verifiable in 52% of cases. EGFR T790M was detected concordantly in 6/7 patients with allele frequencies from 0.1% to 27%. In one patient, the T790M mutation was exclusively detectable with the UMI assay. Our data demonstrate that both assays are applicable as multi-biomarker NGS tools enabling the simultaneous detection of primary EGFR driver and resistance mutations. However, for mutations with low allelic frequencies the use of NGS panels with UMI facilitates a more sensitive and reliable detection. PMID:29719623

Structural effects of clinically observed mutations in JAK2 exons 13-15: comparison with V617F and exon 12 mutations

PubMed Central

Lee, Tai-Sung; Ma, Wanlong; Zhang, Xi; Kantarjian, Hagop; Albitar, Maher

2009-01-01

Background The functional relevance of many of the recently detected JAK2 mutations, except V617F and exon 12 mutants, in patients with chronic myeloproliferative neoplasia (MPN) has been significantly overlooked. To explore atomic-level explanations of the possible mutational effects from those overlooked mutants, we performed a set of molecular dynamics simulations on clinically observed mutants, including newly discovered mutations (K539L, R564L, L579F, H587N, S591L, H606Q, V617I, V617F, C618R, L624P, whole exon 14-deletion) and control mutants (V617C, V617Y, K603Q/N667K). Results Simulation results are consistent with all currently available clinical/experimental evidence. The simulation-derived putative interface, not possibly obtained from static models, between the kinase (JH1) and pseudokinase (JH2) domains of JAK2 provides a platform able to explain the mutational effect for all mutants, including presumably benign control mutants, at the atomic level. Conclusion The results and analysis provide structural bases for mutational mechanisms of JAK2, may advance the understanding of JAK2 auto-regulation, and have the potential to lead to therapeutic approaches. Together with recent mutation profiling results demonstrating the breadth of clinically observed JAK2 mutations, our findings suggest that molecular testing/diagnostics of JAK2 should extend beyond V617F and exon 12 mutations, and perhaps should encompass most of the pseudo-kinase domain-coding region. PMID:19744331

Screening mosaic F1 females for mutations affecting zebrafish heart induction and patterning.

PubMed

Alexander, J; Stainier, D Y; Yelon, D

1998-01-01

The genetic pathways underlying the induction and anterior-posterior patterning of the heart are poorly understood. The recent emergence of the zebrafish model system now allows a classical genetic approach to such challenging problems in vertebrate development. Two large-scale screens for mutations affecting zebrafish embryonic development have recently been completed; among the hundreds of mutations identified were several that affect specific aspects of cardiac morphogenesis, differentiation, and function. However, very few mutations affecting induction and/or anterior-posterior patterning of the heart were identified. We hypothesize that a directed approach utilizing molecular markers to examine these particular steps of heart development will uncover additional such mutations. To test this hypothesis, we are conducting two parallel screens for mutations that affect either the induction or the anterior-posterior patterning of the zebrafish heart. As an indicator of cardiac induction, we examine expression of nkx2.5, the earliest known marker of precardiac mesoderm; to assess anterior-posterior patterning, we distinguish ventricle from atrium with antibodies that recognize different myosin heavy chain isoforms. In order to expedite the examination of a large number of mutations, we are screening the haploid progeny of mosaic F1 females. In these ongoing screens, we have identified four mutations that affect nkx2.5 expression as well as 21 that disrupt either ventricular or atrial development and thus far have recovered several of these mutations, demonstrating the value of our approach. Future analysis of these and other cardiac mutations will provide further insight into the processes of induction and anterior-posterior patterning of the heart.

Mutation site and context dependent effects of ESR1 mutation in genome-edited breast cancer cell models.

PubMed

Bahreini, Amir; Li, Zheqi; Wang, Peilu; Levine, Kevin M; Tasdemir, Nilgun; Cao, Lan; Weir, Hazel M; Puhalla, Shannon L; Davidson, Nancy E; Stern, Andrew M; Chu, David; Park, Ben Ho; Lee, Adrian V; Oesterreich, Steffi

2017-05-23

Mutations in the estrogen receptor alpha (ERα) 1 gene (ESR1) are frequently detected in ER+ metastatic breast cancer, and there is increasing evidence that these mutations confer endocrine resistance in breast cancer patients with advanced disease. However, their functional role is not well-understood, at least in part due to a lack of ESR1 mutant models. Here, we describe the generation and characterization of genome-edited T47D and MCF7 breast cancer cell lines with the two most common ESR1 mutations, Y537S and D538G. Genome editing was performed using CRISPR and adeno-associated virus (AAV) technologies to knock-in ESR1 mutations into T47D and MCF7 cell lines, respectively. Various techniques were utilized to assess the activity of mutant ER, including transactivation, growth and chromatin-immunoprecipitation (ChIP) assays. The level of endocrine resistance was tested in mutant cells using a number of selective estrogen receptor modulators (SERMs) and degraders (SERDs). RNA sequencing (RNA-seq) was employed to study gene targets of mutant ER. Cells with ESR1 mutations displayed ligand-independent ER activity, and were resistant to several SERMs and SERDs, with cell line and mutation-specific differences with respect to magnitude of effect. The SERD AZ9496 showed increased efficacy compared to other drugs tested. Wild-type and mutant cell co-cultures demonstrated a unique evolution of mutant cells under estrogen deprivation and tamoxifen treatment. Transcriptome analysis confirmed ligand-independent regulation of ERα target genes by mutant ERα, but also identified novel target genes, some of which are involved in metastasis-associated phenotypes. Despite significant overlap in the ligand-independent genes between Y537S and D538G, the number of mutant ERα-target genes shared between the two cell lines was limited, suggesting context-dependent activity of the mutant receptor. Some genes and phenotypes were unique to one mutation within a given cell line, suggesting a mutation-specific effect. Taken together, ESR1 mutations in genome-edited breast cancer cell lines confer ligand-independent growth and endocrine resistance. These biologically relevant models can be used for further mechanistic and translational studies, including context-specific and mutation site-specific analysis of the ESR1 mutations.

A novel missense mutation in the NDP gene in a child with Norrie disease and severe neurological involvement including infantile spasms.

PubMed

Lev, Dorit; Weigl, Yuval; Hasan, Mariana; Gak, Eva; Davidovich, Michael; Vinkler, Chana; Leshinsky-Silver, Esther; Lerman-Sagie, Tally; Watemberg, Nathan

2007-05-01

Norrie disease (ND) is a rare X-linked recessive disorder characterized by congenital blindness and in some cases, mental retardation and deafness. Other neurological complications, particularly epilepsy, are rare. We report on a novel mutation identified in a patient with ND and profound mental retardation. The patient was diagnosed at the age of 6 months due to congenital blindness. At the age of 8 months he developed infantile spasms, which were diagnosed at 11 months as his EEG demonstrated hypsarrhythmia. Mutation analysis of the ND gene (NDP) of the affected child and his mother revealed a novel missense mutation at position c.134T > A resulting in amino acid change at codon V45E. To the best of our knowledge, such severe neurological involvement has not been previously reported in ND patients. The severity of the phenotype may suggest the functional importance of this site of the NDP gene.

Fibrinogen storage disease in a Chinese boy with de novo fibrinogen Aguadilla mutation: Incomplete response to carbamazepine and ursodeoxycholic acid.

PubMed

Zhang, Mei-Hong; Knisely, A S; Wang, Neng-Li; Gong, Jing-Yu; Wang, Jian-She

2016-08-12

Fibrinogen storage disease (FSD) is a rare autosomal-dominant disorder caused by mutation in FGG, encoding the fibrinogen gamma chain. Here we report the first Han Chinese patient with FSD, caused by de novo fibrinogen Aguadilla mutation, and his response to pharmacologic management. Epistaxis and persistent clinical-biochemistry test-result abnormalities prompted liver biopsy in a boy, with molecular study of FGG in him and his parents. He was treated with the autophagy enhancer carbamazepine, reportedly effective in FSD, and with ursodeoxycholic acid thereafter. Inclusion bodies in hepatocellular cytoplasm stained immune-histochemically for fibrinogen. Selective analysis of FGG found the heterozygous mutation c.1201C > T (p.Arg401Trp), absent in both parents. Over more than one year's follow-up, transaminase and gamma-glutamyl transpeptidase activities have lessened but not normalized. This report expands the epidemiology of FSD and demonstrates idiosyncrasy in response to oral carbamazepine and/or ursodeoxycholic acid in FSD.

Analysis of the genetic phylogeny of multifocal prostate cancer identifies multiple independent clonal expansions in neoplastic and morphologically normal prostate tissue.

PubMed

Cooper, Colin S; Eeles, Rosalind; Wedge, David C; Van Loo, Peter; Gundem, Gunes; Alexandrov, Ludmil B; Kremeyer, Barbara; Butler, Adam; Lynch, Andrew G; Camacho, Niedzica; Massie, Charlie E; Kay, Jonathan; Luxton, Hayley J; Edwards, Sandra; Kote-Jarai, ZSofia; Dennis, Nening; Merson, Sue; Leongamornlert, Daniel; Zamora, Jorge; Corbishley, Cathy; Thomas, Sarah; Nik-Zainal, Serena; O'Meara, Sarah; Matthews, Lucy; Clark, Jeremy; Hurst, Rachel; Mithen, Richard; Bristow, Robert G; Boutros, Paul C; Fraser, Michael; Cooke, Susanna; Raine, Keiran; Jones, David; Menzies, Andrew; Stebbings, Lucy; Hinton, Jon; Teague, Jon; McLaren, Stuart; Mudie, Laura; Hardy, Claire; Anderson, Elizabeth; Joseph, Olivia; Goody, Victoria; Robinson, Ben; Maddison, Mark; Gamble, Stephen; Greenman, Christopher; Berney, Dan; Hazell, Steven; Livni, Naomi; Fisher, Cyril; Ogden, Christopher; Kumar, Pardeep; Thompson, Alan; Woodhouse, Christopher; Nicol, David; Mayer, Erik; Dudderidge, Tim; Shah, Nimish C; Gnanapragasam, Vincent; Voet, Thierry; Campbell, Peter; Futreal, Andrew; Easton, Douglas; Warren, Anne Y; Foster, Christopher S; Stratton, Michael R; Whitaker, Hayley C; McDermott, Ultan; Brewer, Daniel S; Neal, David E

2015-04-01

Genome-wide DNA sequencing was used to decrypt the phylogeny of multiple samples from distinct areas of cancer and morphologically normal tissue taken from the prostates of three men. Mutations were present at high levels in morphologically normal tissue distant from the cancer, reflecting clonal expansions, and the underlying mutational processes at work in morphologically normal tissue were also at work in cancer. Our observations demonstrate the existence of ongoing abnormal mutational processes, consistent with field effects, underlying carcinogenesis. This mechanism gives rise to extensive branching evolution and cancer clone mixing, as exemplified by the coexistence of multiple cancer lineages harboring distinct ERG fusions within a single cancer nodule. Subsets of mutations were shared either by morphologically normal and malignant tissues or between different ERG lineages, indicating earlier or separate clonal cell expansions. Our observations inform on the origin of multifocal disease and have implications for prostate cancer therapy in individual cases.

Coats' disease of the retina (unilateral retinal telangiectasis) caused by somatic mutation in the NDP gene: a role for norrin in retinal angiogenesis.

PubMed

Black, G C; Perveen, R; Bonshek, R; Cahill, M; Clayton-Smith, J; Lloyd, I C; McLeod, D

1999-10-01

Coats' disease is characterized by abnormal retinal vascular development (so-called 'retinal telangiectasis') which results in massive intraretinal and subretinal lipid accumulation (exudative retinal detachment). The classical form of Coats' disease is almost invariably isolated, unilateral and seen in males. A female with a unilateral variant of Coats' disease gave birth to a son affected by Norrie disease. Both carried a missense mutation within the NDP gene on chromosome Xp11.2. Subsequently analysis of the retinas of nine enucleated eyes from males with Coats' disease demonstrated in one a somatic mutation in the NDP gene which was not present within non-retinal tissue. We suggest that Coats' telangiectasis is secondary to somatic mutation in the NDP gene which results in a deficiency of norrin (the protein product of the NDP gene) within the developing retina. This supports recent observations that the protein is critical for normal retinal vasculogenesis.

Blinded study determination of high sensitivity and specificity microchip electrophoresis-SSCP/HA to detect mutations in the p53 gene.

PubMed

Hestekin, Christa N; Lin, Jennifer S; Senderowicz, Lionel; Jakupciak, John P; O'Connell, Catherine; Rademaker, Alfred; Barron, Annelise E

2011-11-01

Knowledge of the genetic changes that lead to disease has grown and continues to grow at a rapid pace. However, there is a need for clinical devices that can be used routinely to translate this knowledge into the treatment of patients. Use in a clinical setting requires high sensitivity and specificity (>97%) in order to prevent misdiagnoses. Single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are two DNA-based, complementary methods for mutation detection that are inexpensive and relatively easy to implement. However, both methods are most commonly detected by slab gel electrophoresis, which can be labor-intensive, time-consuming, and often the methods are unable to produce high sensitivity and specificity without the use of multiple analysis conditions. Here, we demonstrate the first blinded study using microchip electrophoresis (ME)-SSCP/HA. We demonstrate the ability of ME-SSCP/HA to detect with 98% sensitivity and specificity >100 samples from the p53 gene exons 5-9 in a blinded study in an analysis time of <10 min. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genetic homogeneity of Pelizaeus-Merzbacher disease: Tight linkage to the proteolipoprotein locus in 16 affected families

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

Boespflug-Tanguy, O.; Mimault, C.; Cavagna, A.

1994-09-01

Among the numerous leukodystrophies that have an early onset and no biochemical markers, Pelizaeus-Merzbacher disease (PMD) is one that can be identified using strict clinical criteria and demonstrating an abnormal formation of myelin that is restricted to the CNS in electrophysiological studies and brain magnetic resonance imaging (MRI). In PMD, 12 different base substitutions and one total deletion of the genomic region containing the PLP gene have been reported, but, despite extensive analysis, PLP exon mutations have been found in only 10%-25% of the families analyzed. To test the genetic homogeneity of this disease, the authors have carried out linkagemore » analysis with polymorphic markers of the PLP genomic region in 16 families selected on strict diagnostic criteria of PMD. They observed a tight linkage of the PMD locus with markers of the PLP gene (cDNA PLP, exon IV polymorphism) and of the Xq22 region (DXS17, DXS94, and DXS287), whereas the markers located more proximally (DXYS1X and DXS3) or distally (DXS11) were not linked to the PMD locus. Multipoint analysis gave a maximal location score for the PMD locus (13.98) and the PLP gene (8.32) in the same interval between DXS94 and DXS287, suggesting that in all families PMD is linked to the PLP locus. Mutations of the extraexonic PLP gene sequences or of another unknown close gene could be involved in PMD. In an attempt to identify molecular defects of this genomic region that are responsible for PMD, these results meant that RFLP analysis could be used to improve genetic counseling for the numerous affected families in which a PLP exon mutation could not be demonstrated. 39 refs., 2 figs., 2 tabs.« less

A novel homozygous mutation in the FSHR gene is causative for primary ovarian insufficiency.

PubMed

Liu, Hongli; Xu, Xiaofei; Han, Ting; Yan, Lei; Cheng, Lei; Qin, Yingying; Liu, Wen; Zhao, Shidou; Chen, Zi-Jiang

2017-12-01

To identify the potential FSHR mutation in a Chinese woman with primary ovarian insufficiency (POI). Genetic and functional studies. University-based reproductive medicine center. A POI patient, her family members, and another 192 control women with regular menstruation. Ovarian biopsy was performed in the patient. Sanger sequencing was carried out for the patient, her sister, and parents. The novel variant identified was further confirmed with the use of control subjects. Sanger sequencing and genotype analysis to identify the potential variant of the FSHR gene; hematoxylin and eosin staining of the ovarian section to observe the follicular development; Western blotting and immunofluorescence to detect FSH receptor (FSHR) expression; and cyclic adenosine monophosphate (cAMP) assay to monitor FSH-induced signaling. Histologic examination of the ovaries in the patient revealed follicular development up to the early antral stage. Mutational screening and genotype analysis of the FSHR gene identified a novel homozygous mutation c.175C>T (p.R59X) in exon 2, which was inherited in the autosomal recessive mode from her heterozygous parents but was absent in her sister and the 192 control women. Functional studies demonstrated that in vitro the nonsense mutation caused the loss of full-length FSHR expression and that p.R59X mutant showed no response to FSH stimulation in the cAMP level. The mutation p.R59X in FSHR is causative for POI by means of arresting folliculogenesis. Copyright © 2017 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Pan-Cancer Analysis of Mutation Hotspots in Protein Domains.

PubMed

Miller, Martin L; Reznik, Ed; Gauthier, Nicholas P; Aksoy, Bülent Arman; Korkut, Anil; Gao, Jianjiong; Ciriello, Giovanni; Schultz, Nikolaus; Sander, Chris

2015-09-23

In cancer genomics, recurrence of mutations in independent tumor samples is a strong indicator of functional impact. However, rare functional mutations can escape detection by recurrence analysis owing to lack of statistical power. We enhance statistical power by extending the notion of recurrence of mutations from single genes to gene families that share homologous protein domains. Domain mutation analysis also sharpens the functional interpretation of the impact of mutations, as domains more succinctly embody function than entire genes. By mapping mutations in 22 different tumor types to equivalent positions in multiple sequence alignments of domains, we confirm well-known functional mutation hotspots, identify uncharacterized rare variants in one gene that are equivalent to well-characterized mutations in another gene, detect previously unknown mutation hotspots, and provide hypotheses about molecular mechanisms and downstream effects of domain mutations. With the rapid expansion of cancer genomics projects, protein domain hotspot analysis will likely provide many more leads linking mutations in proteins to the cancer phenotype. Copyright © 2015 Elsevier Inc. All rights reserved.

A novel ATTR L32V mutation causes familial amyloid polyneuropathy in a Bolivian family.

PubMed

Martínez-Ulloa, Pedro L; Vallejo, Manuela; Corral, Iñigo; García-Barragán, Nuria; Alcazar, Alberto; Martínez-Alonso, Emma; Martínez-Poles, Javier; Pian, Hector; Jiménez-Escrig, Adriano

2017-09-01

We report a new transthyretin (ATTR) gene c.272C>G mutation and variant protein, p.Leu32Val, in a kindred of Bolivian origin with a rapid progressive peripheral neuropathy and cardiomyopathy. Three individuals from a kindred with peripheral nerve and cardiac amyloidosis were examined. Analysis of the TTR gene was performed by Sanger direct sequencing. Neuropathologic examination was obtained on the index patient with mass spectrometry study of the ATTR deposition. Direct DNA sequence analysis of exons 2, 3, and 4 of the TTR gene demonstrated a c.272 C>G mutation in exon 2 (p.L32V). Sural nerve biopsy revealed massive amyloid deposition in the perineurium, endoneurium and vasa nervorum. Mass spectrometric analyses of ATTR immunoprecipitated from nerve biopsy showed the presence of both wild-type and variant proteins. The observed mass results for the wild-type and variant proteins were consistent with the predicted values calculated from the genetic analysis data. The ATTR L32V is associated with a severe course. This has implications for treatment of affected individuals and counseling of family members. © 2017 Peripheral Nerve Society.

Severe congenital muscular dystrophy in a Mexican family with a new nonsense mutation (R2578X) in the laminin alpha-2 gene.

PubMed

Coral-Vazquez, Ramon M; Rosas-Vargas, Haydee; Meza-Espinosa, Pedro; Mendoza, Irma; Huicochea, Juan C; Ramon, Guillermo; Salamanca, Fabio

2003-01-01

The congenital muscular dystrophies (CMDs) are a heterogeneous group of autosomal recessive disorders. Approximately one half of cases diagnosed with classic CMD show primary deficiency of the laminin alpha2 chain of merosin. Complete absence of this protein is usually associated with a severe phenotype characterized by drastic muscle weakness and characteristic changes in white matter in cerebral magnetic resonance imaging (MRI). Here we report an 8-month-old Mexican female infant, from a consanguineous family, with classical CMD. Serum creatine kinase was elevated, muscle biopsy showed dystrophic changes, and there were abnormalities in brain MRI. Immunofluorescence analysis demonstrated the complete absence of laminin alpha2. In contrast, expression of alpha-, beta-, gamma-, and delta-sarcoglycans and dystrophin, all components of the dystrophin-glycoprotein complex, appeared normal. A homozygous C long right arrow T substitution at position 7781 that generated a stop codon in the G domain of the protein was identified by mutation analysis of the laminin alpha2 gene ( LAMA2). Sequence analysis on available DNA samples of the family showed that parents and other relatives were carriers of the mutation.

Unique volatolomic signatures of TP53 and KRAS in lung cells

PubMed Central

Davies, M P A; Barash, O; Jeries, R; Peled, N; Ilouze, M; Hyde, R; Marcus, M W; Field, J K; Haick, H

2014-01-01

Background: Volatile organic compounds (VOCs) are potential biomarkers for cancer detection in breath, but it is unclear if they reflect specific mutations. To test this, we have compared human bronchial epithelial cell (HBEC) cell lines carrying the KRASV12 mutation, knockdown of TP53 or both with parental HBEC cells. Methods: VOC from headspace above cultured cells were collected by passive sampling and analysed by thermal desorption gas chromatography mass spectrometry (TD-GC–MS) or sensor array with discriminant factor analysis (DFA). Results: In TD-GC–MS analysis, individual compounds had limited ability to discriminate between cell lines, but by applying DFA analysis combinations of 20 VOCs successfully discriminated between all cell types (accuracies 80–100%, with leave-one-out cross validation). Sensor array detection DFA demonstrated the ability to discriminate samples based on their cell type for all comparisons with accuracies varying between 77% and 93%. Conclusions: Our results demonstrate that minimal genetic changes in bronchial airway cells lead to detectable differences in levels of specific VOCs identified by TD-GC–MS or of patterns of VOCs identified by sensor array output. From the clinical aspect, these results suggest the possibility of breath analysis for detection of minimal genetic changes for earlier diagnosis or for genetic typing of lung cancers. PMID:25051409

Use of mutation spectra analysis software.

PubMed

Rogozin, I; Kondrashov, F; Glazko, G

2001-02-01

The study and comparison of mutation(al) spectra is an important problem in molecular biology, because these spectra often reflect on important features of mutations and their fixation. Such features include the interaction of DNA with various mutagens, the function of repair/replication enzymes, and properties of target proteins. It is known that mutability varies significantly along nucleotide sequences, such that mutations often concentrate at certain positions, called "hotspots," in a sequence. In this paper, we discuss in detail two approaches for mutation spectra analysis: the comparison of mutation spectra with a HG-PUBL program, (FTP: sunsite.unc.edu/pub/academic/biology/dna-mutations/hyperg) and hotspot prediction with the CLUSTERM program (www.itba.mi.cnr.it/webmutation; ftp.bionet.nsc.ru/pub/biology/dbms/clusterm.zip). Several other approaches for mutational spectra analysis, such as the analysis of a target protein structure, hotspot context revealing, multiple spectra comparisons, as well as a number of mutation databases are briefly described. Mutation spectra in the lacI gene of E. coli and the human p53 gene are used for illustration of various difficulties of such analysis. Copyright 2001 Wiley-Liss, Inc.

Ebola Virus VP35 Interaction with Dynein LC8 Regulates Viral RNA Synthesis

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

Luthra, Priya; Jordan, David S.; Leung, Daisy W.

2015-03-04

Ebola virus VP35 inhibits alpha/beta interferon production and functions as a viral polymerase cofactor. Previously, the 8-kDa cytoplasmic dynein light chain (LC8) was demonstrated to interact with VP35, but the functional consequences were unclear. Here we demonstrate that the interaction is direct and of high affinity and that binding stabilizes the VP35 N-terminal oligomerization domain and enhances viral RNA synthesis. Mutational analysis demonstrates that VP35 interaction is required for the functional effects of LC8.

Sarcomere protein gene mutations and inherited heart disease: a beta-cardiac myosin heavy chain mutation causing endocardial fibroelastosis and heart failure.

PubMed

Kamisago, Mitsuhiro; Schmitt, Joachim P; McNamara, Dennis; Seidman, Christine; Seidman, J G

2006-01-01

Inherited human cardiomyopathies often lead to heart failure. A common feature of these conditions is that affected individuals can express the disease causing mutations for many years without showing clinical signs of the disease. Previous studies have demonstrated that sarcomere protein gene mutations can cause either dilated cardiomyopathy or hypertrophic cardiomyopathy. Here we demonstrate that the Arg442His missense mutation in beta-cardiac myosin heavy chain (betaMHC) causes dilated cardiomyopathy, endocardial fibroelastosis and heart failure at a very early age. Using standard genetic engineering tools we and others have made murine models by introducing human disease causing mutations into mice. The central hypothesis of these studies has been that by identifying the pathophysiological pathways activated by these mutations we can define enzymatic activities that are modified during the disease process and which may be involved in pathways that involve more common forms of cardiac disease. Murine models bearing different mutant myosins are being used to address whether each disease causing mutant betaMHC activates the same or different cellular pathways. Dissecting the molecular pathways modulated by mutations in sarcomere protein genes as well as other genes has already demonstrated that there are multiple pathways leading to cardiac remodelling and heart failure. Defining the mechanisms by which mutations in the same genes activate different cellular pathways remains an important question.

A rare complex DNA rearrangement in the murine Steel gene results in exon duplication and a lethal phenotype.

PubMed

Chandra, Saurabh; Kapur, Reuben; Chuzhanova, Nadia; Summey, Victoria; Prentice, David; Barker, Jane; Cooper, David N; Williams, David A

2003-11-15

Kit ligand (Kitl), encoded by the Steel (Sl) locus, plays an essential role in hematopoiesis, gametogenesis, and melanogenesis during both embryonic and adult life. We have characterized a new spontaneous mutant of the Sl locus in mice designated KitlSl-20J that arose in the breeding colony at Jackson Laboratories. Heterozygous KitlSl-20J mice display a white belly spot and intercrossing results in an embryonic lethal phenotype in the homozygous state. Analysis of homozygous embryos demonstrated a significant reduction in fetal liver cellularity, colony forming unit-erythroid (CFU-E) progenitors, and a total absence of germ cells. Although expressed in vivo, recombinant mutant protein demonstrated loss of bioactivity that was correlated with lack of receptor binding. Analysis of the Sl gene transcripts in heterozygous KitlSl-20J mice revealed an in-frame tandem duplication of exon 3. A long-range polymerase chain reaction (PCR) strategy using overlapping primers in exon 3 amplified an approximately 7-kilobase (kb) product from DNA isolated from heterozygous KitlSl-20J mice but not from wild-type DNA that contained sequences from both introns 2 and 3 and an inverted intron 2 sequence, suggesting a complex rearrangement as the mechanism of the mutation. "Complexity analysis" of the sequence of the amplified product strongly suggests that local DNA motifs may have contributed to the generation of this spontaneous KitlSl-20J allele, likely mediated by a 2-step process. The KitlSl-20J mutation is a unique KitlSl allele and represents an unusual mechanism of mutation.

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

PubMed

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

2010-02-15

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

Molecular genetic analysis of some mutations in the cystic fibrosis gene in Moldova: Characterization of molecular markers and their linkage to various mutations

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

Gimbovskaya, S.D.; Kalinin, V.N.; Ivashchenko, T.E.

1994-12-01

Sixty-one patients with cystic fibrosis (CF) from Moldova were tested for mutations {Delta}F508, G551D, and R553X. Frequencies of various alleles of the repeated GATT sequence in intron 6B of the GFTR gene, their linkage to other polymorphic markers, and various mutations were determined. The frequency of occurrence of mutation {Delta}F508 was only 25%. An absolute majority of CF patients (80%) had pancreatic insufficiency. Mutations G551D and R553X were not found in our sample. Each of 31 chromosomes with mutation {Delta}F508 carry the 6-GATT allele. Most {open_quotes}non {Delta}F508{close_quotes} (78%) and normal (80%) chromosomes were marked by the 7-GATT allele. Twenty-seven {Delta}F508more » chromosomes (96.4%) belong to haplotype B6, and only one to D6. Most chromosomes with {open_quotes}non {Delta}F508{close_quotes} mutations are associated with haplotypes D7 (26.3%) and C7 (21%). In addition, a significant portion of chromosomes from this subgroup were associated with haplotypes A7 (23.7%), A6 (10.5%), and C6 (2.7%), which are not yet described for mutant chromosomes. The results obtained demonstrate that CF in Moldova is mainly associated with mutations other than {Delta}F508, G551D, and R553X. Severe forms of the disease, with pancreatic insufficiency, are more frequently caused by these mutations; moreover, our data provides strong evidence for the presence of at least seven additional CF mutations in Moldova, apart from {Delta}F508, G551D, and R553X. Some of these are probably not described.« less

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

PubMed Central

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

2011-01-01

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

Correlation of ultra-widefield fundus autofluorescence patterns with the underlying genotype in retinal dystrophies and retinitis pigmentosa.

PubMed

Trichonas, George; Traboulsi, Elias I; Ehlers, Justis P

2017-01-01

Ultra-widefield fundus autofluorescence (UW-FAF) allows the characterization of the peripheral retinal features of vitreoretinal diseases. The purpose of this study was to examine possible genotypic/phenotypic correlations of UW-FAF patterns in patients with a variety of retinal dystrophies and retinitis pigmentosa (RP). An IRB-approved retrospective consecutive case series study was performed of genetically characterized retinal dystrophy or RP patients who underwent UW-FAF imaging. UW-FAF was performed with the Optos 200Tx system. Clinical variables, genotypic analysis, and phenotypic characteristics were reviewed. Seventeen patients were identified who had identified mutations in retinal dystrophy or RP genes and who also had undergone UW-FAF. Three patients had X-linked RP with RPGR mutations. Six patients had autosomal dominant RP (four with RHO mutations and one with a PRPF31 mutation, and one with RDS/PRPH2 mutation). Four patients had autosomal recessive RP (four with USH2A mutations). Three patients had Leber Congenital Amaurosis (LCA) with mutations including CRB1, CEP290, and RPGRIP1. Macular hyperautofluorescence was noted in all patients. A ring of hyperautofluorescence was clear in patients with RHO and USH2A mutations, and patients with USH2A mutations demonstrated a second ring of hyperautofluorescence. In the periphery, patients with RHO or RPGR mutations exhibited hyperautofluorescence with patchy areas of hypoautofluorescence. Patients with USH2A mutations had a distinctive pattern of diffuse and homogeneous peripheral hypoautofluorescence. UW-FAF may provide important information to facilitate diagnosis and further research is needed to better characterize this technology as an imaging biomarker for genotype association in retinal dystrophies and RP.

Exonuclease mutations in DNA polymerase epsilon reveal replication strand specific mutation patterns and human origins of replication

PubMed Central

Shinbrot, Eve; Henninger, Erin E.; Weinhold, Nils; Covington, Kyle R.; Göksenin, A. Yasemin; Schultz, Nikolaus; Chao, Hsu; Doddapaneni, HarshaVardhan; Muzny, Donna M.; Gibbs, Richard A.; Sander, Chris; Pursell, Zachary F.

2014-01-01

Tumors with somatic mutations in the proofreading exonuclease domain of DNA polymerase epsilon (POLE-exo*) exhibit a novel mutator phenotype, with markedly elevated TCT→TAT and TCG→TTG mutations and overall mutation frequencies often exceeding 100 mutations/Mb. Here, we identify POLE-exo* tumors in numerous cancers and classify them into two groups, A and B, according to their mutational properties. Group A mutants are found only in POLE, whereas Group B mutants are found in POLE and POLD1 and appear to be nonfunctional. In Group A, cell-free polymerase assays confirm that mutations in the exonuclease domain result in high mutation frequencies with a preference for C→A mutation. We describe the patterns of amino acid substitutions caused by POLE-exo* and compare them to other tumor types. The nucleotide preference of POLE-exo* leads to increased frequencies of recurrent nonsense mutations in key tumor suppressors such as TP53, ATM, and PIK3R1. We further demonstrate that strand-specific mutation patterns arise from some of these POLE-exo* mutants during genome duplication. This is the first direct proof of leading strand-specific replication by human POLE, which has only been demonstrated in yeast so far. Taken together, the extremely high mutation frequency and strand specificity of mutations provide a unique identifier of eukaryotic origins of replication. PMID:25228659

R102Q mutation shifts the salt-bridge network and reduces the structural flexibility of human neuronal calcium sensor-1 protein.

PubMed

Zhu, Yuzhen; Wu, Ying; Luo, Yin; Zou, Yu; Ma, Buyong; Zhang, Qingwen

2014-11-20

Neuronal calcium sensor-1 (NCS-1) protein has a variety of different neuronal functions and interacts with multiple binding partners mostly through a large solvent-exposed hydrophobic crevice (HC). A single R102Q mutation in human NCS-1 protein was demonstrated to be associated with autism disease. Solution NMR study reported that this R102Q mutant had long-range chemical shift effects on the HC and the C-terminal tail (L3). To understand the influence of the R102Q mutation on the HC and L3 of NCS-1, we have investigated the conformational dynamics and the structural flexibility of wild type (WT) NCS-1 and its R102Q mutant by conducting extensive all-atom molecular dynamics (MD) simulations. On the basis of six independent 450 ns MD simulations, we have found that the R102Q mutation in NCS-1 protein (1) dramatically reduces the flexibility of loops L2 and L3, (2) facilitates L3 in a more extended state to occupy the hydrophobic crevice to a larger extent, (3) significantly affects the intersegment salt bridges, and (4) changes the subspace of the free energy landscape of NCS-1 protein. Analysis of the salt bridge network in both WT and the R102Q variant demonstrates that the R102Q-mutation-induced salt bridge alternations play a critical role on the reduced flexibility of L2 and L3. These results reveal the important role of salt bridges on the structural properties of NCS-1 protein and that R102Q mutation disables the dynamic relocation of C-terminus, which may block the binding of NCS-1 protein to its receptors. This study may provide structural insights into the autistic spectrum disorder associated with R102Q mutation.

An analysis of the prognostic value of IDH1 (isocitrate dehydrogenase 1) mutation in Polish glioma patients.

PubMed

Lewandowska, Marzena Anna; Furtak, Jacek; Szylberg, Tadeusz; Roszkowski, Krzysztof; Windorbska, Wiesława; Rytlewska, Joanna; Jóźwicki, Wojciech

2014-02-01

IDH1 (isocitrate dehydrogenase 1) is a potential biomarker and drug target. Genomic and epigenetic data on astrocytoma have demonstrated that the IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Furthermore, recent studies have also indicated that a mutant IDH1 inhibitor induced demethylation of histone H3K9me3 and expression of genes associated with gliogenic differentiation. As the presence of the p.R132H mutation in the IDH1 gene seems to be a more powerful prognostic marker than O(6)-methylguanine-DNA methyltransferase promoter status, we evaluated the presence of IDH1 mutation in Polish patients with astrocytoma, glioblastoma, oligoastrocytoma, ganglioglioma, oligodendroglioma, and ependymoma. The IDH1 mutation status at codon 132 was determined using a mouse monoclonal antibody specific for the R132H mutation, direct sequencing, and Co-amplification at Lower Denaturation Temperature (COLD) polymerase chain reaction (PCR) high-resolution melting-curve analysis (HRM). Wild-type (WT) IDH1 was detected in cases with a World Health Organization (WHO) grade I astrocytoma. The IDH1 c.G395A; p.R132H mutation was observed in 56 and 94 % of grade II and grade III astrocytoma cases, respectively. Significant differences in the median overall survival were observed in astrocytoma patients grouped on the basis of the presence of IDH1 mutation: survival was 24 months longer in grade II astrocytoma and 12 months longer in glioblastoma. Overall survival was compared between grade II astrocytoma patients with low or high expression of the mutant protein. Interestingly, lower R132H expression correlated with better overall survival. Our results indicate the usefulness of assessing the R132H IDH1 mutation in glioma patients: the presence or absence of the R132H mutation can help pathologists to distinguish pilocytic astrocytomas (IDH1 WT) from diffuse ones (R132H IDH1/WT). Moreover, low IDH1 p.R132H expression was related to better prognosis. This clinical implication appears to be important for personalization of prognosis and treatment by oncologists.

Disease-associated mutations identify a novel region in human STING necessary for the control of type I interferon signaling.

PubMed

Melki, Isabelle; Rose, Yoann; Uggenti, Carolina; Van Eyck, Lien; Frémond, Marie-Louise; Kitabayashi, Naoki; Rice, Gillian I; Jenkinson, Emma M; Boulai, Anaïs; Jeremiah, Nadia; Gattorno, Marco; Volpi, Sefano; Sacco, Olivero; Terheggen-Lagro, Suzanne W J; Tiddens, Harm A W M; Meyts, Isabelle; Morren, Marie-Anne; De Haes, Petra; Wouters, Carine; Legius, Eric; Corveleyn, Anniek; Rieux-Laucat, Frederic; Bodemer, Christine; Callebaut, Isabelle; Rodero, Mathieu P; Crow, Yanick J

2017-08-01

Gain-of-function mutations in transmembrane protein 173 (TMEM173) encoding stimulator of interferon genes (STING) underlie a recently described type I interferonopathy called STING-associated vasculopathy with onset in infancy (SAVI). We sought to define the molecular and cellular pathology relating to 3 individuals variably exhibiting the core features of the SAVI phenotype including systemic inflammation, destructive skin lesions, and interstitial lung disease. Genetic analysis, conformational studies, in vitro assays and ex vivo flow-cytometry were performed. Molecular and in vitro data demonstrate that the pathology in these patients is due to amino acid substitutions at positions 206, 281, and 284 of the human STING protein. These mutations confer cGAMP-independent constitutive activation of type I interferon signaling through TBK1 (TANK-binding kinase), independent from the alternative STING pathway triggered by membrane fusion of enveloped RNA viruses. This constitutive activation was abrogated by ex vivo treatment with the janus kinase 1/2 inhibitor ruxolitinib. Structural analysis indicates that the 3 disease-associated mutations at positions 206, 281, and 284 of the STING protein define a novel cluster of amino acids with functional importance in the regulation of type I interferon signaling. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Skeletal muscle biopsy analysis in reducing body myopathy and other FHL1-related disorders.

PubMed

Malfatti, Edoardo; Olivé, Montse; Taratuto, Ana Lía; Richard, Pascale; Brochier, Guy; Bitoun, Marc; Gueneau, Lucie; Laforêt, Pascal; Stojkovic, Tanya; Maisonobe, Thierry; Monges, Soledad; Lubieniecki, Fabiana; Vasquez, Gabriel; Streichenberger, Nathalie; Lacène, Emmanuelle; Saccoliti, Maria; Prudhon, Bernard; Alexianu, Marilena; Figarella-Branger, Dominique; Schessl, Joachim; Bonnemann, Carsten; Eymard, Bruno; Fardeau, Michel; Bonne, Gisèle; Romero, Norma Beatriz

2013-09-01

FHL1 mutations have been associated with various disorders that include reducing body myopathy (RBM), Emery-Dreifuss-like muscular dystrophy, isolated hypertrophic cardiomyopathy, and some overlapping conditions. We report a detailed histochemical, immunohistochemical, electron microscopic, and immunoelectron microscopic analyses of muscle biopsies from 18 patients carrying mutations in FHL1: 14 RBM patients (Group 1), 3 Emery-Dreifuss muscular dystrophy patients (Group 2), and 1 patient with hypertrophic cardiomyopathy and muscular hypertrophy (Group 2). Group 1 muscle biopsies consistently showed RBs associated with cytoplasmic bodies. The RBs showed prominent FHL1 immunoreactivity whereas desmin, αB-crystallin, and myotilin immunoreactivity surrounded RBs. By electron microscopy, RBs were composed of electron-dense tubulofilamentous material that seemed to spread progressively between the myofibrils and around myonuclei. By immunoelectron microscopy, FHL1 protein was found exclusively inside RBs. Group 2 biopsies showed mild dystrophic abnormalities without RBs; only minor nonspecific myofibrillar abnormalities were observed under electron microscopy. Molecular analysis revealed missense mutations in the second FHL1 LIM domain in Group 1 patients and ins/del or missense mutations within the fourth FHL1 LIM domain in Group 2 patients. Our findings expand the morphologic features of RBM, clearly demonstrate the localization of FHL1 in RBs, and further illustrate major morphologic differences among different FHL1-related myopathies.

A comprehensive approach to identification of pathogenic FANCA variants in Fanconi anemia patients and their families.

PubMed

Kimble, Danielle C; Lach, Francis P; Gregg, Siobhan Q; Donovan, Frank X; Flynn, Elizabeth K; Kamat, Aparna; Young, Alice; Vemulapalli, Meghana; Thomas, James W; Mullikin, James C; Auerbach, Arleen D; Smogorzewska, Agata; Chandrasekharappa, Settara C

2018-02-01

Fanconi anemia (FA) is a rare recessive DNA repair deficiency resulting from mutations in one of at least 22 genes. Two-thirds of FA families harbor mutations in FANCA. To genotype patients in the International Fanconi Anemia Registry (IFAR) we employed multiple methodologies, screening 216 families for FANCA mutations. We describe identification of 57 large deletions and 261 sequence variants, in 159 families. All but seven families harbored distinct combinations of two mutations demonstrating high heterogeneity. Pathogenicity of the 18 novel missense variants was analyzed functionally by determining the ability of the mutant cDNA to improve the survival of a FANCA-null cell line when treated with MMC. Overexpressed pathogenic missense variants were found to reside in the cytoplasm, and nonpathogenic in the nucleus. RNA analysis demonstrated that two variants (c.522G > C and c.1565A > G), predicted to encode missense variants, which were determined to be nonpathogenic by a functional assay, caused skipping of exons 5 and 16, respectively, and are most likely pathogenic. We report 48 novel FANCA sequence variants. Defining both variants in a large patient cohort is a major step toward cataloging all FANCA variants, and permitting studies of genotype-phenotype correlations. © Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Differential sensitivity of melanoma cell lines with BRAFV600E mutation to the specific Raf inhibitor PLX4032

PubMed Central

2010-01-01

Blocking oncogenic signaling induced by the BRAFV600E mutation is a promising approach for melanoma treatment. We tested the anti-tumor effects of a specific inhibitor of Raf protein kinases, PLX4032/RG7204, in melanoma cell lines. PLX4032 decreased signaling through the MAPK pathway only in cell lines with the BRAFV600E mutation. Seven out of 10 BRAFV600E mutant cell lines displayed sensitivity based on cell viability assays and three were resistant at concentrations up to 10 μM. Among the sensitive cell lines, four were highly sensitive with IC50 values below 1 μM, and three were moderately sensitive with IC50 values between 1 and 10 μM. There was evidence of MAPK pathway inhibition and cell cycle arrest in both sensitive and resistant cell lines. Genomic analysis by sequencing, genotyping of close to 400 oncogeninc mutations by mass spectrometry, and SNP arrays demonstrated no major differences in BRAF locus amplification or in other oncogenic events between sensitive and resistant cell lines. However, metabolic tracer uptake studies demonstrated that sensitive cell lines had a more profound inhibition of FDG uptake upon exposure to PLX4032 than resistant cell lines. In conclusion, BRAFV600E mutant melanoma cell lines displayed a range of sensitivities to PLX4032 and metabolic imaging using PET probes can be used to assess sensitivity. PMID:20406486

Variants of the D{sub 5} dopamine receptor gene found in patients with schizophrenia: Identification of a nonsense mutation and multiple missense changes

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

Sobell, J.L.; Lind, T.J.; Sommer, S.S.

To determine whether mutations in the D{sub 5} dopamine receptor (D{sub 5}DR) gene are associated with schizophrenia, the gene was examined in 78 unrelated schizophrenic individuals. After amplification by the polymerase chain reaction, products were examined by dideoxy fingerprinting (ddF), a highly sensitive screening method related to single strand conformational polymorphism analysis. All samples with unusual ddF patterns were sequenced to precisely identify the sequence change. In the 156 D{sub 5}DR alleles examined, nine sequence changes were identified. Four of the nine did not affect protein structure; of these, three were silent changes and one was a transition in themore » 3{prime} untranslated region. The remaining five sequence changes result in protein alterations: of these, one is a missense change in a non-conserved amino acid, 3 are missense changes in amino acids that are conserved in some dopamine D{sub 5} receptors and the last is a nonsense mutation. To investigate whether the nonsense mutation was associated with schizophrenia, 400 additional schizophrenic cases of western European descent and 1914 ethnically-similar controls were screened for the change. One additional schizophrenic carrier was identified and verified by direct genomic sequencing (allele frequency: .0013), but eight carriers also were found and confirmed among the non-schizophrenics (allele frequency: .0021)(p>.25). The gene was re-examined in all newly identified carriers of the nonsense mutation by direct sequencing and/or ddF in search of additional mutations. None were identified. Family studies also were conducted to investigate possible cosegregation of the mutation with other neuropsychiatric diseases, but this was not demonstrated. Thus, the mutation does not appear to be associated with an increased risk of schizophrenia nor does an initial analysis suggest cosegregation with other neuropsychiatric disorders or symptom complexes.« less

Dominant Drop mutants are gain-of-function alleles of the muscle segment homeobox gene (msh) whose overexpression leads to the arrest of eye development.

PubMed

Mozer, B A

2001-05-15

Dominant Drop (Dr) mutations are nearly eyeless and have additional recessive phenotypes including lethality and patterning defects in eye and sensory bristles due to cis-regulatory lesions in the cell cycle regulator string (stg). Genetic analysis demonstrates that the dominant small eye phenotype is the result of separate gain-of-function mutations in the closely linked muscle segment homeobox (msh) gene, encoding a homeodomain transcription factor required for patterning of muscle and nervous system. Reversion of the Dr(Mio) allele was coincident with the generation of lethal loss-of-function mutations in msh in cis, suggesting that the dominant eye phenotype is the result of ectopic expression. Molecular genetic analysis revealed that two dominant Dr alleles contain lesions upstream of the msh transcription start site. In the Dr(Mio) mutant, a 3S18 retrotransposon insertion is the target of second-site mutations (P-element insertions or deletions) which suppress the dominant eye phenotype following reversion. The pattern of 3S18 expression and the absence of msh in eye imaginal discs suggest that transcriptional activation of the msh promoter accounts for ectopic expression. Dr dominant mutations arrest eye development by blocking the progression of the morphogenetic furrow leading to photoreceptor cell loss via apoptosis. Gal4-mediated ubiquitous expression of msh in third-instar larvae was sufficient to arrest the morphogenetic furrow in the eye imaginal disc and resulted in lethality prior to eclosion. Dominant mutations in the human msx2 gene, one of the vertebrate homologs of msh, are associated with craniosynostosis, a disease affecting cranial development. The Dr mutations are the first example of gain-of-function mutations in the msh/msx gene family identified in a genetically tractible model organism and may serve as a useful tool to identify additional genes that regulate this class of homeodomain proteins. Copyright 2001 Academic Press.

Analysis of mutational characteristics of the drug-resistant gene katG in multi-drug resistant Mycobacterium tuberculosis L-form among patients with pneumoconiosis complicated with tuberculosis.

PubMed

Lu, Jun; Jiang, Shan; Liu, Qian-Ying; Ma, Shuai; Li, Ying; Li, Chao-Pin

2014-05-01

The aim of the present study was to investigate the mutational characteristics of drug‑resistant genetic mutations in the katG gene to isoniazid (INH) in multi‑drug resistant Mycobacterium tuberculosis (MTB) L‑form among patients with pneumoconiosis complicated with tuberculosis (TB), in order to reduce the occurrence of drug resistance in patients, and gain further insight into the mechanisms underlying drug resistance in MDR‑TB L‑form. A total of 114 clinically isolated strains of MTB L‑forms were collected. The MDR‑TB L‑forms were identified using a conventional antimicrobial susceptibility test (AST). The DNA genomes were extracted, the target genes were amplified by polymerase chain reaction technology and the hotspot mutational regions in the katG gene were analyzed by direct sequencing. The results of AST analysis demonstrated that there were 31 strains of MDR‑TB L‑forms in 114 clinical isolates. The mutation rate of katG was 61.29% (19/31) in INH‑resistant isolates, mainly concentrated in codon 315 (Ser315Thr, 48.39% and Ser315Asn, 9.68%) and 431 (Ala431Val, 3.23%). Base substitutions were identified, however, no multisite mutations were found. No mutations in katG were identified in 10 INH‑sensitive strains that were randomly selected. INH‑resistance was more severe in MDR‑TB L‑form isolates among patients with pneumoconiosis complicated with TB. The substitution of highly conserved amino acids encoded by the katG gene resulted in the molecular mechanisms responsible for INH resistance in MDR‑TB L‑form isolates. It was also verified that the katG gene was in diversiform. The katG Ser315Thr mutation is one of the main causes of resistance to INH in MDR‑TB L-form isolates.

Autosomal recessive POLR1D mutation with decrease of TCOF1 mRNA is responsible for Treacher Collins syndrome.

PubMed

Schaefer, Elise; Collet, Corinne; Genevieve, David; Vincent, Marie; Lohmann, Dietmar R; Sanchez, Elodie; Bolender, Chantal; Eliot, Marie-Madeleine; Nürnberg, Gudrun; Passos-Bueno, Maria-Rita; Wieczorek, Dagmar; van Maldergem, Lionel; Doray, Bérénice

2014-09-01

Treacher Collins syndrome is a mandibulofacial dysostosis caused by mutations in genes involved in ribosome biogenesis and synthesis. TCOF1 mutations are observed in ~80% of the patients and are inherited in an autosomal dominant manner. Recently, two other genes have been reported in <2% of patients--POLR1D in patients with autosomal dominant inheritance, and POLR1C in patients with autosomal recessive inheritance. We performed direct sequencing of TCOF1, POLR1C, and POLR1D in two unrelated consanguineous families. The four affected children shared the same homozygous mutation in POLR1D (c.163C>G, p.Leu55Val). This mutation is localized in a region encoding the dimerization domain of the RNA polymerase. It is supposed that this mutation impairs RNA polymerase, resulting in a lower amount of mature dimeric ribosomes. A functional analysis of the transcripts of TCOF1 by real-time quantitative reverse transcription-polymerase chain reaction was performed in the first family, demonstrating a 50% reduction in the index case, compatible with this hypothesis. This is the first report of POLR1D mutation being responsible for an autosomal recessive inherited Treacher Collins syndrome. These results reinforce the concept of genetic heterogeneity of Treacher Collins syndrome and underline the importance of combining clinical expertise and familial molecular analyses for appropriate genetic counseling.

The Sequences of 1504 Mutants in the Model Rice Variety Kitaake Facilitate Rapid Functional Genomic Studies.

PubMed

Li, Guotian; Jain, Rashmi; Chern, Mawsheng; Pham, Nikki T; Martin, Joel A; Wei, Tong; Schackwitz, Wendy S; Lipzen, Anna M; Duong, Phat Q; Jones, Kyle C; Jiang, Liangrong; Ruan, Deling; Bauer, Diane; Peng, Yi; Barry, Kerrie W; Schmutz, Jeremy; Ronald, Pamela C

2017-06-01

The availability of a whole-genome sequenced mutant population and the cataloging of mutations of each line at a single-nucleotide resolution facilitate functional genomic analysis. To this end, we generated and sequenced a fast-neutron-induced mutant population in the model rice cultivar Kitaake ( Oryza sativa ssp japonica ), which completes its life cycle in 9 weeks. We sequenced 1504 mutant lines at 45-fold coverage and identified 91,513 mutations affecting 32,307 genes, i.e., 58% of all rice genes. We detected an average of 61 mutations per line. Mutation types include single-base substitutions, deletions, insertions, inversions, translocations, and tandem duplications. We observed a high proportion of loss-of-function mutations. We identified an inversion affecting a single gene as the causative mutation for the short-grain phenotype in one mutant line. This result reveals the usefulness of the resource for efficient, cost-effective identification of genes conferring specific phenotypes. To facilitate public access to this genetic resource, we established an open access database called KitBase that provides access to sequence data and seed stocks. This population complements other available mutant collections and gene-editing technologies. This work demonstrates how inexpensive next-generation sequencing can be applied to generate a high-density catalog of mutations. © 2017 American Society of Plant Biologists. All rights reserved.

The Sequences of 1,504 Mutants in the Model Rice Variety Kitaake Facilitate Rapid Functional Genomic Studies

DOE PAGES

Li, Guotian; Jain, Rashmi; Chern, Mawsheng; ...

2017-06-02

The availability of a whole-genome sequenced mutant population and the cataloging of mutations of each line at a single-nucleotide resolution facilitate functional genomic analysis. To this end, we generated and sequenced a fast-neutron-induced mutant population in the model rice cultivar Kitaake (Oryza sativa ssp japonica), which completes its life cycle in 9 weeks. We sequenced 1504 mutant lines at 45-fold coverage and identified 91,513 mutations affecting 32,307 genes, i.e., 58% of all rice genes. We detected an average of 61 mutations per line. Mutation types include single-base substitutions, deletions, insertions, inversions, translocations, and tandem duplications. We observed a high proportionmore » of loss-of-function mutations. We identified an inversion affecting a single gene as the causative mutation for the short-grain phenotype in one mutant line. This result reveals the usefulness of the resource for efficient, cost-effective identification of genes conferring specific phenotypes. To facilitate public access to this genetic resource, we established an open access database called KitBase that provides access to sequence data and seed stocks. This population complements other available mutant collections and gene-editing technologies. In conclusion, this work demonstrates how inexpensive next-generation sequencing can be applied to generate a high-density catalog of mutations.« less

Racial disparities in patient survival and tumor mutation burden, and the association between tumor mutation burden and cancer incidence rate.

PubMed

Zhang, Wensheng; Edwards, Andrea; Flemington, Erik K; Zhang, Kun

2017-10-20

The causes underlying racial disparities in cancer are multifactorial. In addition to socioeconomic issues, biological factors may contribute to these inequities, especially in disease incidence and patient survival. To date, there have been few studies that relate the disparities in these aspects to genetic aberrations. In this work, we studied the impacts of race on the patient survival and tumor mutation burden using the data released by the Cancer Genome Atlas (TCGA). The potential relationship between mutation burden and disease incidence is further inferred by an integrative analysis of TCGA data and the data from the Surveillance, Epidemiology, and End Results (SEER) Program. The results show that disparities are present (p < 0.05) in patient survival of five cancers, such as head and neck squamous cell carcinoma. The numbers of tumor driver mutations are differentiated (p < 0.05) over the racial groups in five cancers, such as lung adenocarcinoma. By treating a specific cancer type and a racial group as an "experimental unit", driver mutation numbers demonstrate a significant (r = 0.46, p < 0.002) positive correlation with cancer incidence rates, especially when the five cancers with mutational disparities are exclusively focused (r = 0.88, p < 0.00002). These results enrich our understanding of racial disparities in cancer and carcinogenic process.

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

Parallel Evolution of Polydactyly Traits in Chinese and European Chickens.

PubMed

Zhang, Zebin; Nie, Changsheng; Jia, Yaxiong; Jiang, Runshen; Xia, Haijian; Lv, Xueze; Chen, Yu; Li, Junying; Li, Xianyao; Ning, Zhonghua; Xu, Guiyun; Chen, Jilan; Yang, Ning; Qu, Lujiang

2016-01-01

Polydactyly is one of the most common hereditary congenital limb malformations in chickens and other vertebrates. The zone of polarizing activity regulatory sequence (ZRS) is critical for the development of polydactyly. The causative mutation of polydactyly in the Silkie chicken has been mapped to the ZRS; however, the causative mutations of other chicken breeds are yet to be established. To understand whether the same mutation decides the polydactyly phenotype in other chicken breeds, we detected the single-nucleotide polymorphism in 26 different chicken breeds, specifically, 24 Chinese indigenous breeds and 2 European breeds. The mutation was found to have fully penetrated chickens with polydactyly in China, indicating that it is causative for polydactyly in Chinese indigenous chickens. In comparison, the mutation showed no association with polydactyly in Houdan chickens, which originate from France, Europe. Based on the different morphology of polydactyly in Chinese and European breeds, we assumed that the trait might be attributable to different genetic foundations. Therefore, we subsequently performed genome-wide association analysis (GWAS) to locate the region associated with polydactyly. As a result, a ~0.39 Mb genomic region on GGA2p was identified. The region contains six candidate genes, with the causative mutation found in Chinese indigenous breeds also being located in this region. Our results demonstrate that polydactyly in chickens from China and Europe is caused by two independent mutation events that are closely located in the chicken genome.

The Near Naked Hairless (HrN) Mutation Disrupts Hair Formation but is not Due to a Mutation in the Hairless Coding Region

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

Liu, Yutao; Das, Suchita; Olszewski, Robert Edward

Near naked hairless (HrN) is a semi-dominant mutation that arose spontaneously and was suggested by allelism testing to be an allele of mouse Hairless (Hr). HrN mice differ from other Hr mutants in that hair loss appears as the postnatal coat begins to emerge, as opposed to failure to initiate the first postnatal hair cycle, and that the mutation displays semi-dominant inheritance. We sequenced the Hr cDNA in HrN/HrN mice and characterized the pathological and molecular phenotypes to identify the basis for hair loss in this model. HrN/HrN mice exhibit dystrophic hairs that are unable to consistently emerge from themore » hair follicle, while HrN/+ mice display a sparse coat of hair and a milder degree of follicular dystrophy than their homozygous littermates. DNA microarray analysis of cutaneous gene expression demonstrates that numerous genes are downregulated in HrN/HrN mice, primarily genes important for hair structure. By contrast, Hr expression is significantly increased. Sequencing the Hr coding region, intron-exon boundaries, 5'- and 3'- UTR and immediate upstream region did not reveal the underlying mutation. Therefore HrN does not appear to be an allele of Hr but may result from a mutation in a closely linked gene or from a regulatory mutation in Hr.« less

Identification of a novel homozygous mutation in MYO3A in a Chinese family with DFNB30 non-syndromic hearing impairment.

PubMed

Qu, Ronggui; Sang, Qing; Xu, Yao; Feng, Ruizhi; Jin, Li; He, Lin; Wang, Lei

2016-05-01

Hearing loss is a common sensory impairment. Several genetic loci or genes responsible for non-syndrome hearing loss have been identified, including the well-known deafness genes GJB2, MT-RNR1 and SLC26A4. MYO3A belongs to the myosin superfamily. Previously only three mutations in this gene have been found in an Isreali family with DFNB30, in which patients demonstrated progressive hearing loss. In this study, we characterized a consanguineous Kazakh family with congenital hearing loss. By targeted sequence capture and next-generation sequencing, we identified a homozygous mutation and did bioinformatics analysis to this mutation. A homozygous mutation, MYO3A:c.1841C>T (p.S614F), was identified to be responsible for the disease. Ser614 is located in the motor domain of MYO3A that is highly conserved among different species. Molecular modeling predicts that the conserved Ser614 may play an important role in maintaining the stability of β-sheet and the interaction between neighboring β-strand. This is the second report on MYO3A mutations in deafness and the first report in China. The finding help facilitate establishing a better relationship between MYO3A mutation and hearing phenotypes. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

The Influence of Polyploidy on the Evolution of Yeast Grown in a Sub-Optimal Carbon Source

PubMed Central

Scott, Amber L.; Richmond, Phillip A.; Dowell, Robin D.; Selmecki, Anna M.

2017-01-01

Abstract Polyploidization events have occurred during the evolution of many fungi, plant, and animal species and are thought to contribute to speciation and tumorigenesis, however little is known about how ploidy level contributes to adaptation at the molecular level. Here we integrate whole genome sequencing, RNA expression analysis, and relative fitness of ∼100 evolved clones at three ploidy levels. Independent haploid, diploid, and tetraploid populations were grown in a low carbon environment for 250 generations. We demonstrate that the key adaptive mutation in the evolved clones is predicted by a gene expression signature of just five genes. All of the adaptive mutations identified encompass a narrow set of genes, however the tetraploid clones gain a broader spectrum of adaptive mutations than haploid or diploid clones. While many of the adaptive mutations occur in genes that encode proteins with known roles in glucose sensing and transport, we discover mutations in genes with no canonical role in carbon utilization (IPT1 and MOT3), as well as identify novel dominant mutations in glucose signal transducers thought to only accumulate recessive mutations in carbon limited environments (MTH1 and RGT1). We conclude that polyploid cells explore more genotypic and phenotypic space than lower ploidy cells. Our study provides strong evidence for the beneficial role of polyploidization events that occur during the evolution of many species and during tumorigenesis. PMID:28957510

Biochemical analysis of respiratory function in cybrid cell lines harbouring mitochondrial DNA mutations

PubMed Central

2004-01-01

We analysed key biochemical features that reflect the balance between glycolysis and glucose oxidation in cybrids (cytoplasmic hybrids) harbouring a representative sample of mitochondrial DNA point mutations and deletions. The cybrids analysed had the same 143B cell nuclear background and were isogenic for the mitochondrial background. The 143B cell line and its ρ0 counterpart were used as controls. All cells analysed were in a dynamic state, and cell number, time of plating, culture medium, extracellular volume and time of harvest and assay were strictly controlled. Intra- and extra-cellular lactate and pyruvate levels were measured in homoplasmic wild-type and mutant cells, and correlated with rates of ATP synthesis and O2 consumption. In all mutant cell lines, except those with the T8993C mutation in the ATPase 6 gene, glycolysis was increased even under conditions of low glucose, as demonstrated by increased levels of extracellular lactate and pyruvate. Extracellular lactate levels were strictly and inversely correlated with rates of ATP synthesis and O2 consumption. These results show increased glycolysis and defective oxidative phosphorylation, irrespective of the type or site of the point mutation or deletion in the mitochondrial genome. The different biochemical consequences of the T8993C mutation suggest a uniquely different pathogenic mechanism for this mutation. However, the distinct clinical features associated with some of these mutations still remain to be elucidated. PMID:15324306

Characterization of R132H mutation-specific IDH1 antibody binding in brain tumors.

PubMed

Capper, David; Weissert, Susanne; Balss, Jörg; Habel, Antje; Meyer, Jochen; Jäger, Diana; Ackermann, Ulrike; Tessmer, Claudia; Korshunov, Andrey; Zentgraf, Hanswalter; Hartmann, Christian; von Deimling, Andreas

2010-01-01

Heterozygous point mutations of isocitrate dehydrogenase (IDH)1 codon 132 are frequent in grade II and III gliomas. Recently, we reported an antibody specific for the IDH1R132H mutation. Here we investigate the capability of this antibody to differentiate wild type and mutated IDH1 protein in central nervous system (CNS) tumors by Western blot and immunohistochemistry. Results of protein analysis are correlated to sequencing data. In Western blot, anti-IDH1R132H mouse monoclonal antibody mIDH1R132H detected a specific band only in mutated tumors. Immunohistochemistry of 345 primary brain tumors demonstrated a strong cytoplasmic and weaker nuclear staining in 122 cases. Correlation with direct sequencing of 186 cases resulted in consensus of 177 cases. Genetic retesting of cases with conflicting findings resulted in a match of 186/186 cases, with all discrepancies resolving in favor of immunohistochemistry. Intriguing is the ability of mIDH1R132H to detect single infiltrating tumor cells. The very high frequency and the distribution of this mutation among specific brain tumor entities allow the highly sensitive and specific discrimination of various tumors by immunohistochemistry, such as anaplastic astrocytoma from primary glioblastoma or diffuse astrocytoma World Health Organization (WHO) grade II from pilocytic astrocytoma or ependymoma. Noteworthy is the discrimination of the infiltrating edge of tumors with IDH1 mutation from reactive gliosis.

TP53 mutation and survival in aggressive B cell lymphoma.

PubMed

Zenz, Thorsten; Kreuz, Markus; Fuge, Maxi; Klapper, Wolfram; Horn, Heike; Staiger, Annette M; Winter, Doris; Helfrich, Hanne; Huellein, Jennifer; Hansmann, Martin-Leo; Stein, Harald; Feller, Alfred; Möller, Peter; Schmitz, Norbert; Trümper, Lorenz; Loeffler, Markus; Siebert, Reiner; Rosenwald, Andreas; Ott, German; Pfreundschuh, Michael; Stilgenbauer, Stephan

2017-10-01

TP53 is mutated in 20-25% of aggressive B-cell lymphoma (B-NHL). To date, no studies have addressed the impact of TP53 mutations in prospective clinical trial cohorts. To evaluate the impact of TP53 mutation to current risk models in aggressive B-NHL, we investigated TP53 gene mutations within the RICOVER-60 trial. Of 1,222 elderly patients (aged 61-80 years) enrolled in the study and randomized to six or eight cycles of CHOP-14 with or without Rituximab (NCT00052936), 265 patients were analyzed for TP53 mutations. TP53 mutations were demonstrated in 63 of 265 patients (23.8%). TP53 mutation was associated with higher LDH (65% vs. 37%; p < 0.001), higher international prognostic index-Scores (IPI 4/5 27% vs. 12%; p = 0.025) and B-symptoms (41% vs. 24%; p = 0.011). Patients with TP53 mutation were less likely to obtain a complete remission CR/CRu (CR unconfirmed) 61.9% (mut) vs. 79.7% (wt) (p = 0.007). TP53 mutations were associated with decreased event-free (EFS), progression-free (PFS) and overall survival (OS) (median observation time of 40.2 months): the 3 year EFS, PFS and OS were 42% (vs. 60%; p = 0.012), 42% (vs. 67.5%; p < 0.001) and 50% (vs. 76%; p < 0.001) for the TP53 mutation group. In a Cox proportional hazard analysis adjusting for IPI-factors and treatment arms, TP53 mutation was shown to be an independent predictor of EFS (HR 1.5), PFS (HR 2.0) and OS (HR 2.3; p < 0.001). TP53 mutations are independent predictors of survival in untreated patients with aggressive CD20+ lymphoma. TP53 mutations should be considered for risk models in DLBCL and strategies to improve outcome for patients with mutant TP53 must be developed. © 2017 UICC.

Phenotype variability and allelic heterogeneity in KMT2B-Associated disease.

PubMed

Kawarai, Toshitaka; Miyamoto, Ryosuke; Nakagawa, Eiji; Koichihara, Reiko; Sakamoto, Takashi; Mure, Hideo; Morigaki, Ryoma; Koizumi, Hidetaka; Oki, Ryosuke; Montecchiani, Celeste; Caltagirone, Carlo; Orlacchio, Antonio; Hattori, Ayako; Mashimo, Hideaki; Izumi, Yuishin; Mezaki, Takahiro; Kumada, Satoko; Taniguchi, Makoto; Yokochi, Fusako; Saitoh, Shinji; Goto, Satoshi; Kaji, Ryuji

2018-04-05

Mutations in Lysine-Specific Histone Methyltransferase 2B gene (KMT2B) have been reported to be associated with complex early-onset dystonia. Almost all reported KMT2B mutations occurred de novo in the paternal germline or in the early development of the patient. We describe clinico-genetic features on four Japanese patients with novel de novo mutations and demonstrate the phenotypic spectrum of KMT2B mutations. We performed genetic studies, including trio-based whole exome sequencing (WES), in a cohort of Japanese patients with a seemingly sporadic early-onset generalized combined dystonia. Potential effects by the identified nucleotide variations were evaluated biologically. Genotype-phenotype correlations were also investigated. Four patients had de novo heterozygous mutations in KMT2B, c.309delG, c.1656dupC, c.3325_3326insC, and c.5636delG. Biological analysis of KMT2B mRNA levels showed a reduced expression of mutant transcript frame. All patients presented with motor milestone delay, microcephaly, mild psychomotor impairment, childhood-onset generalized dystonia and superimposed choreoathetosis or myoclonus. One patient cannot stand due to axial hypotonia associated with cerebellar dysfunction. Three patients had bilateral globus pallidal deep brain stimulation (DBS) with excellent or partial response. We further demonstrate the allelic heterogeneity and phenotypic variations of KMT2B-associated disease. Haploinsufficiency is one of molecular pathomechanisms underlying the disease. Cardinal clinical features include combined dystonia accompanying mild psychomotor disability. Cerebellum would be affected in KMT2B-associated disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

Usefulness of BCOR gene mutation as a prognostic factor in acute myeloid leukemia with intermediate cytogenetic prognosis.

PubMed

Terada, Kazuki; Yamaguchi, Hiroki; Ueki, Toshimitsu; Usuki, Kensuke; Kobayashi, Yutaka; Tajika, Kenji; Gomi, Seiji; Kurosawa, Saiko; Saito, Riho; Furuta, Yutaka; Miyadera, Keiki; Tokura, Taichiro; Marumo, Atushi; Omori, Ikuko; Sakaguchi, Masahiro; Fujiwara, Yusuke; Yui, Shunsuke; Ryotokuji, Takeshi; Arai, Kunihito; Kitano, Tomoaki; Wakita, Satoshi; Fukuda, Takahiro; Inokuchi, Koiti

2018-04-16

BCOR gene is a transcription regulatory factor that plays an essential role in normal hematopoiesis. The wider introduction of next-generation sequencing technology has led to reports in recent years of mutations in the BCOR gene in acute myeloid leukemia (AML), but the related clinical characteristics and prognosis are not sufficiently understood. We investigated the clinical characteristics and prognosis of 377 de novo AML cases with BCOR or BCORL1 mutation. BCOR or BCORL1 gene mutations were found in 28 cases (7.4%). Among cases aged 65 years or below that were also FLT3-ITD-negative and in the intermediate cytogenetic prognosis group, BCOR or BCORL1 gene mutations were observed in 11% of cases (12 of 111 cases), and this group had significantly lower 5-year overall survival (OS) (13.6% vs. 55.0%, P=0.0021) and relapse-free survival (RFS) (14.3% vs. 44.5%, P=0.0168) compared to cases without BCOR or BCORL1 gene mutations. Multivariate analysis demonstrated that BCOR mutations were an independent unfavorable prognostic factor (P=0.0038, P=0.0463) for both OS and RFS. In cases of AML that are FLT3-ITD-negative, aged 65 years or below, and in the intermediate cytogenetic prognosis group, which are considered to have relatively favorable prognosis, BCOR gene mutations appear to be an important prognostic factor. This article is protected by copyright. All rights reserved. © 2018 Wiley Periodicals, Inc.

Not all SCID pigs are created equally: Two independent mutations in the Artemis gene cause Severe Combined Immunodeficiency (SCID) in pigs

PubMed Central

Waide, Emily H; Dekkers, Jack CM; Ross, Jason W; Rowland, Raymond RR; Wyatt, Carol R; Ewen, Catherine L; Evans, Alyssa B; Thekkoot, Dinesh M; Boddicker, Nicholas J; Serão, Nick VL; Ellinwood, N Matthew; Tuggle, Christopher K

2017-01-01

Mutations in over 30 genes are known to result in impairment of the adaptive immune system, causing a group of disorders collectively known as severe combined immunodeficiency (SCID). SCID disorders are split into groups based on their presence and/or functionality of B, T, and NK cells. Piglets from a line of Yorkshire pigs at Iowa State University were shown to be affected by T− B− NK+ SCID, representing the first example of naturally occurring SCID in pigs. Here, we present evidence for two spontaneous mutations as the molecular basis for this SCID phenotype. Flow cytometry analysis of thymocytes showed an increased frequency of immature T cells in SCID pigs. Fibroblasts from these pigs were more sensitive to ionizing radiation than non-SCID piglets, eliminating the RAG1 and RAG2 genes. Genetic and molecular analyses showed two mutations were present in the Artemis gene, which in homozygous or compound heterozygous state cause the immunodeficient phenotype. Rescue of SCID fibroblast radiosensitivity by human Artemis protein demonstrated that the identified Artemis mutations are the direct cause of this cellular phenotype. The work presented here reveals two mutations in the Artemis gene that cause T− B− NK+ SCID in pigs. The SCID pig can be an important biomedical model, but these mutations would be undesirable in commercial pig populations. The identified mutations and associated genetic tests can be used to address both of these issues. PMID:26320255

P16 UV mutations in human skin epithelial tumors.

PubMed

Soufir, N; Molès, J P; Vilmer, C; Moch, C; Verola, O; Rivet, J; Tesniere, A; Dubertret, L; Basset-Seguin, N

1999-09-23

The p16 gene expresses two alternative transcripts (p16alpha and p16beta) involved in tumor suppression via the retinoblastoma (Rb) or p53 pathways. Disruption of these pathways can occur through inactivation of p16 or p53, or activating mutations of cyclin dependant kinase 4 gene (Cdk4). We searched for p16, Cdk4 and p53 gene mutations in 20 squamous cell carcinomas (SSCs), 1 actinic keratosis (AK), and 28 basal cell carcinomas (BCCs), using PCR-SSCP. A deletion and methylation analysis of p16 was also performed. Six different mutations (12%) were detected in exon 2 of p16 (common to p16alpha and p16beta), in five out of 21 squamous lesions (24%) (one AK and four SCCs) and one out of 28 BCCs (3.5%). These included four (66%) ultraviolet (UV)-type mutations (two tandems CC : GG to TT : AA transitions and two C : G to T : A transitions at dipyrimidic site) and two transversions. P53 mutations were present in 18 samples (37%), mostly of UV type. Of these, only two (one BCC and one AK) harboured simultaneously mutations of p16, but with no consequence on p16beta transcript. Our data demonstrate for the first time the presence of p16 UV induced mutations in non melanoma skin cancer, particularly in the most aggressive SCC type, and support that p16 and p53 are involved in two independent pathways in skin carcinogenesis.

Saccharomyces cerevisiae Msh2-Msh3 acts in repair of base-base mispairs.

PubMed

Harrington, Jill M; Kolodner, Richard D

2007-09-01

DNA mismatch repair is thought to act through two subpathways involving the recognition of base-base and insertion/deletion mispairs by the Msh2-Msh6 heterodimer and the recognition of insertion/deletion mispairs by the Msh2-Msh3 heterodimer. Here, through genetic and biochemical approaches, we describe a previously unidentified role of the Msh2-Msh3 heterodimer in the recognition of base-base mispairs and the suppression of homology-mediated duplication and deletion mutations. Saccharomyces cerevisiae msh3 mutants did not show an increase in the rate of base substitution mutations by the CAN1 forward mutation assay compared to the rate for the wild type but did show an altered spectrum of base substitution mutations, including an increased accumulation of base pair changes from GC to CG and from AT to TA; msh3 mutants also accumulated homology-mediated duplication and deletion mutations. The mutation spectrum of mlh3 mutants paralleled that of msh3 mutants, suggesting that the Mlh1-Mlh3 heterodimer may also play a role in the repair of base-base mispairs and in the suppression of homology-mediated duplication and deletion mutations. Mispair binding analysis with purified Msh2-Msh3 and DNA substrates derived from CAN1 sequences found to be mutated in vivo demonstrated that Msh2-Msh3 exhibited robust binding to specific base-base mispairs that was consistent with functional mispair binding.

Saccharomyces cerevisiae Msh2-Msh3 Acts in Repair of Base-Base Mispairs▿ †

PubMed Central

Harrington, Jill M.; Kolodner, Richard D.

2007-01-01

DNA mismatch repair is thought to act through two subpathways involving the recognition of base-base and insertion/deletion mispairs by the Msh2-Msh6 heterodimer and the recognition of insertion/deletion mispairs by the Msh2-Msh3 heterodimer. Here, through genetic and biochemical approaches, we describe a previously unidentified role of the Msh2-Msh3 heterodimer in the recognition of base-base mispairs and the suppression of homology-mediated duplication and deletion mutations. Saccharomyces cerevisiae msh3 mutants did not show an increase in the rate of base substitution mutations by the CAN1 forward mutation assay compared to the rate for the wild type but did show an altered spectrum of base substitution mutations, including an increased accumulation of base pair changes from GC to CG and from AT to TA; msh3 mutants also accumulated homology-mediated duplication and deletion mutations. The mutation spectrum of mlh3 mutants paralleled that of msh3 mutants, suggesting that the Mlh1-Mlh3 heterodimer may also play a role in the repair of base-base mispairs and in the suppression of homology-mediated duplication and deletion mutations. Mispair binding analysis with purified Msh2-Msh3 and DNA substrates derived from CAN1 sequences found to be mutated in vivo demonstrated that Msh2-Msh3 exhibited robust binding to specific base-base mispairs that was consistent with functional mispair binding. PMID:17636021

Genetics and Prognostication in Splenic Marginal Zone Lymphoma: Revelations from Deep Sequencing

PubMed Central

Gibson, Jane; Wang, Jun; Walewska, Renata; Parker, Helen; Parker, Anton; Davis, Zadie; Gardiner, Anne; McIver-Brown, Neil; Kalpadakis, Christina; Xochelli, Aliki; Anagnostopoulos, Achilles; Fazi, Claudia; de Castro, David Gonzalez; Dearden, Claire; Pratt, Guy; Rosenquist, Richard; Ashton-Key, Margaret; Forconi, Francesco; Collins, Andrew; Ghia, Paolo; Matutes, Estella; Pangalis, Gerassimos; Stamatopoulos, Kostas; Oscier, David; Strefford, Jonathan C

2015-01-01

Purpose Mounting evidence supports the clinical significance of gene mutations and immunogenetic features in common mature B-cell malignancies. Experimental Design We undertook a detailed characterization of the genetic background of splenic marginal zone lymphoma (SMZL), using targeted re-sequencing and explored potential clinical implications in a multinational cohort of 175 SMZL patients. Results We identified recurrent mutations in TP53 (16%), KLF2 (12%), NOTCH2 (10%), TNFAIP3 (7%), MLL2 (11%), MYD88 (7%) and ARID1A (6%), all genes known to be targeted by somatic mutation in SMZL. KLF2 mutations were early, clonal events, enriched in patients with del(7q) and IGHV1-2*04 B-cell receptor immunoglobulins, and were associated with a short median time-to-first-treatment (0.12 vs. 1.11 yrs; P=0.01). In multivariate analysis mutations in NOTCH2 (HR 2.12, 95%CI 1.02-4.4, P=0.044) and 100% germline IGHV gene identity (HR 2.19, 95%CI 1.05-4.55, P=0.036) were independent markers of short time-to-first-treatment, while TP53 mutations were an independent marker of short overall survival (HR 2.36, 95% CI 1.08-5.2, P=0.03). Conclusion We identify key associations between gene mutations and clinical outcome, demonstrating for the first time that NOTCH2 and TP53 gene mutations are independent markers of reduced treatment-free and overall survival, respectively. PMID:25779943

Identification of a structurally novel BTK mutation that drives ibrutinib resistance in CLL.

PubMed

Sharma, Shruti; Galanina, Natalie; Guo, Ailin; Lee, Jimmy; Kadri, Sabah; Van Slambrouck, Charles; Long, Bradley; Wang, Weige; Ming, Mei; Furtado, Larissa V; Segal, Jeremy P; Stock, Wendy; Venkataraman, Girish; Tang, Wei-Jen; Lu, Pin; Wang, Yue Lynn

2016-10-18

Ibrutinib (ibr), a first-in-class Bruton tyrosine kinase (BTK) inhibitor, has demonstrated high response rates in both relapsed/refractory and treatment naïve chronic lymphocytic leukemia (CLL). However, about 25% of patients discontinue ibrutinib therapy at a median follow-up of 20 months and many patients discontinue the treatment due to leukemia progression or Richter transformation. Mutations affecting the C481 residue of BTK disrupt ibrutinib binding and have been characterized by us and others as the most common mechanism of ibrutinib resistance. Thus far, all described BTK mutations are located in its kinase domain and mutations outside this domain have never been described. Herein, we report a patient whose CLL progressed, was salvaged with ibrutinib and then relapsed. Serial analysis of samples throughout patient's clinical course identified a structurally novel mutation (BTKT316A) in the SH2 domain, but not kinase domain, of Bruton tyrosine kinase which was associated with disease relapse. Functionally, cells carrying BTKT316A show resistance to ibrutinib at both cellular and molecular levels to a similar extent as BTKC481S. Our study lends further insight into the diverse mechanisms of ibrutinib resistance that has important implications for the development of next-generation BTK inhibitors as well as mutation detection in relapsed patients.

Oligosyndactylism Mice Have an Inversion of Chromosome 8

PubMed Central

Wise, Thomas L.; Pravtcheva, Dimitrina D.

2004-01-01

The radiation-induced mutation Oligosyndactylism (Os) is associated with limb and kidney defects in heterozygotes and with mitotic arrest and embryonic lethality in homozygotes. We reported that the cell cycle block in Os and in the 94-A/K transgene-induced mutations is due to disruption of the Anapc10 (Apc10/Doc1) gene. To understand the genetic basis of the limb and kidney abnormalities in Os mice we characterized the structural changes of chromosome 8 associated with this mutation. We demonstrate that the Os chromosome 8 has suffered two breaks that are 5 cM (∼10 Mb) apart and the internal fragment delineated by the breaks is in an inverted orientation on the mutant chromosome. While sequences in proximity to the distal break are present in an abnormal Os-specific Anapc10 hybrid transcript, transcription of these sequences in normal mice is low and difficult to detect. Transfer of the Os mutation onto an FVB/N background indicated that the absence of dominant effects in 94-A/K mice is not due to strain background effects on the mutation. Further analysis of this mutation will determine if a gene interrupted by the break or a long-range effect of the rearrangement on neighboring genes is responsible for the dominant effects of Os. PMID:15611179

Study of base pair mutations in proline-rich homeodomain (PRH)-DNA complexes using molecular dynamics.

PubMed

Jalili, Seifollah; Karami, Leila; Schofield, Jeremy

2013-06-01

Proline-rich homeodomain (PRH) is a regulatory protein controlling transcription and gene expression processes by binding to the specific sequence of DNA, especially to the sequence 5'-TAATNN-3'. The impact of base pair mutations on the binding between the PRH protein and DNA is investigated using molecular dynamics and free energy simulations to identify DNA sequences that form stable complexes with PRH. Three 20-ns molecular dynamics simulations (PRH-TAATTG, PRH-TAATTA and PRH-TAATGG complexes) in explicit solvent water were performed to investigate three complexes structurally. Structural analysis shows that the native TAATTG sequence forms a complex that is more stable than complexes with base pair mutations. It is also observed that upon mutation, the number and occupancy of the direct and water-mediated hydrogen bonds decrease. Free energy calculations performed with the thermodynamic integration method predict relative binding free energies of 0.64 and 2 kcal/mol for GC to AT and TA to GC mutations, respectively, suggesting that among the three DNA sequences, the PRH-TAATTG complex is more stable than the two mutated complexes. In addition, it is demonstrated that the stability of the PRH-TAATTA complex is greater than that of the PRH-TAATGG complex.

Identification of somatic mutations in non-small cell lung carcinomas using whole-exome sequencing

PubMed Central

Liu, Pengyuan; Morrison, Carl; Wang, Liang; Xiong, Donghai; Vedell, Peter; Cui, Peng; Hua, Xing; Ding, Feng; Lu, Yan; James, Michael; Ebben, John D.; Xu, Haiming; Adjei, Alex A.; Head, Karen; Andrae, Jaime W.; Tschannen, Michael R.; Jacob, Howard; Pan, Jing; Zhang, Qi; Van den Bergh, Francoise; Xiao, Haijie; Lo, Ken C.; Patel, Jigar; Richmond, Todd; Watt, Mary-Anne; Albert, Thomas; Selzer, Rebecca; Anderson, Marshall; Wang, Jiang; Wang, Yian; Starnes, Sandra; Yang, Ping; You, Ming

2012-01-01

Lung cancer is the leading cause of cancer-related death, with non-small cell lung cancer (NSCLC) being the predominant form of the disease. Most lung cancer is caused by the accumulation of genomic alterations due to tobacco exposure. To uncover its mutational landscape, we performed whole-exome sequencing in 31 NSCLCs and their matched normal tissue samples. We identified both common and unique mutation spectra and pathway activation in lung adenocarcinomas and squamous cell carcinomas, two major histologies in NSCLC. In addition to identifying previously known lung cancer genes (TP53, KRAS, EGFR, CDKN2A and RB1), the analysis revealed many genes not previously implicated in this malignancy. Notably, a novel gene CSMD3 was identified as the second most frequently mutated gene (next to TP53) in lung cancer. We further demonstrated that loss of CSMD3 results in increased proliferation of airway epithelial cells. The study provides unprecedented insights into mutational processes, cellular pathways and gene networks associated with lung cancer. Of potential immediate clinical relevance, several highly mutated genes identified in our study are promising druggable targets in cancer therapy including ALK, CTNNA3, DCC, MLL3, PCDHIIX, PIK3C2B, PIK3CG and ROCK2. PMID:22510280

Prenatal diagnosis of glycogen storage disease type 1a by single stranded conformation polymorphism (SSCP).

PubMed

Parvari, R; Hershkovitz, E; Carmi, R; Moses, S

1996-09-01

Glycogen storage disease type 1a (GSD 1a), a severe metabolic disorder, is caused by the absence of glucose-6-phosphatase (G6Pase) activity. Diagnosis is currently established by demonstrating the lack of G6Pase activity in the patient's liver specimen. Enzymatic diagnosis cannot be performed in chorionic villi or amniocytes as G6Pase is active only in the liver, kidney, and intestinal mucosa. Recent cloning of the G6Pase gene and subsequent identification of the mutations causing GSD 1a have led to the possibility of performing DNA-based diagnosis in chorionic villus samples (CVS) or amniocytes. Here we report the first DNA-based prenatal diagnosis in two families in whom GSD 1a patients were diagnosed. In one Jewish family with a previously identified R83C mutation, single-stranded conformation polymorphism (SSCP) analysis of the DNA extracted from CVS showed a homozygous R83C mutant pattern. As a result, the pregnancy was terminated and the diagnosis was confirmed on DNA analysis of the aborted fetus. In another family of Arabic extraction in which a V166G mutation has been identified in one of the siblings, SSCP analysis performed on DNA extracted from CVS presented the pattern of a normal control. The pregnancy was carried to term and a healthy baby was born. Thus, once mutations causing the disease are identified, prenatal diagnosis of GSD 1a is possible. SSCP analysis of DNA prepared from CVS is reliable, simple and fast, making it a suitable method for prenatal diagnosis.

The role of molecular genetic analysis in the diagnosis of primary ciliary dyskinesia.

PubMed

Kim, Raymond H; A Hall, David; Cutz, Ernest; Knowles, Michael R; Nelligan, Kathleen A; Nykamp, Keith; Zariwala, Maimoona A; Dell, Sharon D

2014-03-01

Primary ciliary dyskinesia (PCD) is an autosomal recessive genetic disorder of motile cilia. The diagnosis of PCD has previously relied on ciliary analysis with transmission electron microscopy or video microscopy. However, patients with PCD may have normal ultrastructural appearance, and ciliary analysis has limited accessibility. Alternatively, PCD can be diagnosed by demonstrating biallelic mutations in known PCD genes. Genetic testing is emerging as a diagnostic tool to complement ciliary analysis where interpretation and access may delay diagnosis. To determine the diagnostic yield of genetic testing of patients with a confirmed or suspected diagnosis of PCD in a multiethnic urban center. Twenty-eight individuals with confirmed PCD on transmission electron microscopy of ciliary ultrastructure and 24 individuals with a probable diagnosis of PCD based on a classical PCD phenotype and low nasal nitric oxide had molecular analysis of 12 genes associated with PCD. Of 49 subjects who underwent ciliary biopsy, 28 (57%) were diagnosed with PCD through an ultrastructural defect. Of the 52 individuals who underwent molecular genetic analysis, 22 (42%) individuals had two mutations in known PCD genes. Twenty-four previously unreported mutations in known PCD genes were observed. Combining both diagnostic modalities of biopsy and molecular genetics, the diagnostic yield increased to 69% compared with 57% based on biopsy alone. The diagnosis of PCD is challenging and has traditionally relied on ciliary biopsy, which is unreliable as the sole criterion for a definitive diagnosis. Molecular genetic analysis can be used as a complementary test to increase the diagnostic yield.

Mutation detection using ENDO1: application to disease diagnostics in humans and TILLING and Eco-TILLING in plants.

PubMed

Triques, Karine; Piednoir, Elodie; Dalmais, Marion; Schmidt, Julien; Le Signor, Christine; Sharkey, Mark; Caboche, Michel; Sturbois, Bénédicte; Bendahmane, Abdelhafid

2008-04-23

Most enzymatic mutation detection methods are based on the cleavage of heteroduplex DNA by a mismatch-specific endonuclease at mismatch sites and the analysis of the digestion product on a DNA sequencer. Important limitations of these methods are the availability of a mismatch-specific endonuclease, their sensitivity in detecting one allele in pool of DNA, the cost of the analysis and the ease by which the technique could be implemented in a standard molecular biology laboratory. The co-agroinfiltration of ENDO1 and p19 constructs into N. benthamiana leaves allowed high level of transient expression of a mismatch-specific and sensitive endonuclease, ENDO1 from Arabidopsis thaliana. We demonstrate the broad range of uses of the produced enzyme in detection of mutations. In human, we report the diagnosis of the G1691A mutation in Leiden factor-V gene associated with venous thrombosis and the fingerprinting of HIV-1 quasispecies in patients subjected to antiretroviral treatments. In plants, we report the use of ENDO1 system for detection of mutant alleles of Retinoblastoma-related gene by TILLING in Pisum sativum and discovery of natural sequence variations by Eco-TILLING in Arabidopsis thaliana. We introduce a cost-effective tool based on a simplified purification protocol of a mismatch-specific and sensitive endonuclease, ENDO1. Especially, we report the successful applications of ENDO1 in mutation diagnostics in humans, fingerprinting of complex population of viruses, and in TILLING and Eco-TILLING in plants.

MERRF/MELAS overlap syndrome due to the m.3291T>C mutation.

PubMed

Liu, Kaiming; Zhao, Hui; Ji, Kunqian; Yan, Chuanzhu

2014-03-01

We report the case of a 19-year-old Chinese female harboring the m.3291T>C mutation in the MT-TL1 gene encoding the mitochondrial transfer RNA for leucine. She presented with a complex phenotype characterized by progressive cerebellar ataxia, frequent myoclonus seizures, recurrent stroke-like episodes, migraine-like headaches with nausea and vomiting, and elevated resting lactate blood level. It is known that the myoclonus epilepsy with ragged-red fibers (MERRF) is characterized by cerebellar ataxia and myoclonus epilepsy, while that the mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is characterized by recurrent stroke-like episodes, migraine-like headaches, and elevated resting lactate blood level. So the patient's clinical manifestations suggest the presence of a MERRF/MELAS overlap syndrome. Muscle biopsy of the patient showed the presence of numerous scattered ragged-red fibers, some cytochrome c oxidase-deficient fibers, and several strongly succinate dehygrogenase-reactive vessels, suggestive of a mitochondrial disorder. Direct sequencing of the complete mitochondrial genome of the proband revealed no mutations other than the T-to-C transition at nucleotide position 3291. Restriction fragment length polymorphism analysis of the proband and her family revealed maternal inheritance of the mutation in a heteroplasmic manner. The analysis of aerobic respiration and glycolysis demonstrated that the fibroblasts from the patient had mitochondrial dysfunction. Our results suggest that the m.3291T>C is pathogenic. This study is the first to describe the m.3291T>C mutation in association with the MERRF/MELAS overlap syndrome.

Role of CFTR mutation analysis in the diagnostic algorithm for cystic fibrosis.

PubMed

Ratkiewicz, Michelle; Pastore, Matthew; McCoy, Karen Sharrock; Thompson, Rohan; Hayes, Don; Sheikh, Shahid Ijaz

2017-04-01

The cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation identification is being used with increased frequency to aid in the diagnosis of cystic fibrosis (CF) in those suspected with CF. Aim of this study was to identify diagnostic outcomes when CFTR mutational analysis was used in CF diagnosis. CFTR mutational analysis results were also compared with sweat chloride results. This study was done on all patients at our institution who had CFTR mutation analysis over a sevenyear period since August 2006. A total of 315 patients underwent CFTR mutational analysis. Fifty-one (16.2%) patients had two mutations identified. Among them 32 had positive sweat chloride levels (≥60 mmol/L), while seven had borderline sweat chloride levels (40-59 mmol/L). An additional 70 patients (22.3%) had only one mutation identified. Among them eight had positive sweat chloride levels, and 17 had borderline sweat chloride levels. Fifty-five patients (17.5%) without CFTR mutations had either borderline (n=45) or positive (n=10) sweat chloride results. Three patients with a CF phenotype had negative CFTR analysis but elevated sweat chloride levels. In eighty-three patients (26.4%) CFTR mutational analysis was done without corresponding sweat chloride testing. Although CFTR mutation analysis has improved the diagnostic capability for CF, its use either as the first step or the only test to diagnose CFTR dysfunction should be discouraged and CF diagnostic guidelines need to be followed.

Novel loss-of-function variants in DIAPH1 associated with syndromic microcephaly, blindness, and early onset seizures.

PubMed

Al-Maawali, Almundher; Barry, Brenda J; Rajab, Anna; El-Quessny, Malak; Seman, Ann; Coury, Stephanie Newton; Barkovich, A James; Yang, Edward; Walsh, Christopher A; Mochida, Ganeshwaran H; Stoler, Joan M

2016-02-01

Exome sequencing identified homozygous loss-of-function variants in DIAPH1 (c.2769delT; p.F923fs and c.3145C>T; p.R1049X) in four affected individuals from two unrelated consanguineous families. The affected individuals in our report were diagnosed with postnatal microcephaly, early-onset epilepsy, severe vision impairment, and pulmonary symptoms including bronchiectasis and recurrent respiratory infections. A heterozygous DIAPH1 mutation was originally reported in one family with autosomal dominant deafness. Recently, however, a homozygous nonsense DIAPH1 mutation (c.2332C4T; p.Q778X) was reported in five siblings in a single family affected by microcephaly, blindness, early onset seizures, developmental delay, and bronchiectasis. The role of DIAPH1 was supported using parametric linkage analysis, RNA and protein studies in their patients' cell lines and further studies in human neural progenitors cells and a diap1 knockout mouse. In this report, the proband was initially brought to medical attention for profound metopic synostosis. Additional concerns arose when his head circumference did not increase after surgical release at 5 months of age and he was diagnosed with microcephaly and epilepsy at 6 months of age. Clinical exome analysis identified a homozygous DIAPH1 mutation. Another homozygous DIAPH1 mutation was identified in the research exome analysis of a second family with three siblings presenting with a similar phenotype. Importantly, no hearing impairment is reported in the homozygous affected individuals or in the heterozygous carrier parents in any of the families demonstrating the autosomal recessive microcephaly phenotype. These additional families provide further evidence of the likely causal relationship between DIAPH1 mutations and a neurodevelopmental disorder. © 2016 Wiley Periodicals, Inc.

Information theory-based analysis of CYP2C19, CYP2D6 and CYP3A5 splicing mutations.

PubMed

Rogan, Peter K; Svojanovsky, Stan; Leeder, J Steven

2003-04-01

Several mutations are known or suspected to affect mRNA splicing of CYP2C19, CYP2D6 and CYP3A5 genes; however, little experimental evidence exists to support these conclusions. The present study applies mathematical models that measure changes in information content of splice sites in these genes to demonstrate the relationship between the predicted phenotypes of these variants to the corresponding genotypes. Based on information analysis, the CYP2C19*2 variant activates a new cryptic site 40 nucleotides downstream of the natural splice site. CYP2C19*7 abolishes splicing at the exon 5 donor site. The CYP2D6*4 allele similarly inactivates splicing at the acceptor site of exon 4 and activates a new cryptic site one nucleotide downstream of the natural acceptor. CYP2D6*11 inactivates the acceptor site of exon 2. The CYP3A5*3 allele activates a new cryptic site 236 nucleotides upstream of the exon 4 natural acceptor site. CYP3A5*5 inactivates the exon 5 donor site and CYP3A5*6 strengthens a site upstream of the natural donor site, resulting in skipping of exon 7. Other previously described missense and nonsense mutations at terminal codons of exons in these genes affected splicing. CYP2D6*8 and CYP2D6*14 both decrease the strength of the exon 3 donor site, producing transcripts lacking this exon. The results of information analysis are consistent with the poor metabolizer phenotypes observed in patients with these mutations, and illustrate the potential value of these mathematical models to quantitatively evaluate the functional consequences of new mutations suspected of altering mRNA splicing.

Congenital hypogonadotropic hypogonadism with split hand/foot malformation: a clinical entity with a high frequency of FGFR1 mutations

PubMed Central

Villanueva, Carine; Jacobson-Dickman, Elka; Xu, Cheng; Manouvrier, Sylvie; Dwyer, Andrew A.; Sykiotis, Gerasimos P.; Beenken, Andrew; Liu, Yang; Tommiska, Johanna; Hu, Youli; Tiosano, Dov; Gerard, Marion; Leger, Juliane; Drouin-Garraud, Valérie; Lefebvre, Hervé; Polak, Michel; Carel, Jean-Claude; Phan-Hug, Franziska; Hauschild, Michael; Plummer, Lacey; Rey, Jean-Pierre; Raivio, Taneli; Bouloux, Pierre; Sidis, Yisrael; Mohammadi, Moosa; de Roux, Nicolas; Pitteloud, Nelly

2014-01-01

Purpose Congenital hypogonadotropic hypogonadism (CHH) and split hand/foot malformation (SHFM) are two rare genetic conditions. Here we report a clinical entity comprising CHH and SHFM. Methods We identified patients with CHH and SHFM through international collaboration. Probands and available family members underwent phenotyping and screening for FGFR1 mutations. The impact of identified mutations was assessed by sequence- and structure-based predictions, and/or functional assays. Results We identified 8 probands with CHH with (n=3, Kallmann Syndrome) or without anosmia (n=5) and SHFM, 7 of whom (88%) harbor FGFR1 mutations: one individual is homozygous for p.V429E; six individuals are heterozygous for p.G348R, p.G485R, p.Q594*, p.E670A, p.V688L, and p.L712P. All mutations were predicted to be loss-of-function by in silico analysis. Probands with FGFR1 mutations have severe GnRH deficiency (absent puberty and/or cryptorchidism and/or micropenis). SHFM in both hands and feet was only observed in the patient with the homozygous p.V429E mutation; V429 maps to the FRS2α binding domain of FGFR1, and functional studies of the p.V429E mutation demonstrated that it decreased recruitment and phosphorylation of FRS2α to FG FR 1 , thereby resulting in reduced MAPK signaling. Conclusion FGFR1 should be prioritized for genetic testing in patients with CHH and SHFM, because the likelihood of a mutation increases from 10% in the general CHH population to 88%. PMID:25394172

Mutation exposed: a neutral explanation for extreme base composition of an endosymbiont genome.

PubMed

Wernegreen, Jennifer J; Funk, Daniel J

2004-12-01

The influence of neutral mutation pressure versus selection on base composition evolution is a subject of considerable controversy. Yet the present study represents the first explicit population genetic analysis of this issue in prokaryotes, the group in which base composition variation is most dramatic. Here, we explore the impact of mutation and selection on the dynamics of synonymous changes in Buchnera aphidicola, the AT-rich bacterial endosymbiont of aphids. Specifically, we evaluated three forms of evidence. (i) We compared the frequencies of directional base changes (AT-->GC vs. GC-->AT) at synonymous sites within and between Buchnera species, to test for selective preference versus effective neutrality of these mutational categories. Reconstructed mutational changes across a robust intraspecific phylogeny showed a nearly 1:1 AT-->GC:GC-->AT ratio. Likewise, stationarity of base composition among Buchnera species indicated equal rates of AT-->GC and GC-->AT substitutions. The similarity of these patterns within and between species supported the neutral model. (ii) We observed an equivalence of relative per-site AT mutation rate and current AT content at synonymous sites, indicating that base composition is at mutational equilibrium. (iii) We demonstrated statistically greater equality in the frequency of mutational categories in Buchnera than in parallel mammalian studies that documented selection on synonymous sites. Our results indicate that effectively neutral mutational pressure, rather than selection, represents the major force driving base composition evolution in Buchnera. Thus they further corroborate recent evidence for the critical role of reduced N(e) in the molecular evolution of bacterial endosymbionts.

[Mutation analysis of the PAH gene in children with phenylketonuria from the Qinghai area of China].

PubMed

He, Jiang; Wang, Hui-Zhen; Xu, Fa-Liang; Yang, Xi; Wang, Rui; Zou, Hong-Yun; Yu, Wu-Zhong

2015-11-01

To study the mutation characteristics of the phenylalanine hydroxylase (PAH) gene in children with phenylketonuria (PKU) from the Qinghai area of China, in order to provide basic information for genetic counseling and prenatal diagnosis. Mutations of the PAH gene were detected in the promoter and exons 1-13 and their flanking intronic sequences of PAH gene by PCR and DNA sequencing in 49 children with PKU and their parents from the Qinghai area of China. A total of 30 different mutations were detected in 80 out of 98 mutant alleles (82%), including 19 missense (63%), 5 nonsense (17%), 3 splice-site (10%) and 3 deletions (10%). Most mutations were detected in exons 3, 6, 7, 11 and intron 4 of PAH gene. The most frequent mutations were p.R243Q (19%), IVS4-1G>A (9%), p.Y356X (7%) and p.EX6-96A>G(5%). Two novel mutations p.N93fsX5 (c.279-282delCATC) and p.G171E (c.512G>A) were found. p.H64fsX9(c.190delC) was documented for the second time in Chinese PAH gene. The mutation spectrum of the gene PAH in the Qinghai population was similar to that in other populations in North China while significantly different from that in the populations from some provinces in southern China, Japan and Europe. The mutations of PAH gene in the Qinghai area of China demonstrate a unique diversity, complexity and specificity.

Identification and characterization of a novel XK splice site mutation in a patient with McLeod syndrome.

PubMed

Arnaud, Lionel; Salachas, François; Lucien, Nicole; Maisonobe, Thierry; Le Pennec, Pierre-Yves; Babinet, Jérôme; Cartron, Jean-Pierre

2009-03-01

McLeod syndrome is a rare X-linked neuroacanthocytosis syndrome with hematologic, muscular, and neurologic manifestations. McLeod syndrome is caused by mutations in the XK gene whose product is expressed at the red blood cell (RBC) surface but whose function is currently unknown. A variety of XK mutations has been reported but no clear phenotype-genotype correlation has been found, especially for the point mutations affecting splicing sites. A man suspected of neuroacanthocytosis was evaluated by neurologic examination, electromyography, muscle biopsy, muscle computed tomography, and cerebral magnetic resonance imaging. The McLeod RBC phenotype was disclosed by blood smear and immunohematology analyses and then confirmed at the biochemical level by Western blot analysis. The responsible XK mutation was characterized at the mRNA level by reverse transcription-polymerase chain reaction (PCR), identified by genomic DNA sequencing, and verified by allele-specific PCR. A novel XK splice site mutation (IVS1-1G>A) has been identified in a McLeod patient who has developed hematologic, neuromuscular, and neurologic symptoms. This is the first reported example of a XK point mutation affecting the 3' acceptor splice site of Intron 1, and it was demonstrated that this mutation indeed induces aberrant splicing of XK RNA and lack of XK protein at the RBC membrane. The detailed characterization at the molecular biology level of this novel XK splice site mutation associated with the clinical description of the patient contributes to a better understanding of the phenotype-genotype correlation in the McLeod syndrome.

Molecular analysis of hyperoxaluria type 1 in Italian patients reveals eight new mutations in the alanine: glyoxylate aminotransferase gene.

PubMed

Pirulli, D; Puzzer, D; Ferri, L; Crovella, S; Amoroso, A; Ferrettini, C; Marangella, M; Mazzola, G; Florian, F

1999-06-01

Systematic screening using the SSCP technique followed by sequencing of bands with abnormal mobility derived from the AGXT exons of 15 unrelated Italian patients with primary hyperoxaluria type 1 (PH1) allowed us to characterize both the mutant alleles in each individual. Eight new mutations were identified: C155del, C156ins, G244T, C252T, GAG408ins, G468A, G588A and G1098del. This study demonstrates both the effectiveness of the screening strategy chosen to identify all the mutant alleles and the high degree of allelic heterogeneity in PH1.

Molecular Clock of Neutral Mutations in a Fitness-Increasing Evolutionary Process

PubMed Central

Iijima, Leo; Suzuki, Shingo; Hashimoto, Tomomi; Oyake, Ayana; Kobayashi, Hisaka; Someya, Yuki; Narisawa, Dai; Yomo, Tetsuya

2015-01-01

The molecular clock of neutral mutations, which represents linear mutation fixation over generations, is theoretically explained by genetic drift in fitness-steady evolution or hitchhiking in adaptive evolution. The present study is the first experimental demonstration for the molecular clock of neutral mutations in a fitness-increasing evolutionary process. The dynamics of genome mutation fixation in the thermal adaptive evolution of Escherichia coli were evaluated in a prolonged evolution experiment in duplicated lineages. The cells from the continuously fitness-increasing evolutionary process were subjected to genome sequencing and analyzed at both the population and single-colony levels. Although the dynamics of genome mutation fixation were complicated by the combination of the stochastic appearance of adaptive mutations and clonal interference, the mutation fixation in the population was simply linear over generations. Each genome in the population accumulated 1.6 synonymous and 3.1 non-synonymous neutral mutations, on average, by the spontaneous mutation accumulation rate, while only a single genome in the population occasionally acquired an adaptive mutation. The neutral mutations that preexisted on the single genome hitchhiked on the domination of the adaptive mutation. The successive fixation processes of the 128 mutations demonstrated that hitchhiking and not genetic drift were responsible for the coincidence of the spontaneous mutation accumulation rate in the genome with the fixation rate of neutral mutations in the population. The molecular clock of neutral mutations to the fitness-increasing evolution suggests that the numerous neutral mutations observed in molecular phylogenetic trees may not always have been fixed in fitness-steady evolution but in adaptive evolution. PMID:26177190

Molecular Clock of Neutral Mutations in a Fitness-Increasing Evolutionary Process.

PubMed

Kishimoto, Toshihiko; Ying, Bei-Wen; Tsuru, Saburo; Iijima, Leo; Suzuki, Shingo; Hashimoto, Tomomi; Oyake, Ayana; Kobayashi, Hisaka; Someya, Yuki; Narisawa, Dai; Yomo, Tetsuya

2015-07-01

The molecular clock of neutral mutations, which represents linear mutation fixation over generations, is theoretically explained by genetic drift in fitness-steady evolution or hitchhiking in adaptive evolution. The present study is the first experimental demonstration for the molecular clock of neutral mutations in a fitness-increasing evolutionary process. The dynamics of genome mutation fixation in the thermal adaptive evolution of Escherichia coli were evaluated in a prolonged evolution experiment in duplicated lineages. The cells from the continuously fitness-increasing evolutionary process were subjected to genome sequencing and analyzed at both the population and single-colony levels. Although the dynamics of genome mutation fixation were complicated by the combination of the stochastic appearance of adaptive mutations and clonal interference, the mutation fixation in the population was simply linear over generations. Each genome in the population accumulated 1.6 synonymous and 3.1 non-synonymous neutral mutations, on average, by the spontaneous mutation accumulation rate, while only a single genome in the population occasionally acquired an adaptive mutation. The neutral mutations that preexisted on the single genome hitchhiked on the domination of the adaptive mutation. The successive fixation processes of the 128 mutations demonstrated that hitchhiking and not genetic drift were responsible for the coincidence of the spontaneous mutation accumulation rate in the genome with the fixation rate of neutral mutations in the population. The molecular clock of neutral mutations to the fitness-increasing evolution suggests that the numerous neutral mutations observed in molecular phylogenetic trees may not always have been fixed in fitness-steady evolution but in adaptive evolution.

Host-specific parvovirus evolution in nature is recapitulated by in vitro adaptation to different carnivore species.

PubMed

Allison, Andrew B; Kohler, Dennis J; Ortega, Alicia; Hoover, Elizabeth A; Grove, Daniel M; Holmes, Edward C; Parrish, Colin R

2014-11-01

Canine parvovirus (CPV) emerged as a new pandemic pathogen of dogs in the 1970s and is closely related to feline panleukopenia virus (FPV), a parvovirus of cats and related carnivores. Although both viruses have wide host ranges, analysis of viral sequences recovered from different wild carnivore species, as shown here, demonstrated that>95% were derived from CPV-like viruses, suggesting that CPV is dominant in sylvatic cycles. Many viral sequences showed host-specific mutations in their capsid proteins, which were often close to sites known to control binding to the transferrin receptor (TfR), the host receptor for these carnivore parvoviruses, and which exhibited frequent parallel evolution. To further examine the process of host adaptation, we passaged parvoviruses with alternative backgrounds in cells from different carnivore hosts. Specific mutations were selected in several viruses and these differed depending on both the background of the virus and the host cells in which they were passaged. Strikingly, these in vitro mutations recapitulated many specific changes seen in viruses from natural populations, strongly suggesting they are host adaptive, and which were shown to result in fitness advantages over their parental virus. Comparison of the sequences of the transferrin receptors of the different carnivore species demonstrated that many mutations occurred in and around the apical domain where the virus binds, indicating that viral variants were likely selected through their fit to receptor structures. Some of the viruses accumulated high levels of variation upon passage in alternative hosts, while others could infect multiple different hosts with no or only a few additional mutations. Overall, these studies demonstrate that the evolutionary history of a virus, including how long it has been circulating and in which hosts, as well as its phylogenetic background, has a profound effect on determining viral host range.

Host-Specific Parvovirus Evolution in Nature Is Recapitulated by In Vitro Adaptation to Different Carnivore Species

PubMed Central

Allison, Andrew B.; Kohler, Dennis J.; Ortega, Alicia; Hoover, Elizabeth A.; Grove, Daniel M.; Holmes, Edward C.; Parrish, Colin R.

2014-01-01

Canine parvovirus (CPV) emerged as a new pandemic pathogen of dogs in the 1970s and is closely related to feline panleukopenia virus (FPV), a parvovirus of cats and related carnivores. Although both viruses have wide host ranges, analysis of viral sequences recovered from different wild carnivore species, as shown here, demonstrated that >95% were derived from CPV-like viruses, suggesting that CPV is dominant in sylvatic cycles. Many viral sequences showed host-specific mutations in their capsid proteins, which were often close to sites known to control binding to the transferrin receptor (TfR), the host receptor for these carnivore parvoviruses, and which exhibited frequent parallel evolution. To further examine the process of host adaptation, we passaged parvoviruses with alternative backgrounds in cells from different carnivore hosts. Specific mutations were selected in several viruses and these differed depending on both the background of the virus and the host cells in which they were passaged. Strikingly, these in vitro mutations recapitulated many specific changes seen in viruses from natural populations, strongly suggesting they are host adaptive, and which were shown to result in fitness advantages over their parental virus. Comparison of the sequences of the transferrin receptors of the different carnivore species demonstrated that many mutations occurred in and around the apical domain where the virus binds, indicating that viral variants were likely selected through their fit to receptor structures. Some of the viruses accumulated high levels of variation upon passage in alternative hosts, while others could infect multiple different hosts with no or only a few additional mutations. Overall, these studies demonstrate that the evolutionary history of a virus, including how long it has been circulating and in which hosts, as well as its phylogenetic background, has a profound effect on determining viral host range. PMID:25375184

Coevolutionary analysis enabled rational deregulation of allosteric enzyme inhibition in Corynebacterium glutamicum for lysine production.

PubMed

Chen, Zhen; Meyer, Weiqian; Rappert, Sugima; Sun, Jibin; Zeng, An-Ping

2011-07-01

Product feedback inhibition of allosteric enzymes is an essential issue for the development of highly efficient microbial strains for bioproduction. Here we used aspartokinase from Corynebacterium glutamicum (CgAK), a key enzyme controlling the biosynthesis of industrially important aspartate family amino acids, as a model to demonstrate a fast and efficient approach to the deregulation of allostery. In the last 50 years many researchers and companies have made considerable efforts to deregulate this enzyme from allosteric inhibition by lysine and threonine. However, only a limited number of positive mutants have been identified so far, almost exclusively by random mutation and selection. In this study, we used statistical coupling analysis of protein sequences, a method based on coevolutionary analysis, to systematically clarify the interaction network within the regulatory domain of CgAK that is essential for allosteric inhibition. A cluster of interconnected residues linking different inhibitors' binding sites as well as other regions of the protein have been identified, including most of the previously reported positions of successful mutations. Beyond these mutation positions, we have created another 14 mutants that can partially or completely desensitize CgAK from allosteric inhibition, as shown by enzyme activity assays. The introduction of only one of the inhibition-insensitive CgAK mutations (here Q298G) into a wild-type C. glutamicum strain by homologous recombination resulted in an accumulation of 58 g/liter L-lysine within 30 h of fed-batch fermentation in a bioreactor.

Coevolutionary Analysis Enabled Rational Deregulation of Allosteric Enzyme Inhibition in Corynebacterium glutamicum for Lysine Production ▿

PubMed Central

Chen, Zhen; Meyer, Weiqian; Rappert, Sugima; Sun, Jibin; Zeng, An-Ping

2011-01-01

Product feedback inhibition of allosteric enzymes is an essential issue for the development of highly efficient microbial strains for bioproduction. Here we used aspartokinase from Corynebacterium glutamicum (CgAK), a key enzyme controlling the biosynthesis of industrially important aspartate family amino acids, as a model to demonstrate a fast and efficient approach to the deregulation of allostery. In the last 50 years many researchers and companies have made considerable efforts to deregulate this enzyme from allosteric inhibition by lysine and threonine. However, only a limited number of positive mutants have been identified so far, almost exclusively by random mutation and selection. In this study, we used statistical coupling analysis of protein sequences, a method based on coevolutionary analysis, to systematically clarify the interaction network within the regulatory domain of CgAK that is essential for allosteric inhibition. A cluster of interconnected residues linking different inhibitors' binding sites as well as other regions of the protein have been identified, including most of the previously reported positions of successful mutations. Beyond these mutation positions, we have created another 14 mutants that can partially or completely desensitize CgAK from allosteric inhibition, as shown by enzyme activity assays. The introduction of only one of the inhibition-insensitive CgAK mutations (here Q298G) into a wild-type C. glutamicum strain by homologous recombination resulted in an accumulation of 58 g/liter l-lysine within 30 h of fed-batch fermentation in a bioreactor. PMID:21531824

Quantitative analysis of brain atrophy in patients with xeroderma pigmentosum group A carrying the founder mutation in Japan.

PubMed

Ueda, Takehiro; Kanda, Fumio; Nishiyama, Masahiro; Nishigori, Chikako; Toda, Tatsushi

2017-10-15

Xeroderma pigmentosum (XP) is an inherited congenital disease presenting with dermatological and neurological manifestations. In Japan, XP complementation group A (XP-A) is most frequently observed in eight clinical subtypes, and the homozygous founder mutation, IVS3-1G>C in XPA, suffer from severe manifestations including progressive brain atrophy since childhood. In this study, we used magnetic resonance imaging (MRI) and applied volumetric analysis to elucidate the start and the progression of the brain atrophy in these patients. Twelve Japanese patients with XP-A carrying the founder mutation and seven controls were included. MRI was performed for each patient once or more. Three-dimensional T1 weighted images were segmented to gray matter, white matter, and cerebrospinal fluid, and each volume was calculated. Conventional MRI demonstrated progressive whole brain atrophy in patients with XP-A. Moreover, volumetric analysis showed that reductions of total gray matter volumes (GMV) and total brain volumes (TBV) started at the age of five. The slope of reduction was similar in all cases. The GMV and TBV values in controls were higher than those in XP-A cases after the age of five. This is the first quantitative report presenting with the progression of brain atrophy in patients with XP-A. It is revealed that the brain atrophy started from early childhood in Japanese patients with XP-A carrying the homozygous founder mutation. Copyright © 2017 Elsevier B.V. All rights reserved.

Atypical fibroxanthoma and pleomorphic dermal sarcoma harbor frequent NOTCH1/2 and FAT1 mutations and similar DNA copy number alteration profiles.

PubMed

Griewank, Klaus G; Wiesner, Thomas; Murali, Rajmohan; Pischler, Carina; Müller, Hansgeorg; Koelsche, Christian; Möller, Inga; Franklin, Cindy; Cosgarea, Ioana; Sucker, Antje; Schadendorf, Dirk; Schaller, Jörg; Horn, Susanne; Brenn, Thomas; Mentzel, Thomas

2018-03-01

Atypical fibroxanthomas and pleomorphic dermal sarcomas are tumors arising in sun-damaged skin of elderly patients. They have differing prognoses and are currently distinguished using histological criteria, such as invasion of deeper tissue structures, necrosis and lymphovascular or perineural invasion. To investigate the as-yet poorly understood genetics of these tumors, 41 atypical fibroxanthomas and 40 pleomorphic dermal sarcomas were subjected to targeted next-generation sequencing approaches as well as DNA copy number analysis by comparative genomic hybridization. In an analysis of the entire coding region of 341 oncogenes and tumor suppressor genes in 13 atypical fibroxanthomas using an established hybridization-based next-generation sequencing approach, we found that these tumors harbor a large number of mutations. Gene alterations were identified in more than half of the analyzed samples in FAT1, NOTCH1/2, CDKN2A, TP53, and the TERT promoter. The presence of these alterations was verified in 26 atypical fibroxanthoma and 35 pleomorphic dermal sarcoma samples by targeted amplicon-based next-generation sequencing. Similar mutation profiles in FAT1, NOTCH1/2, CDKN2A, TP53, and the TERT promoter were identified in both atypical fibroxanthoma and pleomorphic dermal sarcoma. Activating RAS mutations (G12 and G13) identified in 3 pleomorphic dermal sarcoma were not found in atypical fibroxanthoma. Comprehensive DNA copy number analysis demonstrated a wide array of different copy number gains and losses, with similar profiles in atypical fibroxanthoma and pleomorphic dermal sarcoma. In summary, atypical fibroxanthoma and pleomorphic dermal sarcoma are highly mutated tumors with recurrent mutations in FAT1, NOTCH1/2, CDKN2A, TP53, and the TERT promoter, and a range of DNA copy number alterations. These findings suggest that atypical fibroxanthomas and pleomorphic dermal sarcomas are genetically related, potentially representing two ends of a common tumor spectrum and distinguishing these entities is at present still best performed using histological criteria.

A familial case of achondrogenesis type II caused by a dominant COL2A1 mutation and "patchy" expression in the mosaic father.

PubMed

Forzano, F; Lituania, M; Viassolo, A; Superti-Furga, V; Wildhardt, G; Zabel, B; Faravelli, F

2007-12-01

Achondrogenesis type II (ACG2) is the most severe disorder that can be produced by dominant mutations in COL2A1. We report on four pregnancies of an apparently healthy, nonconsanguineous young couple. The father had scoliosis as a child, and has slight body disproportion with short trunk. The first child was born at 32 weeks and died neonatally. In the second pregnancy, short limbs and fetal hygroma were noted on ultrasound at 17 weeks' gestation. Similar findings were observed in the third fetus. Clinical, radiological, and histological evaluation of the fetuses after termination of the pregnancies showed findings consistent with ACG2. Molecular analysis of genomic DNA extracted from amniotic cells of the second and third fetuses revealed heterozygosity for a 10370G > T missense mutation (G346V) in the COL2A1 gene. This mutation was also found in the father, as a mosaic. The couple had a fourth pregnancy, and at 11 weeks fetal hydrops with a septated cystic hygroma were obvious. DNA from CVS demonstrated the same COL2A1 mutation. (c) 2007 Wiley-Liss, Inc.

Identification of novel missense mutations in the Norrie disease gene associated with one X-linked and four sporadic cases of familial exudative vitreoretinopathy.

PubMed

Shastry, B S; Hejtmancik, J F; Trese, M T

1997-01-01

X-linked Familial Exudative Vitreoretinopathy (XLFEVR) is a hereditary eye disorder that affects both the retina and the vitreous body. It is characterized by an abnormal vascularization of the peripheral retina. It has been previously shown by linkage and candidate gene analysis that XLFEVR and Norrie disease are allelic. In this report we describe four novel mutations (R41K, H42R, K58N, and Y120C) in the Norrie disease gene associated with one X-linked and four sporadic cases of FEVR. One mutation (H42R) was found to be segregating with the disease in three generations (X-linked family), and the others are sporadic. These sequence alterations changed the encoded amino acids in the Norrie disease protein and were not found in 17 unaffected family members or in 36 randomly selected normal individuals. This study provides additional evidence that mutations in the same gene can result in FEVR and Norrie disease. It also demonstrates that it may be beneficial for clinical diagnosis to screen for mutations in the Norrie disease gene in sporadic FEVR cases.

Pitfalls in lung cancer molecular pathology: how to limit them in routine practice?

PubMed

Ilie, M; Hofman, P

2012-01-01

New treatment options in advanced non-small cell lung carcinoma (NSCLC) targeting activating epidermal growth factor receptor (EGFR) gene mutations and other genetic alterations demonstrated the clinical significance of the molecular features of specific subsets of tumors. Therefore, the development of personalized medicine has stimulated the routine integration into pathology departments of somatic mutation testing. However, clinical mutation testing must be optimized and standardized with regard to histological profile, type of samples, pre-analytical steps, methodology and result reporting. Routine molecular testing in NSCLC is currently moving beyond EGFR mutational analysis. Recent progress of targeted therapies will require molecular testing for a wide panel of mutations for a personalized molecular diagnosis. As a consequence, efficient testing of multiple molecular abnormalities is an urgent requirement in thoracic oncology. Moreover, increasingly limited tumor sample becomes a major challenge for molecular pathology. Continuous efforts should be made for safe, effective and specific molecular analyses. This must be based on close collaboration between the departments involved in the management of lung cancer. In this review we explored the practical issues and pitfalls surrounding the routine implementation of molecular testing in NSCLC in a pathology laboratory.

Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons.

PubMed

Ludtmann, Marthe H R; Arber, Charles; Bartolome, Fernando; de Vicente, Macarena; Preza, Elisavet; Carro, Eva; Houlden, Henry; Gandhi, Sonia; Wray, Selina; Abramov, Andrey Y

2017-05-26

Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

PANRETINAL DEGENERATION ASSOCIATED WITH LONG-TERM HYDROXYCHLOROQUINE USE AND HETEROZYGOUS USH2A MUTATION.

PubMed

Katsman, Diana; Sanfilippo, Christian; Sarraf, David

2017-01-01

To report a case of bilateral panretinal degeneration in a patient with long-term hydroxychloroquine exposure and positive for a heterozygous mutation in the USH2A gene. Retrospective case report. Multimodal imaging including spectral-domain optical coherence tomography, fundus autofluorescence, and fluorescein angiography was performed and the results are presented. Electroretinography findings are also described. The authors report a 39-year-old patient with a history of hydroxychloroquine therapy for 20 years (cumulative dose of 2,774 g). Multimodal retinal imaging demonstrated bilateral paracentral outer retinal atrophy with spectral-domain optical coherence tomography and characteristic of hydroxychloroquine toxicity. Full-field electroretinography showed bilateral panretinal depression of the rod and cone responses. Mutational analysis revealed that the patient was a carrier for an autosomal recessive mutation in the USH2A gene. We report a case of panretinal degeneration but with features characteristic of hydroxychloroquine retinopathy in a patient who was found to be a heterozygous carrier of the USH2A gene, a cause of recessive retinitis pigmentosa without hearing loss. Carrier status for a retinal degenerative mutation may have rendered this patient more susceptible to the retinotoxic effects of long-term hydroxychloroquine therapy.

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

PubMed

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

2012-11-01

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

Clinical significance of monitoring ESR1 mutations in circulating cell-free DNA in estrogen receptor positive breast cancer patients.

PubMed

Takeshita, Takashi; Yamamoto, Yutaka; Yamamoto-Ibusuki, Mutsuko; Inao, Toko; Sueta, Aiko; Fujiwara, Saori; Omoto, Yoko; Iwase, Hirotaka

2016-05-31

The measurement of circulating cell-free DNA (cfDNA) may transform the management of breast cancer patients. We aimed to investigate the clinical significance of sequential measurements of ESR1 mutations in primary breast cancer (PBC) and metastatic breast cancer (MBC) patients. ESR1 mutations ratio in the PBC groups was used as the minimum cutoff for determining increases in cfDNA ESR1 mutation ratio. An increase in cfDNA ESR1 mutations was found in 13 samples of cfDNA from 12 (28.6%) out of 42 MBC patients. A total of 10 (83.3%) out of 12 MBC patients with increase cfDNA ESR1 mutations showed a poor response to treatment. In survival analysis, increase cfDNA ESR1 mutations may predict a shorter duration of post-endocrine-therapy effectiveness (P = 0.0033). A total of 119 patients (253 plasma samples) with breast carcinoma were enrolled in this study. Cases were selected if archival plasma samples were available from PBC before and after treatment and from MBC gathered more than twice at the time of progression. cfDNA was isolated from the 77 PBC patients (154 plasma samples) and from the 42 MBC patients (99 plasma samples). To investigate any changes in each cfDNA ESR1 mutation before and after treatment, we analyzed the difference with cfDNA ESR1 mutations ratio in the first blood sample using droplet digital polymerase chain reaction (ddPCR). We demonstrate that ddPCR monitoring of the recurrent ESR1 mutation in cfDNA of MBC patients is a feasible and useful method of providing relevant predictive information.

Clinical significance of monitoring ESR1 mutations in circulating cell-free DNA in estrogen receptor positive breast cancer patients

PubMed Central

Takeshita, Takashi; Yamamoto, Yutaka; Yamamoto-Ibusuki, Mutsuko; Inao, Toko; Sueta, Aiko; Fujiwara, Saori; Omoto, Yoko; Iwase, Hirotaka

2016-01-01

Background The measurement of circulating cell-free DNA (cfDNA) may transform the management of breast cancer patients. We aimed to investigate the clinical significance of sequential measurements of ESR1 mutations in primary breast cancer (PBC) and metastatic breast cancer (MBC) patients. Results ESR1 mutations ratio in the PBC groups was used as the minimum cutoff for determining increases in cfDNA ESR1 mutation ratio. An increase in cfDNA ESR1 mutations was found in 13 samples of cfDNA from 12 (28.6%) out of 42 MBC patients. A total of 10 (83.3%) out of 12 MBC patients with increase cfDNA ESR1 mutations showed a poor response to treatment. In survival analysis, increase cfDNA ESR1 mutations may predict a shorter duration of post-endocrine-therapy effectiveness (P = 0.0033). Methods A total of 119 patients (253 plasma samples) with breast carcinoma were enrolled in this study. Cases were selected if archival plasma samples were available from PBC before and after treatment and from MBC gathered more than twice at the time of progression. cfDNA was isolated from the 77 PBC patients (154 plasma samples) and from the 42 MBC patients (99 plasma samples). To investigate any changes in each cfDNA ESR1 mutation before and after treatment, we analyzed the difference with cfDNA ESR1 mutations ratio in the first blood sample using droplet digital polymerase chain reaction (ddPCR). Conclusions We demonstrate that ddPCR monitoring of the recurrent ESR1 mutation in cfDNA of MBC patients is a feasible and useful method of providing relevant predictive information. PMID:27102299

Spectrum of APC and MUTYH germ-line mutations in Russian patients with colorectal malignancies.

PubMed

Yanus, G A; Akhapkina, T A; Ivantsov, A O; Preobrazhenskaya, E V; Aleksakhina, S N; Bizin, I V; Sokolenko, A P; Mitiushkina, N V; Kuligina, E Sh; Suspitsin, E N; Venina, A R; Holmatov, M M; Zaitseva, O A; Yatsuk, O S; Pashkov, D V; Belyaev, A M; Togo, A V; Imyanitov, E N; Iyevleva, A G

2018-05-01

Distribution of cancer-predisposing mutations demonstrates significant interethnic variations. This study aimed to evaluate patterns of APC and MUTYH germ-line mutations in Russian patients with colorectal malignancies. APC gene defects were identified in 26/38 (68%) subjects with colon polyposis; 8/26 (31%) APC mutations were associated with 2 known mutational hotspots (p.E1309Dfs*4 [n = 5] and p.Q1062fs* [n = 3]), while 6/26 (23%) mutations were novel (p.K73Nfs*6, p.S254Hfs*12, p.S1072Kfs*9, p.E1547Kfs*11, p.L1564X and p.C1263Wfs*22). Biallelic mutations in MUTYH gene were detected in 3/12 (25%) remaining subjects with polyposis and in 6/90 (6.7%) patients with colorectal cancer (CRC) carrying KRAS p.G12C substitution, but not in 231 early-onset CRC cases negative for KRAS p.G12C allele. In addition to known European founder alleles p.Y179C and p.G396D, this study revealed a recurrent character of MUTYH p.R245H germ-line mutation. Besides that, 3 novel pathogenic MUTYH alleles (p.L111P, p.R245S and p.Q293X) were found. Targeted next-generation sequencing of 7 APC/MUTYH mutation-negative DNA samples identified novel potentially pathogenic POLD1 variant (p.L460R) in 1 patient and known low-penetrant cancer-associated allele CHEK2 p.I157T in 3 patients. The analysis of 1120 healthy subjects revealed 15 heterozygous carriers of recurrent MUTYH mutations, thus the expected incidence of MUTYH-associated polyposis in Russia is likely to be 1:23 000. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Age-related clinical and biological features of PTEN abnormalities in T-cell acute lymphoblastic leukaemia.

PubMed

Tesio, M; Trinquand, A; Ballerini, P; Hypolite, G; Lhermitte, L; Petit, A; Ifrah, N; Baruchel, A; Dombret, H; Macintyre, E; Asnafi, V

2017-12-01

The tumour suppressor gene PTEN is commonly altered in T-cell acute lymphoblastic leukaemia but its prognostic impact is still debated. We screened a cohort of 573 fully characterised adult and paediatric T-cell acute lymphoblastic leukaemia (T-ALL) patients for genomic PTEN abnormalities. PTEN-inactivating mutations and/or deletions were identified in 91 cases (16%), including 18% of paediatric (49/277) and 14% of adult cases (42/296). Thirty-four patients harboured only mutations, 12 cases demonstrated only large deletions and 9 only microdeletions. About 36 patients had combined alterations. Different mechanisms of PTEN inactivation predicted differences in the clinical outcome for both adult and paediatric patients treated according to the GRAALL03/05 and FRALLE2000 protocols. Whereas large deletions predicted lower 5-year overall survival (P=0.0053 in adults, P=0.001 in children) and disease-free survival (P=0.0009 in adults, P=0.0002 in children), mutations were not associated with a worse prognosis. The prognostic impact of PTEN loss is therefore linked to the underlying type of genomic abnormality, both in adult and paediatric T-ALLs, demonstrating that detailed analysis of the type of abnormality type would be useful to refine risk stratification.

Lysophosphatidic Acid Initiates Epithelial to Mesenchymal Transition and Induces β-Catenin-mediated Transcription in Epithelial Ovarian Carcinoma*

PubMed Central

Burkhalter, Rebecca J.; Westfall, Suzanne D.; Liu, Yueying; Stack, M. Sharon

2015-01-01

During tumor progression, epithelial ovarian cancer (EOC) cells undergo epithelial-to-mesenchymal transition (EMT), which influences metastatic success. Mutation-dependent activation of Wnt/β-catenin signaling has been implicated in gain of mesenchymal phenotype and loss of differentiation in several solid tumors; however, similar mutations are rare in most EOC histotypes. Nevertheless, evidence for activated Wnt/β-catenin signaling in EOC has been reported, and immunohistochemical analysis of human EOC tumors demonstrates nuclear staining in all histotypes. This study addresses the hypothesis that the bioactive lipid lysophosphatidic acid (LPA), prevalent in the EOC microenvironment, functions to regulate EMT in EOC. Our results demonstrate that LPA induces loss of junctional β-catenin, stimulates clustering of β1 integrins, and enhances the conformationally active population of surface β1 integrins. Furthermore, LPA treatment initiates nuclear translocation of β-catenin and transcriptional activation of Wnt/β-catenin target genes resulting in gain of mesenchymal marker expression. Together these data suggest that LPA initiates EMT in ovarian tumors through β1-integrin-dependent activation of Wnt/β-catenin signaling, providing a novel mechanism for mutation-independent activation of this pathway in EOC progression. PMID:26175151

Next-Generation Sequencing of Matched Primary and Metastatic Rectal Adenocarcinomas Demonstrates Minimal Mutation Gain and Concordance to Colonic Adenocarcinomas.

PubMed

Crumley, Suzanne M; Pepper, Kristi L; Phan, Alexandria T; Olsen, Randall J; Schwartz, Mary R; Portier, Bryce P

2016-06-01

-Colorectal carcinoma is the third most common cause of cancer death in males and females in the United States. Rectal adenocarcinoma can have distinct therapeutic and surgical management from colonic adenocarcinoma owing to its location and anatomic considerations. -To determine the oncologic driver mutations and better understand the molecular pathogenesis of rectal adenocarcinoma in relation to colon adenocarcinoma. -Next-generation sequencing was performed on 20 cases of primary rectal adenocarcinoma with a paired lymph node or solid organ metastasis by using an amplicon-based assay of more than 2800 Catalogue of Somatic Mutations in Cancer (COSMIC)-identified somatic mutations. -Next-generation sequencing data were obtained on both the primary tumor and metastasis from 16 patients. Most rectal adenocarcinoma cases demonstrated identical mutations in the primary tumor and metastasis (13 of 16, 81%). The mutations identified, listed in order of frequency, included TP53, KRAS, APC, FBXW7, GNAS, FGFR3, BRAF, NRAS, PIK3CA, and SMAD4. -The somatic mutations identified in our rectal adenocarcinoma cohort showed a strong correlation to those previously characterized in colonic adenocarcinoma. In addition, most rectal adenocarcinomas harbored identical somatic mutations in both the primary tumor and metastasis. These findings demonstrate evidence that rectal adenocarcinoma follows a similar molecular pathogenesis as colonic adenocarcinoma and that sampling either the primary or metastatic lesion is valid for initial evaluation of somatic mutations and selection of possible targeted therapy.

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.

Next generation sequencing of carcinoma of unknown primary reveals novel combinatorial strategies in a heterogeneous mutational landscape

PubMed Central

Subbiah, Ishwaria M.; Tsimberidou, Apostolia; Subbiah, Vivek; Janku, Filip; Roy-Chowdhuri, Sinchita; Hong, David S.

2017-01-01

Background Advanced carcinoma of unknown primary (CUP) has limited effective therapeutic options given the phenotypic and genotypic diversity. To identify future novel therapeutic strategies we conducted an exploratory analysis of next-generation sequencing (NGS) of relapsed, refractory CUP. Methods We identified patients in our phase I clinic where archival tissue was available for a targeted NGS CLIA-certified assay. Results Of 17 patients tested, 15 (88%) demonstrated genomic alterations (median 2 aberrations; range 0–8, total 59 alterations). Nine (53%) patients had altered cell signaling including the PI3K/AKT/MTOR (n=5, 29%) and MAPK pathways (n=3,18%); 7 (41%) patients demonstrated ≥1 alterations in tumor suppressor genes (TP53 in 5 patients), 8 (47%) had impaired epigenetic regulation and DNA methylation, 8 (47%) had aberrant cell cycle regulation, commonly in the cyclin dependent kinases. Ten (59%) patients had alterations in transcriptional regulators. Concurrent mutations affecting cell cycle regulation were noted to occur with aberrant epigenetic regulation (n=6, 35%) and MAPK/PI3K pathway (n=5, 29%). Conclusion Every patient had a unique molecular profile with no two patients demonstrating an identical panel of mutations. We identify two emerging novel combinatorial strategies targeting impaired cell cycle arrest, first with epigenetic modifiers and, second, with MAPK/PI3K pathway inhibition. PMID:28781987

Advanced cell-based modeling of the royal disease: characterization of the mutated F9 mRNA.

PubMed

Martorell, L; Luce, E; Vazquez, J L; Richaud-Patin, Y; Jimenez-Delgado, S; Corrales, I; Borras, N; Casacuberta-Serra, S; Weber, A; Parra, R; Altisent, C; Follenzi, A; Dubart-Kupperschmitt, A; Raya, A; Vidal, F; Barquinero, J

2017-11-01

Essentials The Royal disease (RD) is a form of hemophilia B predicted to be caused by a splicing mutation. We generated an iPSC-based model of the disease allowing mechanistic studies at the RNA level. F9 mRNA analysis in iPSC-derived hepatocyte-like cells showed the predicted abnormal splicing. Mutated F9 mRNA level was very low but we also found traces of wild type transcripts. Background The royal disease is a form of hemophilia B (HB) that affected many descendants of Queen Victoria in the 19th and 20th centuries. It was found to be caused by the mutation F9 c.278-3A>G. Objective To generate a physiological cell model of the disease and to study F9 expression at the RNA level. Methods Using fibroblasts from skin biopsies of a previously identified hemophilic patient bearing the F9 c.278-3A>G mutation and his mother, we generated induced pluripotent stem cells (iPSCs). Both the patient's and mother's iPSCs were differentiated into hepatocyte-like cells (HLCs) and their F9 mRNA was analyzed using next-generation sequencing (NGS). Results and Conclusion We demonstrated the previously predicted aberrant splicing of the F9 transcript as a result of an intronic nucleotide substitution leading to a frameshift and the generation of a premature termination codon (PTC). The F9 mRNA level in the patient's HLCs was significantly reduced compared with that of his mother, suggesting that mutated transcripts undergo nonsense-mediated decay (NMD), a cellular mechanism that degrades PTC-containing mRNAs. We also detected small proportions of correctly spliced transcripts in the patient's HLCs, which, combined with genetic variability in splicing and NMD machineries, could partially explain some clinical variability among affected members of the European royal families who had lifespans above the average. This work allowed the demonstration of the pathologic consequences of an intronic mutation in the F9 gene and represents the first bona fide cellular model of HB allowing the study of rare mutations at the RNA level. © 2017 International Society on Thrombosis and Haemostasis.

Tobacco Induced Mutations: A Fun, Visually Impressive Experiment

ERIC Educational Resources Information Center

Milholland, Rebecca B. R.; Hines, Stefani D.

2004-01-01

A modified version "Tobacco Induced Mutations" of Ames assay experiment provides a meaningful context for students to learn about the concept of mutations by using a known carcinogen that is tobacco. This experiment shows toxicological concept of the dose/response relationship and visually demonstrates when a mutation have occurred in bacteria…

Crizotinib-Resistant Mutants of EML4-ALK Identified Through an Accelerated Mutagenesis Screen

PubMed Central

Zhang, Sen; Wang, Frank; Keats, Jeffrey; Zhu, Xiaotian; Ning, Yaoyu; Wardwell, Scott D; Moran, Lauren; Mohemmad, Qurish K; Anjum, Rana; Wang, Yihan; Narasimhan, Narayana I; Dalgarno, David; Shakespeare, William C; Miret, Juan J; Clackson, Tim; Rivera, Victor M

2011-01-01

Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non-small-cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non-small-cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK-positive non-small-cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib's narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance. PMID:22034911

Mutation Analysis in Classical Phenylketonuria Patients Followed by Detecting Haplotypes Linked to Some PAH Mutations.

PubMed

Dehghanian, Fatemeh; Silawi, Mohammad; Tabei, Seyed M B

2017-02-01

Deficiency of phenylalanine hydroxylase (PAH) enzyme and elevation of phenylalanine in body fluids cause phenylketonuria (PKU). The gold standard for confirming PKU and PAH deficiency is detecting causal mutations by direct sequencing of the coding exons and splicing involved sequences of the PAH gene. Furthermore, haplotype analysis could be considered as an auxiliary approach for detecting PKU causative mutations before direct sequencing of the PAH gene by making comparisons between prior detected mutation linked-haplotypes and new PKU case haplotypes with undetermined mutations. In this study, 13 unrelated classical PKU patients took part in the study detecting causative mutations. Mutations were identified by polymerase chain reaction (PCR) and direct sequencing in all patients. After that, haplotype analysis was performed by studying VNTR and PAHSTR markers (linked genetic markers of the PAH gene) through application of PCR and capillary electrophoresis (CE). Mutation analysis was performed successfully and the detected mutations were as follows: c.782G>A, c.754C>T, c.842C>G, c.113-115delTCT, c.688G>A, and c.696A>G. Additionally, PAHSTR/VNTR haplotypes were detected to discover haplotypes linked to each mutation. Mutation detection is the best approach for confirming PAH enzyme deficiency in PKU patients. Due to the relatively large size of the PAH gene and high cost of the direct sequencing in developing countries, haplotype analysis could be used before DNA sequencing and mutation detection for a faster and cheaper way via identifying probable mutated exons.

Identification of a novel loss-of-function PHEX mutation, Ala720Ser, in a sporadic case of adult-onset hypophosphatemic osteomalacia.

PubMed

Goljanek-Whysall, Katarzyna; Tridimas, Andreas; McCormick, Rachel; Russell, Nicki-Jayne; Sloman, Melissa; Sorani, Alan; Fraser, William D; Hannan, Fadil M

2018-01-01

Adults presenting with sporadic hypophosphatemia and elevations in circulating fibroblast growth factor-23 (FGF23) concentrations are usually investigated for an acquired disorder of FGF23 excess such as tumor induced osteomalacia (TIO). However, in some cases the underlying tumor is not detected, and such patients may harbor other causes of FGF23 excess. Indeed, coding-region and 3'UTR mutations of phosphate-regulating neutral endopeptidase (PHEX), which encodes a cell-surface protein that regulates circulating FGF23 concentrations, can lead to alterations in phosphate homeostasis, which are not detected until adulthood. Here, we report an adult female who presented with hypophosphatemic osteomalacia and raised serum FGF23 concentrations. The patient and her parents, who were her only first-degree relatives, had no history of rickets. The patient was thus suspected of having TIO. However, no tumor had been identified following extensive localization studies. Mutational analysis of the PHEX coding-region and 3'UTR was undertaken, and this revealed the patient to be heterozygous for a novel germline PHEX mutation (c.2158G>T; p.Ala720Ser). In vitro studies involving the expression of WT and mutant PHEX proteins in HEK293 cells demonstrated the Ala720Ser mutation to impair trafficking of PHEX, with ~20% of the mutant protein being expressed at the cell surface, compared to ~80% cell surface expression for WT PHEX (p<0.05). Thus, our studies have identified a pathogenic PHEX mutation in a sporadic case of adult-onset hypophosphatemic osteomalacia, and these findings highlight a role for PHEX gene analysis in some cases of suspected TIO, particularly when no tumor has been identified. Copyright © 2017 Elsevier Inc. All rights reserved.

Mutagenicity and Potential Carcinogenicity of Thiopurine Treatment in Patients with Inflammatory Bowel Disease

PubMed Central

Nguyen, Truc; Vacek, Pamela M.; O’Neill, Patrick; Colletti, Richard B.; Finette, Barry A.

2009-01-01

The thiopurines, azathioprine and 6-mercaptopurine, are effective immune-modulators and cytotoxic agents extensively used in the treatment of autoimmune diseases, graft rejection, and cancer. There is compelling epidemiologic evidence that thiopurine treatment increases the risk for a variety of tumors by mechanisms that are unclear. We investigated the in vivo mutagenicity of long-term thiopurine treatment by determining the frequency and spectra of somatic mutation events at the HPRT locus in peripheral T lymphocytes as well as the prevalence of mutant clonal proliferation in a cross-sectional analysis of data from 119 children and adults with inflammatory bowel disease (IBD). Analyses of variance and regression were performed to assess relationships among the frequency and spectra of HPRT mutations with disease, duration of illness, duration of treatment and total therapeutic dose of azathioprine and 6-mercaptopurine. We observed a significant increase in the frequency of somatic mutations in 56 subjects treated with thiopurines for IBD compared to 63 subjects not treated with thiopurines. This increase was related to both total dose (p<0.001) and duration of treatment (p<0.001). Comparative mutation spectra analysis of 1,020 mutant isolates revealed a significant increase in the proportion of all transitions (p <0.001), in particular G:C to A:T transitions (p<0.001). Combined analyses of two signatures for mutant clonality, HPRT mutation and TCRβ CDR3 region unique gene sequence also demonstrated a significant thiopurine-dependent increase in mutant cell clonal proliferation (p<0.001). These findings provide in vivo evidence for mutation induction as a potential carcinogenic mechanism associated with chronic thiopurine intervention. PMID:19706768

Elevated heart rate triggers action potential alternans and sudden death. translational study of a homozygous KCNH2 mutation.

PubMed

Schweigmann, Ulrich; Biliczki, Peter; Ramirez, Rafael J; Marschall, Christoph; Takac, Ina; Brandes, Ralf P; Kotzot, Dieter; Girmatsion, Zenawit; Hohnloser, Stefan H; Ehrlich, Joachim R

2014-01-01

Long QT syndrome (LQTS) leads to arrhythmic events and increased risk for sudden cardiac death (SCD). Homozygous KCNH2 mutations underlying LQTS-2 have previously been termed "human HERG knockout" and typically express severe phenotypes. We studied genotype-phenotype correlations of an LQTS type 2 mutation identified in the homozygous index patient from a consanguineous Turkish family after his brother died suddenly during febrile illness. Clinical work-up, DNA sequencing, mutagenesis, cell culture, patch-clamp, in silico mathematical modelling, protein biochemistry, confocal microscopy were performed. Genetic analysis revealed a homozygous C-terminal KCNH2 mutation (p.R835Q) in the index patient (QTc ∼506 ms with notched T waves). Parents were I° cousins - both heterozygous for the mutation and clinically unremarkable (QTc ∼447 ms, father and ∼396 ms, mother). Heterologous expression of KCNH2-R835Q showed mildly reduced current amplitudes. Biophysical properties of ionic currents were also only nominally changed with slight acceleration of deactivation and more negative V50 in R835Q-currents. Protein biochemistry and confocal microscopy revealed similar expression patterns and trafficking of WT and R835Q, even at elevated temperature. In silico analysis demonstrated mildly prolonged ventricular action potential duration (APD) compared to WT at a cycle length of 1000 ms. At a cycle length of 350 ms M-cell APD remained stable in WT, but displayed APD alternans in R835Q. Kv11.1 channels affected by the C-terminal R835Q mutation display mildly modified biophysical properties, but leads to M-cell APD alternans with elevated heart rate and could precipitate SCD under specific clinical circumstances associated with high heart rates.

Dissecting Vancomycin-Intermediate Resistance in Staphylococcus aureus Using Genome-Wide Association

PubMed Central

Alam, Md Tauqeer; Petit, Robert A.; Crispell, Emily K.; Thornton, Timothy A.; Conneely, Karen N.; Jiang, Yunxuan; Satola, Sarah W.; Read, Timothy D.

2014-01-01

Vancomycin-intermediate Staphylococcus aureus (VISA) is currently defined as having minimal inhibitory concentration (MIC) of 4–8 µg/ml. VISA evolves through changes in multiple genetic loci with at least 16 candidate genes identified in clinical and in vitro-selected VISA strains. We report a whole-genome comparative analysis of 49 vancomycin-sensitive S. aureus and 26 VISA strains. Resistance to vancomycin was determined by broth microdilution, Etest, and population analysis profile-area under the curve (PAP-AUC). Genome-wide association studies (GWAS) of 55,977 single-nucleotide polymorphisms identified in one or more strains found one highly significant association (P = 8.78E-08) between a nonsynonymous mutation at codon 481 (H481) of the rpoB gene and increased vancomycin MIC. Additionally, we used a database of public S. aureus genome sequences to identify rare mutations in candidate genes associated with VISA. On the basis of these data, we proposed a preliminary model called ECM+RMCG for the VISA phenotype as a benchmark for future efforts. The model predicted VISA based on the presence of a rare mutation in a set of candidate genes (walKR, vraSR, graSR, and agrA) and/or three previously experimentally verified mutations (including the rpoB H481 locus) with an accuracy of 81% and a sensitivity of 73%. Further, the level of resistance measured by both Etest and PAP-AUC regressed positively with the number of mutations present in a strain. This study demonstrated 1) the power of GWAS for identifying common genetic variants associated with antibiotic resistance in bacteria and 2) that rare mutations in candidate gene, identified using large genomic data sets, can also be associated with resistance phenotypes. PMID:24787619

Detection of novel mutations that cause autosomal dominant retinitis pigmentosa in candidate genes by long-range PCR amplification and next-generation sequencing

PubMed Central

Dias, Miguel de Sousa; Hernan, Imma; Pascual, Beatriz; Borràs, Emma; Mañé, Begoña; Gamundi, Maria José

2013-01-01

Purpose To devise an effective method for detecting mutations in 12 genes (CA4, CRX, IMPDH1, NR2E3, RP9, PRPF3, PRPF8, PRPF31, PRPH2, RHO, RP1, and TOPORS) commonly associated with autosomal dominant retinitis pigmentosa (adRP) that account for more than 95% of known mutations. Methods We used long-range PCR (LR-PCR) amplification and next-generation sequencing (NGS) performed in a GS Junior 454 benchtop sequencing platform. Twenty LR-PCR fragments, between 3,000 and 10,000 bp, containing all coding exons and flanking regions of the 12 genes, were obtained from DNA samples of patients with adRP. Sequencing libraries were prepared with an enzymatic (Fragmentase technology) method. Results Complete coverage of the coding and flanking sequences of the 12 genes assayed was obtained with NGS, with an average sequence depth of 380× (ranging from 128× to 1,077×). Five previous known mutations in the adRP genes were detected with a sequence variation percentage between 35% and 65%. We also performed a parallel sequence analysis of four samples, three of them new patients with index adRP, in which two novel mutations were detected in RHO (p.Asn73del) and PRPF31 (p.Ile109del). Conclusions The results demonstrate that genomic LR-PCR amplification together with NGS is an effective method for analyzing individual patient samples for mutations in a monogenic heterogeneous disease such as adRP. This approach proved effective for the parallel analysis of adRP and has been introduced as routine. Additionally, this approach could be extended to other heterogeneous genetic diseases. PMID:23559859

Calreticulin mutation analysis in non-mutated Janus kinase 2 essential thrombocythemia patients in Chiang Mai University: analysis of three methods and clinical correlations.

PubMed

Rattarittamrong, Ekarat; Tantiworawit, Adisak; Kumpunya, Noppamas; Wongtagan, Ornkamon; Tongphung, Ratchanoo; Phusua, Arunee; Chai-Adisaksopha, Chatree; Hantrakool, Sasinee; Rattanathammethee, Thanawat; Norasetthada, Lalita; Charoenkwan, Pimlak; Lekawanvijit, Suree

2018-03-09

The primary objective was to determine the prevalence of calreticulin (CALR) mutation in patients with non-JAK2V617F mutated essential thrombocythemia (ET). The secondary objectives were to evaluate the accuracy of CALR mutation analysis by high-resolution melting (HRM) analysis and real-time polymerase chain reaction (PCR) compared with DNA sequencing and to compare clinical characteristics of CALR mutated and JAK2V617F mutated ET. This was a prospective cohort study involving ET patients registered at Chiang Mai University in the period September 2015-September 2017 who were aged more than 2 years, and did not harbor JAK2V617F mutation. The presence of CALR mutation was established by DNA sequencing, HRM, and real-time PCR for type 1 and type 2 mutation. Clinical data were compared with that from ET patients with mutated JAK2V617F. Twenty-eight patients were enrolled onto the study. CALR mutations were found in 10 patients (35.7%). Three patients had type 1 mutation, 5 patients had type 2 mutation, 1 patient had type 18 mutation, and 1 patients had novel mutations (c.1093 C-G, c.1098_1131 del, c.1135 G-A). HRM could differentiate between the types of mutation in complete agreement with DNA sequencing. Patients with a CALR mutation showed a significantly greater male predominance and had a higher platelet count when compared with 42 JAK2V617F patients. The prevalence of CALR mutation in JAK2V617F-negative ET in this study is 35.7%. HRM is an effective method of detecting CALR mutation and is a more advantageous method of screening for CALR mutation.

Multiplex sequence analysis demonstrates the competitive growth advantage of the A-to-G mutants of clarithromycin-resistant Helicobacter pylori.

PubMed

Wang, G; Rahman, M S; Humayun, M Z; Taylor, D E

1999-03-01

Clarithromycin resistance in Helicobacter pylori is due to point mutation within the 23S rRNA. We examined the growth rates of different types of site-directed mutants and demonstrated quantitatively the competitive growth advantage of A-to-G mutants over other types of mutants by a multiplex sequencing assay. The results provide a rational explanation of why A-to-G mutants are predominantly observed among clarithromycin-resistant clinical isolates.

Multiplex Sequence Analysis Demonstrates the Competitive Growth Advantage of the A-to-G Mutants of Clarithromycin-Resistant Helicobacter pylori

PubMed Central

Wang, Ge; Rahman, M. Sayeedur; Humayun, M. Zafri; Taylor, Diane E.

1999-01-01

Clarithromycin resistance in Helicobacter pylori is due to point mutation within the 23S rRNA. We examined the growth rates of different types of site-directed mutants and demonstrated quantitatively the competitive growth advantage of A-to-G mutants over other types of mutants by a multiplex sequencing assay. The results provide a rational explanation of why A-to-G mutants are predominantly observed among clarithromycin-resistant clinical isolates. PMID:10049289

Mutation of zebrafish dihydrolipoamide branched-chain transacylase E2 results in motor dysfunction and models maple syrup urine disease

PubMed Central

Friedrich, Timo; Lambert, Aaron M.; Masino, Mark A.; Downes, Gerald B.

2012-01-01

SUMMARY Analysis of zebrafish mutants that demonstrate abnormal locomotive behavior can elucidate the molecular requirements for neural network function and provide new models of human disease. Here, we show that zebrafish quetschkommode (que) mutant larvae exhibit a progressive locomotor defect that culminates in unusual nose-to-tail compressions and an inability to swim. Correspondingly, extracellular peripheral nerve recordings show that que mutants demonstrate abnormal locomotor output to the axial muscles used for swimming. Using positional cloning and candidate gene analysis, we reveal that a point mutation disrupts the gene encoding dihydrolipoamide branched-chain transacylase E2 (Dbt), a component of a mitochondrial enzyme complex, to generate the que phenotype. In humans, mutation of the DBT gene causes maple syrup urine disease (MSUD), a disorder of branched-chain amino acid metabolism that can result in mental retardation, severe dystonia, profound neurological damage and death. que mutants harbor abnormal amino acid levels, similar to MSUD patients and consistent with an error in branched-chain amino acid metabolism. que mutants also contain markedly reduced levels of the neurotransmitter glutamate within the brain and spinal cord, which probably contributes to their abnormal spinal cord locomotor output and aberrant motility behavior, a trait that probably represents severe dystonia in larval zebrafish. Taken together, these data illustrate how defects in branched-chain amino acid metabolism can disrupt nervous system development and/or function, and establish zebrafish que mutants as a model to better understand MSUD. PMID:22046030

Reduced BRCA1 transcript levels in freshly isolated blood leukocytes from BRCA1 mutation carriers is mutation specific.

PubMed

Chehade, Rania; Pettapiece-Phillips, Rachael; Salmena, Leonardo; Kotlyar, Max; Jurisica, Igor; Narod, Steven A; Akbari, Mohammad R; Kotsopoulos, Joanne

2016-08-17

BRCA1 mutation carriers face a high lifetime risk of developing both breast and ovarian cancer. Haploinsufficiency is thought to predispose these women to cancer by reducing the pool of available BRCA1 transcript and protein, thereby compromising BRCA1 function. Whether or not cancer-free BRCA1 mutation carriers have lower messenger (m)RNA transcript levels in peripheral blood leukocytes has not been evaluated. The primary aim of this study was to characterize an association between BRCA1 mutation status and BRCA1 mRNA leukocyte expression levels among healthy women with a BRCA1 mutation. RNA was extracted from freshly isolated peripheral blood leukocytes of 58 cancer-free, female participants (22 BRCA1 mutation carriers and 36 non-carriers). The expression levels of 236 cancer-associated genes, including BRCA1, were quantified using the Human Cancer Reference gene panel from the Nanostring Technologies nCounter Analysis System. Multivariate modeling demonstrated that carrying a BRCA1 mutation was the most significant predictor of BRCA1 mRNA levels. BRCA1 mRNA levels were significantly lower in BRCA1 mutation carriers compared to non-carriers (146.7 counts vs. 175.1 counts; P = 0.002). Samples with BRCA1 mutations within exon 11 had lower BRCA1 mRNA levels than samples with mutations within the 5' and 3' regions of the BRCA1 gene (122.1 counts vs. 138.9 and 168.6 counts, respectively; P = 0.003). Unsupervised hierarchical clustering of gene expression profiles from freshly isolated blood leukocytes revealed that BRCA1 mutation carriers cluster more closely with other BRCA1 mutation carriers than with BRCA1 wild-type samples. Moreover, a set of 17 genes (including BRCA1) previously shown to be involved in carcinogenesis, were differentially expressed between BRCA1 mutation carriers and non-carriers. Overall, these findings support the concept of BRCA1 haploinsufficiency wherein a specific mutation results in dosage-dependent alteration of BRCA1 at the transcriptional level. This study is the first to show a decrease in BRCA1 mRNA expression in freshly isolated blood leukocytes from healthy, unaffected BRCA1 mutation carriers.

CFTR Mutations Spectrum and the Efficiency of Molecular Diagnostics in Polish Cystic Fibrosis Patients

PubMed Central

Ziętkiewicz, Ewa; Rutkiewicz, Ewa; Pogorzelski, Andrzej; Klimek, Barbara; Voelkel, Katarzyna; Witt, Michał

2014-01-01

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR). In light of the strong allelic heterogeneity and regional specificity of the mutation spectrum, the strategy of molecular diagnostics and counseling in CF requires genetic tests to reflect the frequency profile characteristic for a given population. The goal of the study was to provide an updated comprehensive estimation of the distribution of CFTR mutations in Polish CF patients and to assess the effectiveness of INNOLiPA_CFTR tests in Polish population. The analyzed cohort consisted of 738 patients with the clinically confirmed CF diagnosis, prescreened for molecular defects using INNOLiPA_CFTR panels from Innogenetics. A combined efficiency of INNOLiPA CFTR_19 and CFTR_17_TnUpdate tests was 75.5%; both mutations were detected in 68.2%, and one mutation in 14.8% of the affected individuals. The group composed of all the patients with only one or with no mutation detected (109 and 126 individuals, respectively) was analyzed further using a mutation screening approach, i.e. SSCP/HD (single strand conformational polymorphism/heteroduplex) analysis of PCR products followed by sequencing of the coding sequence. As a result, 53 more mutations were found in 97 patients. The overall efficiency of the CF allele detection was 82.5% (7.0% increase compared to INNOLiPA tests alone). The distribution of the most frequent mutations in Poland was assessed. Most of the mutations repetitively found in Polish patients had been previously described in other European populations. The most frequent mutated allele, F508del, represented 54.5% of Polish CF chromosomes. Another eight mutations had frequencies over 1%, 24 had frequencies between 1 and 0.1%; c.2052-2053insA and c.3468+2_3468+3insT were the most frequent non-INNOLiPA mutations. Mutation distribution described herein is also relevant to the Polish diaspora. Our study also demonstrates that the reported efficiency of mutation detection strongly depends on the diagnostic experience of referring health centers. PMID:24586523

CFTR mutations spectrum and the efficiency of molecular diagnostics in Polish cystic fibrosis patients.

PubMed

Ziętkiewicz, Ewa; Rutkiewicz, Ewa; Pogorzelski, Andrzej; Klimek, Barbara; Voelkel, Katarzyna; Witt, Michał

2014-01-01

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane regulator gene (CFTR). In light of the strong allelic heterogeneity and regional specificity of the mutation spectrum, the strategy of molecular diagnostics and counseling in CF requires genetic tests to reflect the frequency profile characteristic for a given population. The goal of the study was to provide an updated comprehensive estimation of the distribution of CFTR mutations in Polish CF patients and to assess the effectiveness of INNOLiPA_CFTR tests in Polish population. The analyzed cohort consisted of 738 patients with the clinically confirmed CF diagnosis, prescreened for molecular defects using INNOLiPA_CFTR panels from Innogenetics. A combined efficiency of INNOLiPA CFTR_19 and CFTR_17_TnUpdate tests was 75.5%; both mutations were detected in 68.2%, and one mutation in 14.8% of the affected individuals. The group composed of all the patients with only one or with no mutation detected (109 and 126 individuals, respectively) was analyzed further using a mutation screening approach, i.e. SSCP/HD (single strand conformational polymorphism/heteroduplex) analysis of PCR products followed by sequencing of the coding sequence. As a result, 53 more mutations were found in 97 patients. The overall efficiency of the CF allele detection was 82.5% (7.0% increase compared to INNOLiPA tests alone). The distribution of the most frequent mutations in Poland was assessed. Most of the mutations repetitively found in Polish patients had been previously described in other European populations. The most frequent mutated allele, F508del, represented 54.5% of Polish CF chromosomes. Another eight mutations had frequencies over 1%, 24 had frequencies between 1 and 0.1%; c.2052-2053insA and c.3468+2_3468+3insT were the most frequent non-INNOLiPA mutations. Mutation distribution described herein is also relevant to the Polish diaspora. Our study also demonstrates that the reported efficiency of mutation detection strongly depends on the diagnostic experience of referring health centers.

Contribution of the TTC21B gene to glomerular and cystic kidney diseases.

PubMed

Bullich, Gemma; Vargas, Iván; Trujillano, Daniel; Mendizábal, Santiago; Piñero-Fernández, Juan Alberto; Fraga, Gloria; García-Solano, José; Ballarín, José; Estivill, Xavier; Torra, Roser; Ars, Elisabet

2017-01-01

The TTC21B gene was initially described as causative of nephronophthisis (NPHP). Recently, the homozygous TTC21B p.P209L mutation has been identified in families with focal segmental glomerulosclerosis (FSGS) and tubulointerstitial lesions. Heterozygous TTC21B variants have been proposed as genetic modifiers in ciliopathies. We aimed to study the causative and modifying role of the TTC21B gene in glomerular and cystic kidney diseases. Mutation analysis of the TTC21B gene was performed by massive parallel sequencing. We studied the causative role of the TTC21B gene in 17 patients with primary diagnosis of FSGS or NPHP and its modifying role in 184 patients with inherited glomerular or cystic kidney diseases. Disease-causing TTC21B mutations were identified in three families presenting nephrotic proteinuria with FSGS and tubulointerstitial lesions in which some family members presented hypertension and myopia. Two families carried the homozygous p.P209L and the third was compound heterozygous for the p.P209L and a novel p.H426D mutation. Rare heterozygous TTC21B variants predicted to be pathogenic were found in five patients. These TTC21B variants were significantly more frequent in renal patients compared with controls (P = 0.0349). Two patients with a heterozygous deleterious TTC21B variant in addition to the disease-causing mutation presented a more severe phenotype than expected. Our results confirm the causal role of the homozygous p.P209L TTC21B mutation in two new families with FSGS and tubulointerstitial disease. We identified a novel TTC21B mutation demonstrating that p.P209L is not the unique causative mutation of this nephropathy. Thus, TTC21B mutation analysis should be considered for the genetic diagnosis of families with FSGS and tubulointerstitial lesions. Finally, we provide evidence that heterozygous deleterious TTC21B variants may act as genetic modifiers of the severity of glomerular and cystic kidney diseases. © The Author 2016. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.

On Statistical Modeling of Sequencing Noise in High Depth Data to Assess Tumor Evolution

NASA Astrophysics Data System (ADS)

Rabadan, Raul; Bhanot, Gyan; Marsilio, Sonia; Chiorazzi, Nicholas; Pasqualucci, Laura; Khiabanian, Hossein

2018-07-01

One cause of cancer mortality is tumor evolution to therapy-resistant disease. First line therapy often targets the dominant clone, and drug resistance can emerge from preexisting clones that gain fitness through therapy-induced natural selection. Such mutations may be identified using targeted sequencing assays by analysis of noise in high-depth data. Here, we develop a comprehensive, unbiased model for sequencing error background. We find that noise in sufficiently deep DNA sequencing data can be approximated by aggregating negative binomial distributions. Mutations with frequencies above noise may have prognostic value. We evaluate our model with simulated exponentially expanded populations as well as data from cell line and patient sample dilution experiments, demonstrating its utility in prognosticating tumor progression. Our results may have the potential to identify significant mutations that can cause recurrence. These results are relevant in the pretreatment clinical setting to determine appropriate therapy and prepare for potential recurrence pretreatment.

Mutations in KPTN Cause Macrocephaly, Neurodevelopmental Delay, and Seizures

PubMed Central

Baple, Emma L.; Maroofian, Reza; Chioza, Barry A.; Izadi, Maryam; Cross, Harold E.; Al-Turki, Saeed; Barwick, Katy; Skrzypiec, Anna; Pawlak, Robert; Wagner, Karin; Coblentz, Roselyn; Zainy, Tala; Patton, Michael A.; Mansour, Sahar; Rich, Phillip; Qualmann, Britta; Hurles, Matt E.; Kessels, Michael M.; Crosby, Andrew H.

2014-01-01

The proper development of neuronal circuits during neuromorphogenesis and neuronal-network formation is critically dependent on a coordinated and intricate series of molecular and cellular cues and responses. Although the cortical actin cytoskeleton is known to play a key role in neuromorphogenesis, relatively little is known about the specific molecules important for this process. Using linkage analysis and whole-exome sequencing on samples from families from the Amish community of Ohio, we have demonstrated that mutations in KPTN, encoding kaptin, cause a syndrome typified by macrocephaly, neurodevelopmental delay, and seizures. Our immunofluorescence analyses in primary neuronal cell cultures showed that endogenous and GFP-tagged kaptin associates with dynamic actin cytoskeletal structures and that this association is lost upon introduction of the identified mutations. Taken together, our studies have identified kaptin alterations responsible for macrocephaly and neurodevelopmental delay and define kaptin as a molecule crucial for normal human neuromorphogenesis. PMID:24239382

Tuning and Switching Enantioselectivity of Asymmetric Carboligation in an Enzyme through Mutational Analysis of a Single Hot Spot.

PubMed

Wechsler, Cindy; Meyer, Danilo; Loschonsky, Sabrina; Funk, Lisa-Marie; Neumann, Piotr; Ficner, Ralf; Brodhun, Florian; Müller, Michael; Tittmann, Kai

2015-12-01

Enantioselective bond making and breaking is a hallmark of enzyme action, yet switching the enantioselectivity of the reaction is a difficult undertaking, and typically requires extensive screening of mutant libraries and multiple mutations. Here, we demonstrate that mutational diversification of a single catalytic hot spot in the enzyme pyruvate decarboxylase gives access to both enantiomers of acyloins acetoin and phenylacetylcarbinol, important pharmaceutical precursors, in the case of acetoin even starting from the unselective wild-type protein. Protein crystallography was used to rationalize these findings and to propose a mechanistic model of how enantioselectivity is controlled. In a broader context, our studies highlight the efficiency of mechanism-inspired and structure-guided rational protein design for enhancing and switching enantioselectivity of enzymatic reactions, by systematically exploring the biocatalytic potential of a single hot spot. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Genetic loss of SH2B3 in acute lymphoblastic leukemia.

PubMed

Perez-Garcia, Arianne; Ambesi-Impiombato, Alberto; Hadler, Michael; Rigo, Isaura; LeDuc, Charles A; Kelly, Kara; Jalas, Chaim; Paietta, Elisabeth; Racevskis, Janis; Rowe, Jacob M; Tallman, Martin S; Paganin, Maddalena; Basso, Giuseppe; Tong, Wei; Chung, Wendy K; Ferrando, Adolfo A

2013-10-03

The SH2B adaptor protein 3 (SH2B3) gene encodes a negative regulator of cytokine signaling with a critical role in the homeostasis of hematopoietic stem cells and lymphoid progenitors. Here, we report the identification of germline homozygous SH2B3 mutations in 2 siblings affected with developmental delay and autoimmunity, one in whom B-precursor acute lymphoblastic leukemia (ALL) developed. Mechanistically, loss of SH2B3 increases Janus kinase-signal transducer and activator of transcription signaling, promotes lymphoid cell proliferation, and accelerates leukemia development in a mouse model of NOTCH1-induced ALL. Moreover, extended mutation analysis showed homozygous somatic mutations in SH2B3 in 2 of 167 ALLs analyzed. Overall, these results demonstrate a Knudson tumor suppressor role for SH2B3 in the pathogenesis of ALL and highlight a possible link between genetic predisposition factors in the pathogenesis of autoimmunity and leukemogenesis.

The nonreceptor protein tyrosine phosphatase corkscrew functions in multiple receptor tyrosine kinase pathways in Drosophila.

PubMed

Perkins, L A; Johnson, M R; Melnick, M B; Perrimon, N

1996-11-25

Corkscrew (csw) encodes a nonreceptor protein tyrosine phosphatase (PTPase) that has been implicated in signaling from the Torso receptor tyrosine kinase (RTK). csw mutations, unlike tor mutations, are associated with zygotic lethality, indicating that Csw plays additional roles during development. We have conducted a detailed phenotypic analysis of csw mutations to identify these additional functions of Csw. Our results indicate that Csw operates positively downstream of other Drosophila RTKs such as the Drosophila epidermal growth factor receptor (DER), the fibroblast growth factor receptor (Breathless), and likely other RTKs. This model is substantiated by specific dosage interactions between csw and DER. It is proposed that Csw is part of the evolutionarily conserved "signaling cassette" that operates downstream of all RTKs. In support of this hypothesis, we demonstrate that SHP-2, a vertebrate PTPase similar to Csw and previously implicated in RTK signaling, encodes the functional vertebrate homologue of Csw.

Novel ELANE Gene Mutation in a Korean Girl with Severe Congenital Neutropenia

PubMed Central

Shim, Ye Jee; Kim, Hee-Jin; Suh, Jang Soo

2011-01-01

Severe congenital neutropenia is a heterozygous group of bone marrow failure syndromes that cause lifelong infections. Mutation of the ELANE gene encoding human neutrophil elastase is the most common genetic alteration. A Korean female pediatric patient was admitted because of recurrent cervical lymphadenitis without abscess formation. She had a past history of omphalitis and isolated neutropenia at birth. The peripheral blood showed a markedly decreased absolute neutrophil count, and the bone marrow findings revealed maturation arrest of myeloid precursors at the promyelocyte to myelocyte stage. Her direct DNA sequencing analysis demonstrated an ELANE gene mutation (c.607G > C; p.Gly203Arg), but her parents were negative for it. She showed only transient response after subcutaneous 15 µg/kg/day of granulocyte colony stimulating factor administration for six consecutive days. During the follow-up observation period, she suffered from subsequent seven febrile illnesses including urinary tract infection, septicemia, and cellulitis. PMID:22148006

Exome Sequence Reveals Mutations in CoA Synthase as a Cause of Neurodegeneration with Brain Iron Accumulation

PubMed Central

Dusi, Sabrina; Valletta, Lorella; Haack, Tobias B.; Tsuchiya, Yugo; Venco, Paola; Pasqualato, Sebastiano; Goffrini, Paola; Tigano, Marco; Demchenko, Nikita; Wieland, Thomas; Schwarzmayr, Thomas; Strom, Tim M.; Invernizzi, Federica; Garavaglia, Barbara; Gregory, Allison; Sanford, Lynn; Hamada, Jeffrey; Bettencourt, Conceição; Houlden, Henry; Chiapparini, Luisa; Zorzi, Giovanna; Kurian, Manju A.; Nardocci, Nardo; Prokisch, Holger; Hayflick, Susan; Gout, Ivan; Tiranti, Valeria

2014-01-01

Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of disorders with progressive extrapyramidal signs and neurological deterioration, characterized by iron accumulation in the basal ganglia. Exome sequencing revealed the presence of recessive missense mutations in COASY, encoding coenzyme A (CoA) synthase in one NBIA-affected subject. A second unrelated individual carrying mutations in COASY was identified by Sanger sequence analysis. CoA synthase is a bifunctional enzyme catalyzing the final steps of CoA biosynthesis by coupling phosphopantetheine with ATP to form dephospho-CoA and its subsequent phosphorylation to generate CoA. We demonstrate alterations in RNA and protein expression levels of CoA synthase, as well as CoA amount, in fibroblasts derived from the two clinical cases and in yeast. This is the second inborn error of coenzyme A biosynthesis to be implicated in NBIA. PMID:24360804