Multiscale mutation clustering algorithm identifies pan-cancer mutational clusters associated with pathway-level changes in gene expression.

PubMed

Poole, William; Leinonen, Kalle; Shmulevich, Ilya; Knijnenburg, Theo A; Bernard, Brady

2017-02-01

Cancer researchers have long recognized that somatic mutations are not uniformly distributed within genes. However, most approaches for identifying cancer mutations focus on either the entire-gene or single amino-acid level. We have bridged these two methodologies with a multiscale mutation clustering algorithm that identifies variable length mutation clusters in cancer genes. We ran our algorithm on 539 genes using the combined mutation data in 23 cancer types from The Cancer Genome Atlas (TCGA) and identified 1295 mutation clusters. The resulting mutation clusters cover a wide range of scales and often overlap with many kinds of protein features including structured domains, phosphorylation sites, and known single nucleotide variants. We statistically associated these multiscale clusters with gene expression and drug response data to illuminate the functional and clinical consequences of mutations in our clusters. Interestingly, we find multiple clusters within individual genes that have differential functional associations: these include PTEN, FUBP1, and CDH1. This methodology has potential implications in identifying protein regions for drug targets, understanding the biological underpinnings of cancer, and personalizing cancer treatments. Toward this end, we have made the mutation clusters and the clustering algorithm available to the public. Clusters and pathway associations can be interactively browsed at m2c.systemsbiology.net. The multiscale mutation clustering algorithm is available at https://github.com/IlyaLab/M2C.

Identify mutation in amyotrophic lateral sclerosis cases using HaloPlex target enrichment system.

PubMed

Liu, Zhi-Jun; Li, Hong-Fu; Tan, Guo-He; Tao, Qing-Qing; Ni, Wang; Cheng, Xue-Wen; Xiong, Zhi-Qi; Wu, Zhi-Ying

2014-12-01

To date, at least 18 causative genes have been identified in amyotrophic lateral sclerosis (ALS). Because of the clinical and genetic heterogeneity, molecular diagnosis for ALS faces great challenges. HaloPlex target enrichment system is a new targeted sequencing approach, which can detect already known mutations or candidate genes. We performed this approach to screen 18 causative genes of ALS, including SOD1, SETX, FUS, ANG, TARDBP, ALS2, FIG4, VAPB, OPTN, DAO, VCP, UBQLN2, SPG11, SIGMAR1, DCTN1, SQSTM1, PFN1, and CHMP2B in 8 ALS probands. Using this approach, we got an average of 9.5 synonymous or missense mutations per sample. After validation by Sanger sequencing, we identified 3 documented SOD1 mutations (p.F21C, p.G148D, and p.C147R) and 1 novel DCTN1 p.G59R mutation in 4 probands. The novel DCTN1 mutation appeared to segregate with the disease in the pedigree and was absent in 200 control subjects. The high throughput and efficiency of this approach indicated that it could be applied to diagnose ALS and other inherited diseases with multiple causative genes in clinical practice. Copyright © 2014 Elsevier Inc. All rights reserved.

Congenital Insensitivity to Pain: Novel SCN9A Missense and In-Frame Deletion Mutations

PubMed Central

Cox, James J; Sheynin, Jony; Shorer, Zamir; Reimann, Frank; Nicholas, Adeline K; Zubovic, Lorena; Baralle, Marco; Wraige, Elizabeth; Manor, Esther; Levy, Jacov; Woods, C Geoffery; Parvari, Ruti

2010-01-01

SCN9A encodes the voltage-gated sodium channel Nav1.7, a protein highly expressed in pain-sensing neurons. Mutations in SCN9A cause three human pain disorders: bi-allelic loss of function mutations result in Channelopathy-associated Insensitivity to Pain (CIP), whereas activating mutations cause severe episodic pain in Paroxysmal Extreme Pain Disorder (PEPD) and Primary Erythermalgia (PE). To date, all mutations in SCN9A that cause a complete inability to experience pain are protein truncating and presumably lead to no protein being produced. Here, we describe the identification and functional characterization of two novel non-truncating mutations in families with CIP: a homozygously-inherited missense mutation found in a consanguineous Israeli Bedouin family (Nav1.7-R896Q) and a five amino acid in-frame deletion found in a sporadic compound heterozygote (Nav1.7-ΔR1370-L1374). Both of these mutations map to the pore region of the Nav1.7 sodium channel. Using transient transfection of PC12 cells we found a significant reduction in membrane localization of the mutant protein compared to the wild type. Furthermore, voltage clamp experiments of mutant-transfected HEK293 cells show a complete loss of function of the sodium channel, consistent with the absence of pain phenotype. In summary, this study has identified critical amino acids needed for the normal subcellular localization and function of Nav1.7. © 2010 Wiley-Liss, Inc. PMID:20635406

Congenital insensitivity to pain: novel SCN9A missense and in-frame deletion mutations.

PubMed

Cox, James J; Sheynin, Jony; Shorer, Zamir; Reimann, Frank; Nicholas, Adeline K; Zubovic, Lorena; Baralle, Marco; Wraige, Elizabeth; Manor, Esther; Levy, Jacov; Woods, C Geoffery; Parvari, Ruti

2010-09-01

SCN9Aencodes the voltage-gated sodium channel Na(v)1.7, a protein highly expressed in pain-sensing neurons. Mutations in SCN9A cause three human pain disorders: bi-allelic loss of function mutations result in Channelopathy-associated Insensitivity to Pain (CIP), whereas activating mutations cause severe episodic pain in Paroxysmal Extreme Pain Disorder (PEPD) and Primary Erythermalgia (PE). To date, all mutations in SCN9A that cause a complete inability to experience pain are protein truncating and presumably lead to no protein being produced. Here, we describe the identification and functional characterization of two novel non-truncating mutations in families with CIP: a homozygously-inherited missense mutation found in a consanguineous Israeli Bedouin family (Na(v)1.7-R896Q) and a five amino acid in-frame deletion found in a sporadic compound heterozygote (Na(v)1.7-DeltaR1370-L1374). Both of these mutations map to the pore region of the Na(v)1.7 sodium channel. Using transient transfection of PC12 cells we found a significant reduction in membrane localization of the mutant protein compared to the wild type. Furthermore, voltage clamp experiments of mutant-transfected HEK293 cells show a complete loss of function of the sodium channel, consistent with the absence of pain phenotype. In summary, this study has identified critical amino acids needed for the normal subcellular localization and function of Na(v)1.7. Copyright 2010 Wiley-Liss, Inc.

Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms

PubMed Central

Milosevic Feenstra, Jelena D.; Nivarthi, Harini; Gisslinger, Heinz; Leroy, Emilie; Rumi, Elisa; Chachoua, Ilyas; Bagienski, Klaudia; Kubesova, Blanka; Pietra, Daniela; Gisslinger, Bettina; Milanesi, Chiara; Jäger, Roland; Chen, Doris; Berg, Tiina; Schalling, Martin; Schuster, Michael; Bock, Christoph; Constantinescu, Stefan N.; Cazzola, Mario

2016-01-01

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed “triple negative.” We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders. PMID:26423830

Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms.

PubMed

Milosevic Feenstra, Jelena D; Nivarthi, Harini; Gisslinger, Heinz; Leroy, Emilie; Rumi, Elisa; Chachoua, Ilyas; Bagienski, Klaudia; Kubesova, Blanka; Pietra, Daniela; Gisslinger, Bettina; Milanesi, Chiara; Jäger, Roland; Chen, Doris; Berg, Tiina; Schalling, Martin; Schuster, Michael; Bock, Christoph; Constantinescu, Stefan N; Cazzola, Mario; Kralovics, Robert

2016-01-21

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed "triple negative." We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders. © 2016 by The American Society of Hematology.

CRISPR/Cas9-mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse.

PubMed

Guan, Yuting; Ma, Yanlin; Li, Qi; Sun, Zhenliang; Ma, Lie; Wu, Lijuan; Wang, Liren; Zeng, Li; Shao, Yanjiao; Chen, Yuting; Ma, Ning; Lu, Wenqing; Hu, Kewen; Han, Honghui; Yu, Yanhong; Huang, Yuanhua; Liu, Mingyao; Li, Dali

2016-05-01

The X-linked genetic bleeding disorder caused by deficiency of coagulator factor IX, hemophilia B, is a disease ideally suited for gene therapy with genome editing technology. Here, we identify a family with hemophilia B carrying a novel mutation, Y371D, in the human F9 gene. The CRISPR/Cas9 system was used to generate distinct genetically modified mouse models and confirmed that the novel Y371D mutation resulted in a more severe hemophilia B phenotype than the previously identified Y371S mutation. To develop therapeutic strategies targeting this mutation, we subsequently compared naked DNA constructs versus adenoviral vectors to deliver Cas9 components targeting the F9 Y371D mutation in adult mice. After treatment, hemophilia B mice receiving naked DNA constructs exhibited correction of over 0.56% of F9 alleles in hepatocytes, which was sufficient to restore hemostasis. In contrast, the adenoviral delivery system resulted in a higher corrective efficiency but no therapeutic effects due to severe hepatic toxicity. Our studies suggest that CRISPR/Cas-mediated in situ genome editing could be a feasible therapeutic strategy for human hereditary diseases, although an efficient and clinically relevant delivery system is required for further clinical studies. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

Statistical Methods for Identifying Sequence Motifs Affecting Point Mutations

PubMed Central

Zhu, Yicheng; Neeman, Teresa; Yap, Von Bing; Huttley, Gavin A.

2017-01-01

Mutation processes differ between types of point mutation, genomic locations, cells, and biological species. For some point mutations, specific neighboring bases are known to be mechanistically influential. Beyond these cases, numerous questions remain unresolved, including: what are the sequence motifs that affect point mutations? How large are the motifs? Are they strand symmetric? And, do they vary between samples? We present new log-linear models that allow explicit examination of these questions, along with sequence logo style visualization to enable identifying specific motifs. We demonstrate the performance of these methods by analyzing mutation processes in human germline and malignant melanoma. We recapitulate the known CpG effect, and identify novel motifs, including a highly significant motif associated with A→G mutations. We show that major effects of neighbors on germline mutation lie within ±2 of the mutating base. Models are also presented for contrasting the entire mutation spectra (the distribution of the different point mutations). We show the spectra vary significantly between autosomes and X-chromosome, with a difference in T→C transition dominating. Analyses of malignant melanoma confirmed reported characteristic features of this cancer, including statistically significant strand asymmetry, and markedly different neighboring influences. The methods we present are made freely available as a Python library https://bitbucket.org/pycogent3/mutationmotif. PMID:27974498

Mutation screening of Chinese Treacher Collins syndrome patients identified novel TCOF1 mutations.

PubMed

Chen, Ying; Guo, Luo; Li, Chen-Long; Shan, Jing; Xu, Hai-Song; Li, Jie-Ying; Sun, Shan; Hao, Shao-Juan; Jin, Lei; Chai, Gang; Zhang, Tian-Yu

2018-04-01

Treacher Collins syndrome (TCS) (OMIM 154500) is a rare congenital craniofacial disorder with an autosomal dominant manner of inheritance in most cases. To date, three pathogenic genes (TCOF1, POLR1D and POLR1C) have been identified. In this study, we conducted mutational analysis on Chinese TCS patients to reveal a mutational spectrum of known causative genes and show phenotype-genotype data to provide more information for gene counselling and future studies on the pathogenesis of TCS. Twenty-two TCS patients were recruited from two tertiary referral centres, and Sanger sequencing for the coding exons and exon-intron boundaries of TCOF1, POLR1D and POLR1C was performed. For patients without small variants, further copy number variations (CNVs) analysis was conducted using high-density SNP array platforms. The Sanger sequencing overall mutation detection rate was as high as 86.3% (19/22) for our cohort. Fifteen TCOF1 pathogenic variants, including ten novel mutations, were identified in nineteen patients. No causative mutations in POLR1D and POLR1C genes and no CNVs mutations were detected. A suspected autosomal dominant inheritance case that implies germinal mosaicism was described. Our study confirmed that TCOF1 was the main disease-causing gene for the Chinese TCS population and revealed its mutation spectrum. We also addressed the need for more studies of mosaicism in TCS cases, which could explain the mechanism of autosomal dominant inheritance in TCS cases and benefit the prevention of TCS.

SEPT9 Mutations and a Conserved 17q25 Sequence in Sporadic and Hereditary Brachial Plexus Neuropathy

PubMed Central

Klein, Christopher J.; Wu, Yanhong; Cunningham, Julie M.; Windebank, Anthony J.; Dyck, P. James B.; Friedenberg, Scott M.; Klein, Diane M.; Dyck, Peter J.

2009-01-01

Background The clinical characteristics of sporadic brachial plexus neuropathy (S-BPN) and hereditary brachial plexus neuropathy (H-BPN) are similar. At times of attack inflammation in brachial plexus nerves has been identified in both conditions. SEPT-9 mutations (Arg88Trp, Ser93Phe, 5UTR-131G to C) occur in some families with H-BPN. These mutations were not found in American H-BPN kindreds with a conserved 500 Kb sequence of DNA at 17q25 (the location of SEPT-9) where a founder mutation has been suggested. Objective To study 17q25 and SEPT-9 in S-BPN (56 patients) and H-BPN (13 kindreds). Methods Allele analysis at 17q25, SEPT-9 DNA sequencing and mRNA analysis from lymphoblast cultures. Results A conserved 17q25 sequence was found in 5 of 13 H-BPN kindreds and one S-BPN patient. This conserved sequence was not found in the family with a SEPT-9 mutation (Arg88Trp) or controls (182). SEPT-9 mRNA expression did not differ between forms of H-BPN and controls. No known mutations of SEPT-9 were found in S-BPN. Conclusions/Relevance Rare S-BPN patients have the same conserved 17q25 sequence found in many American H-BPN kindreds. BPN patients with this conserved sequence do not appear to have SEPT-9 mutations or alterations of its mRNA expression levels in lymphoblast cultures. BPN patients with this conserved sequence may have the most common genetic cause in the Americas by a founder effect mutation. PMID:19204161

Promoting Cas9 degradation reduces mosaic mutations in non-human primate embryos

PubMed Central

Tu, Zhuchi; Yang, Weili; Yan, Sen; Yin, An; Gao, Jinquan; Liu, Xudong; Zheng, Yinghui; Zheng, Jiezhao; Li, Zhujun; Yang, Su; Li, Shihua; Guo, Xiangyu; Li, Xiao-Jiang

2017-01-01

CRISPR-Cas9 is a powerful new tool for genome editing, but this technique creates mosaic mutations that affect the efficiency and precision of its ability to edit the genome. Reducing mosaic mutations is particularly important for gene therapy and precision genome editing. Although the mechanisms underlying the CRSIPR/Cas9-mediated mosaic mutations remain elusive, the prolonged expression and activity of Cas9 in embryos could contribute to mosaicism in DNA mutations. Here we report that tagging Cas9 with ubiquitin-proteasomal degradation signals can facilitate the degradation of Cas9 in non-human primate embryos. Using embryo-splitting approach, we found that shortening the half-life of Cas9 in fertilized zygotes reduces mosaic mutations and increases its ability to modify genomes in non-human primate embryos. Also, injection of modified Cas9 in one-cell embryos leads to live monkeys with the targeted gene modifications. Our findings suggest that modifying Cas9 activity can be an effective strategy to enhance precision genome editing. PMID:28155910

SLC30A9 mutation affecting intracellular zinc homeostasis causes a novel cerebro-renal syndrome.

PubMed

Perez, Yonatan; Shorer, Zamir; Liani-Leibson, Keren; Chabosseau, Pauline; Kadir, Rotem; Volodarsky, Michael; Halperin, Daniel; Barber-Zucker, Shiran; Shalev, Hanna; Schreiber, Ruth; Gradstein, Libe; Gurevich, Evgenia; Zarivach, Raz; Rutter, Guy A; Landau, Daniel; Birk, Ohad S

2017-04-01

A novel autosomal recessive cerebro-renal syndrome was identified in consanguineous Bedouin kindred: neurological deterioration was evident as of early age, progressing into severe intellectual disability, profound ataxia, camptocormia and oculomotor apraxia. Brain MRI was normal. Four of the six affected individuals also had early-onset nephropathy with features of tubulo-interstitial nephritis, hypertension and tendency for hyperkalemia, though none had rapid deterioration of renal function. Genome wide linkage analysis identified an ∼18 Mb disease-associated locus on chromosome 4 (maximal logarithm of odds score 4.4 at D4S2971; θ = 0). Whole exome sequencing identified a single mutation in SLC30A9 within this locus, segregating as expected within the kindred and not found in a homozygous state in 300 Bedouin controls. We showed that SLC30A9 (solute carrier family 30 member 9; also known as ZnT-9) is ubiquitously expressed with high levels in cerebellum, skeletal muscle, thymus and kidney. Confocal analysis of SH-SY5Y cells overexpressing SLC30A9 fused to enhanced green fluorescent protein demonstrated vesicular cytosolic localization associated with the endoplasmic reticulum, not co-localizing with endosomal or Golgi markers. SLC30A9 encodes a putative zinc transporter (by similarity) previously associated with Wnt signalling. However, using dual-luciferase reporter assay in SH-SY5Y cells we showed that Wnt signalling was not affected by the mutation. Based on protein modelling, the identified mutation is expected to affect SLC30A9's highly conserved cation efflux domain, putatively disrupting its transmembrane helix structure. Cytosolic Zn2+ measurements in HEK293 cells overexpressing wild-type and mutant SLC30A9 showed lower zinc concentration within mutant rather than wild-type SLC30A9 cells. This suggests that SLC30A9 has zinc transport properties affecting intracellular zinc homeostasis, and that the molecular mechanism of the disease is through

SLC30A9 mutation affecting intracellular zinc homeostasis causes a novel cerebro-renal syndrome

PubMed Central

Perez, Yonatan; Shorer, Zamir; Liani-Leibson, Keren; Chabosseau, Pauline; Kadir, Rotem; Volodarsky, Michael; Halperin, Daniel; Barber-Zucker, Shiran; Shalev, Hanna; Schreiber, Ruth; Gradstein, Libe; Gurevich, Evgenia; Zarivach, Raz; Rutter, Guy A.; Landau, Daniel

2017-01-01

Abstract A novel autosomal recessive cerebro-renal syndrome was identified in consanguineous Bedouin kindred: neurological deterioration was evident as of early age, progressing into severe intellectual disability, profound ataxia, camptocormia and oculomotor apraxia. Brain MRI was normal. Four of the six affected individuals also had early-onset nephropathy with features of tubulo-interstitial nephritis, hypertension and tendency for hyperkalemia, though none had rapid deterioration of renal function. Genome wide linkage analysis identified an ∼18 Mb disease-associated locus on chromosome 4 (maximal logarithm of odds score 4.4 at D4S2971; θ = 0). Whole exome sequencing identified a single mutation in SLC30A9 within this locus, segregating as expected within the kindred and not found in a homozygous state in 300 Bedouin controls. We showed that SLC30A9 (solute carrier family 30 member 9; also known as ZnT-9) is ubiquitously expressed with high levels in cerebellum, skeletal muscle, thymus and kidney. Confocal analysis of SH-SY5Y cells overexpressing SLC30A9 fused to enhanced green fluorescent protein demonstrated vesicular cytosolic localization associated with the endoplasmic reticulum, not co-localizing with endosomal or Golgi markers. SLC30A9 encodes a putative zinc transporter (by similarity) previously associated with Wnt signalling. However, using dual-luciferase reporter assay in SH-SY5Y cells we showed that Wnt signalling was not affected by the mutation. Based on protein modelling, the identified mutation is expected to affect SLC30A9’s highly conserved cation efflux domain, putatively disrupting its transmembrane helix structure. Cytosolic Zn2+ measurements in HEK293 cells overexpressing wild-type and mutant SLC30A9 showed lower zinc concentration within mutant rather than wild-type SLC30A9 cells. This suggests that SLC30A9 has zinc transport properties affecting intracellular zinc homeostasis, and that the molecular mechanism of the disease is

CCR4 frameshift mutation identifies a distinct group of adult T cell leukaemia/lymphoma with poor prognosis.

PubMed

Yoshida, Noriaki; Miyoshi, Hiroaki; Kato, Takeharu; Sakata-Yanagimoto, Mamiko; Niino, Daisuke; Taniguchi, Hiroaki; Moriuchi, Yukiyoshi; Miyahara, Masaharu; Kurita, Daisuke; Sasaki, Yuya; Shimono, Joji; Kawamoto, Keisuke; Utsunomiya, Atae; Imaizumi, Yoshitaka; Seto, Masao; Ohshima, Koichi

2016-04-01

Adult T cell leukaemia/lymphoma (ATLL) is an intractable T cell neoplasm caused by human T cell leukaemia virus type 1. Next-generation sequencing-based comprehensive mutation studies have revealed recurrent somatic CCR4 mutations in ATLL, although clinicopathological findings associated with CCR4 mutations remain to be delineated. In the current study, 184 cases of peripheral T cell lymphoma, including 113 cases of ATLL, were subjected to CCR4 mutation analysis. This sequence analysis identified mutations in 27% (30/113) of cases of ATLL and 9% (4/44) of cases of peripheral T cell lymphoma not otherwise specified. Identified mutations included nonsense (NS) and frameshift (FS) mutations. No significant differences in clinicopathological findings were observed between ATLL cases stratified by presence of CCR4 mutation. All ATLL cases with CCR4 mutations exhibited cell-surface CCR4 positivity. Semi-quantitative CCR4 protein analysis of immunohistochemical sections revealed higher CCR4 expression in cases with NS mutations of CCR4 than in cases with wild-type (WT) CCR4. Furthermore, among ATLL cases, FS mutation was significantly associated with a poor prognosis, compared with NS mutation and WT CCR4. These results suggest that CCR4 mutation is an important determinant of the clinical course in ATLL cases, and that NS and FS mutations of CCR4 behave differently with respect to ATLL pathophysiology. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Constitutional SAMD9L mutations cause familial myelodysplastic syndrome and transient monosomy 7

PubMed Central

Pastor, Victor B.; Sahoo, Sushree S.; Boklan, Jessica; Schwabe, Georg C.; Saribeyoglu, Ebru; Strahm, Brigitte; Lebrecht, Dirk; Voss, Matthias; Bryceson, Yenan T.; Erlacher, Miriam; Ehninger, Gerhard; Niewisch, Marena; Schlegelberger, Brigitte; Baumann, Irith; Achermann, John C.; Shimamura, Akiko; Hochrein, Jochen; Tedgård, Ulf; Nilsson, Lars; Hasle, Henrik; Boerries, Melanie; Busch, Hauke; Niemeyer, Charlotte M.; Wlodarski, Marcin W.

2018-01-01

Familial myelodysplastic syndromes arise from haploinsufficiency of genes involved in hematopoiesis and are primarily associated with early-onset disease. Here we describe a familial syndrome in seven patients from four unrelated pedigrees presenting with myelodysplastic syndrome and loss of chromosome 7/7q. Their median age at diagnosis was 2.1 years (range, 1–42). All patients presented with thrombocytopenia with or without additional cytopenias and a hypocellular marrow without an increase of blasts. Genomic studies identified constitutional mutations (p.H880Q, p.R986H, p.R986C and p.V1512M) in the SAMD9L gene on 7q21, with decreased allele frequency in hematopoiesis. The non-random loss of mutated SAMD9L alleles was attained via monosomy 7, deletion 7q, UPD7q, or acquired truncating SAMD9L variants p.R1188X and p.S1317RfsX21. Incomplete penetrance was noted in 30% (3/10) of mutation carriers. Long-term observation revealed divergent outcomes with either progression to leukemia and/or accumulation of driver mutations (n=2), persistent monosomy 7 (n=4), and transient monosomy 7 followed by spontaneous recovery with SAMD9L-wildtype UPD7q (n=2). Dysmorphic features or neurological symptoms were absent in our patients, pointing to the notion that myelodysplasia with monosomy 7 can be a sole manifestation of SAMD9L disease. Collectively, our results define a new subtype of familial myelodysplastic syndrome and provide an explanation for the phenomenon of transient monosomy 7. Registered at: www.clinicaltrials.gov; #NCT00047268. PMID:29217778

Constitutional SAMD9L mutations cause familial myelodysplastic syndrome and transient monosomy 7.

PubMed

Pastor, Victor B; Sahoo, Sushree S; Boklan, Jessica; Schwabe, Georg C; Saribeyoglu, Ebru; Strahm, Brigitte; Lebrecht, Dirk; Voss, Matthias; Bryceson, Yenan T; Erlacher, Miriam; Ehninger, Gerhard; Niewisch, Marena; Schlegelberger, Brigitte; Baumann, Irith; Achermann, John C; Shimamura, Akiko; Hochrein, Jochen; Tedgård, Ulf; Nilsson, Lars; Hasle, Henrik; Boerries, Melanie; Busch, Hauke; Niemeyer, Charlotte M; Wlodarski, Marcin W

2018-03-01

Familial myelodysplastic syndromes arise from haploinsufficiency of genes involved in hematopoiesis and are primarily associated with early-onset disease. Here we describe a familial syndrome in seven patients from four unrelated pedigrees presenting with myelodysplastic syndrome and loss of chromosome 7/7q. Their median age at diagnosis was 2.1 years (range, 1-42). All patients presented with thrombocytopenia with or without additional cytopenias and a hypocellular marrow without an increase of blasts. Genomic studies identified constitutional mutations (p.H880Q, p.R986H, p.R986C and p.V1512M) in the SAMD9L gene on 7q21, with decreased allele frequency in hematopoiesis. The non-random loss of mutated SAMD9L alleles was attained via monosomy 7, deletion 7q, UPD7q, or acquired truncating SAMD9L variants p.R1188X and p.S1317RfsX21. Incomplete penetrance was noted in 30% (3/10) of mutation carriers. Long-term observation revealed divergent outcomes with either progression to leukemia and/or accumulation of driver mutations (n=2), persistent monosomy 7 (n=4), and transient monosomy 7 followed by spontaneous recovery with SAMD9L -wildtype UPD7q (n=2). Dysmorphic features or neurological symptoms were absent in our patients, pointing to the notion that myelodysplasia with monosomy 7 can be a sole manifestation of SAMD9L disease. Collectively, our results define a new subtype of familial myelodysplastic syndrome and provide an explanation for the phenomenon of transient monosomy 7. Registered at: www.clinicaltrials.gov; #NCT00047268 . Copyright© 2018 Ferrata Storti Foundation.

Loss-of-Function PCSK9 Mutations Are Not Associated With Alzheimer Disease.

PubMed

Paquette, Martine; Saavedra, Yascara Grisel Luna; Poirier, Judes; Théroux, Louise; Dea, Doris; Baass, Alexis; Dufour, Robert

2018-03-01

Hypercholesterolemia is a major risk factor for the late-onset form of Alzheimer disease (AD). Loss-of-function (LOF) mutations of PCSK9 and PCSK9 inhibitors lower low-density lipoprotein cholesterol (LDL-C) and have been associated with a reduced risk of cardiovascular disease. The aim of this study was to examine the effect of PCSK9 LOF variants on risk and age of onset of AD. A total of 878 participants (410 controls and 468 AD cases) from the Quebec Founder Population were included in the study. Fifty-four (6.2%) participants carried the R46L mutation, whereas 226 (26.2%) participants carried the InsLEU mutation. There was no protective or no deleterious effect of carrying PCSK9 LOF mutations on AD prevalence nor on age of onset, even when stratified by apolipoprotein E epsilon 4 genotype or by gender. Our data indicate that carrying PCSK9 LOF mutations has a neutral effect on neurocognitive health and the prevalence of AD.

Identification of a novel homozygous TRAPPC9 gene mutation causing non-syndromic intellectual disability, speech disorder, and secondary microcephaly.

PubMed

Abbasi, Ansar A; Blaesius, Kathrin; Hu, Hao; Latif, Zahid; Picker-Minh, Sylvie; Khan, Muhammad N; Farooq, Sundas; Khan, Muzammil A; Kaindl, Angela M

2017-12-01

TRAPPC9 gene mutations have been linked recently to autosomal recessive mental retardation 13 (MRT13; MIM#613192) with only eight families reported world-wide. We assessed patients from two consanguineous pedigrees of Pakistani descent with non-syndromic intellectual disability and postnatal microcephaly through whole exome sequencing (WES) and cosegregation analysis. Here we report six further patients from two pedigrees with homozygous TRAPPC9 gene mutations, the novel nonsense mutation c.2065G>T (p.E689*) and the previously identified nonsense mutation c.1423C>T (p.R475*). We provide an overview of previously reported clinical features and highlight common symptoms and variability of MRT13. Common findings are intellectual disability and absent speech, and frequently microcephaly, motor delay and pathological findings on MRI including diminished cerebral white matter volume are present. Mutations in TRAPPC9 should be considered in non-syndromic autosomal recessive intellectual disability with severe speech disorder. © 2017 Wiley Periodicals, Inc.

Mutation Frequency of the Major Frontotemporal Dementia Genes, MAPT, GRN and C9ORF72 in a Turkish Cohort of Dementia Patients.

PubMed

Guven, Gamze; Lohmann, Ebba; Bras, Jose; Gibbs, J Raphael; Gurvit, Hakan; Bilgic, Basar; Hanagasi, Hasmet; Rizzu, Patrizia; Heutink, Peter; Emre, Murat; Erginel-Unaltuna, Nihan; Just, Walter; Hardy, John; Singleton, Andrew; Guerreiro, Rita

2016-01-01

'Microtubule-associated protein tau' (MAPT), 'granulin' (GRN) and 'chromosome 9 open reading frame72' (C9ORF72) gene mutations are the major known genetic causes of frontotemporal dementia (FTD). Recent studies suggest that mutations in these genes may also be associated with other forms of dementia. Therefore we investigated whether MAPT, GRN and C9ORF72 gene mutations are major contributors to dementia in a random, unselected Turkish cohort of dementia patients. A combination of whole-exome sequencing, Sanger sequencing and fragment analysis/Southern blot was performed in order to identify pathogenic mutations and novel variants in these genes as well as other FTD-related genes such as the 'charged multivesicular body protein 2B' (CHMP2B), the 'FUS RNA binding protein' (FUS), the 'TAR DNA binding protein' (TARDBP), the 'sequestosome1' (SQSTM1), and the 'valosin containing protein' (VCP). We determined one pathogenic MAPT mutation (c.1906C>T, p.P636L) and one novel missense variant (c.38A>G, p.D13G). In GRN we identified a probably pathogenic TGAG deletion in the splice donor site of exon 6. Three patients were found to carry the GGGGCC expansions in the non-coding region of the C9ORF72 gene. In summary, a complete screening for mutations in MAPT, GRN and C9ORF72 genes revealed a frequency of 5.4% of pathogenic mutations in a random cohort of 93 Turkish index patients with dementia.

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

Key clinical features to identify girls with CDKL5 mutations.

PubMed

Bahi-Buisson, Nadia; Nectoux, Juliette; Rosas-Vargas, Haydeé; Milh, Mathieu; Boddaert, Nathalie; Girard, Benoit; Cances, Claude; Ville, Dorothée; Afenjar, Alexandra; Rio, Marlène; Héron, Delphine; N'guyen Morel, Marie Ange; Arzimanoglou, Alexis; Philippe, Christophe; Jonveaux, Philippe; Chelly, Jamel; Bienvenu, Thierry

2008-10-01

Mutations in the human X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been shown to cause infantile spasms as well as Rett syndrome (RTT)-like phenotype. To date, less than 25 different mutations have been reported. So far, there are still little data on the key clinical diagnosis criteria and on the natural history of CDKL5-associated encephalopathy. We screened the entire coding region of CDKL5 for mutations in 183 females with encephalopathy with early seizures by denaturing high liquid performance chromatography and direct sequencing, and we identified in 20 unrelated girls, 18 different mutations including 7 novel mutations. These mutations were identified in eight patients with encephalopathy with RTT-like features, five with infantile spasms and seven with encephalopathy with refractory epilepsy. Early epilepsy with normal interictal EEG and severe hypotonia are the key clinical features in identifying patients likely to have CDKL5 mutations. Our study also indicates that these patients clearly exhibit some RTT features such as deceleration of head growth, stereotypies and hand apraxia and that these RTT features become more evident in older and ambulatory patients. However, some RTT signs are clearly absent such as the so called RTT disease profile (period of nearly normal development followed by regression with loss of acquired fine finger skill in early childhood and characteristic intensive eye communication) and the characteristic evolution of the RTT electroencephalogram. Interestingly, in addition to the overall stereotypical symptomatology (age of onset and evolution of the disease) resulting from CDKL5 mutations, atypical forms of CDKL5-related conditions have also been observed. Our data suggest that phenotypic heterogeneity does not correlate with the nature or the position of the mutations or with the pattern of X-chromosome inactivation, but most probably with the functional transcriptional and/or translational consequences of CDKL5

A new mutation identified in SPATA16 in two globozoospermic patients.

PubMed

ElInati, Elias; Fossard, Camille; Okutman, Ozlem; Ghédir, Houda; Ibala-Romdhane, Samira; Ray, Pierre F; Saad, Ali; Hennebicq, Sylvianne; Viville, Stéphane

2016-06-01

The aim of this study is to identify potential genes involved in human globozoopsermia. Nineteen globozoospermic patients (previously screened for DPY19L2 mutations with no causative mutation) were recruited in this study and screened for mutations in genes implicated in human globozoospermia SPATA16 and PICK1. Using the candidate gene approach and the determination of Spata16 partners by Glutathione S-transferase (GST) pull-down four genes were also selected and screened for mutations. We identified a novel mutation of SPATA16: deletion of 22.6 Kb encompassing the first coding exon in two unrelated Tunisian patients who presented the same deletion breakpoints. The two patients shared the same haplotype, suggesting a possible ancestral founder effect for this new deletion. Four genes were selected using the candidate gene approach and the GST pull-down (GOPC, PICK1, AGFG1 and IRGC) and were screened for mutation, but no variation was identified. The present study confirms the pathogenicity of the SPATA16 mutations. The fact that no variation was detected in the coding sequence of AFGF1, GOPC, PICK1 and IRGC does not mean that they are not involved in human globozoospermia. A larger globozoospermic cohort must be studied in order to accelerate the process of identifying new genes involved in such phenotypes. Until sufficient numbers of patients have been screened, AFGF1, GOPC, PICK1 and IRGC should still be considered as candidate genes.

Perturbation of the mutated EGFR interactome identifies vulnerabilities and resistance mechanisms.

PubMed

Li, Jiannong; Bennett, Keiryn; Stukalov, Alexey; Fang, Bin; Zhang, Guolin; Yoshida, Takeshi; Okamoto, Isamu; Kim, Jae-Young; Song, Lanxi; Bai, Yun; Qian, Xiaoning; Rawal, Bhupendra; Schell, Michael; Grebien, Florian; Winter, Georg; Rix, Uwe; Eschrich, Steven; Colinge, Jacques; Koomen, John; Superti-Furga, Giulio; Haura, Eric B

2013-11-05

We hypothesized that elucidating the interactome of epidermal growth factor receptor (EGFR) forms that are mutated in lung cancer, via global analysis of protein-protein interactions, phosphorylation, and systematically perturbing the ensuing network nodes, should offer a new, more systems-level perspective of the molecular etiology. Here, we describe an EGFR interactome of 263 proteins and offer a 14-protein core network critical to the viability of multiple EGFR-mutated lung cancer cells. Cells with acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) had differential dependence of the core network proteins based on the underlying molecular mechanisms of resistance. Of the 14 proteins, 9 are shown to be specifically associated with survival of EGFR-mutated lung cancer cell lines. This included EGFR, GRB2, MK12, SHC1, ARAF, CD11B, ARHG5, GLU2B, and CD11A. With the use of a drug network associated with the core network proteins, we identified two compounds, midostaurin and lestaurtinib, that could overcome drug resistance through direct EGFR inhibition when combined with erlotinib. Our results, enabled by interactome mapping, suggest new targets and combination therapies that could circumvent EGFR TKI resistance.

Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9.

PubMed

Paquet, Dominik; Kwart, Dylan; Chen, Antonia; Sproul, Andrew; Jacob, Samson; Teo, Shaun; Olsen, Kimberly Moore; Gregg, Andrew; Noggle, Scott; Tessier-Lavigne, Marc

2016-05-05

The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases, for example, in human pluripotent stem cells. CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency, which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions, deletions or other mutations (indels). DSBs may also be repaired by homology-directed repair (HDR) using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations. Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ, editing by HDR remains inefficient and can be corrupted by additional indels, preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore, targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations, and establish a method termed 'CORRECT' for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation's incorporation rate and its distance to the DSB, we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation, whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach, we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer's disease-causing mutations in

Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia

PubMed Central

Puente, Xose S.; Pinyol, Magda; Quesada, Víctor; Conde, Laura; Ordóñez, Gonzalo R.; Villamor, Neus; Escaramis, Georgia; Jares, Pedro; Beà, Sílvia; González-Díaz, Marcos; Bassaganyas, Laia; Baumann, Tycho; Juan, Manel; López-Guerra, Mónica; Colomer, Dolors; Tubío, José M. C.; López, Cristina; Navarro, Alba; Tornador, Cristian; Aymerich, Marta; Rozman, María; Hernández, Jesús M.; Puente, Diana A.; Freije, José M. P.; Velasco, Gloria; Gutiérrez-Fernández, Ana; Costa, Dolors; Carrió, Anna; Guijarro, Sara; Enjuanes, Anna; Hernández, Lluís; Yagüe, Jordi; Nicolás, Pilar; Romeo-Casabona, Carlos M.; Himmelbauer, Heinz; Castillo, Ester; Dohm, Juliane C.; de Sanjosé, Silvia; Piris, Miguel A.; de Alava, Enrique; Miguel, Jesús San; Royo, Romina; Gelpí, Josep L.; Torrents, David; Orozco, Modesto; Pisano, David G.; Valencia, Alfonso; Guigó, Roderic; Bayés, Mónica; Heath, Simon; Gut, Marta; Klatt, Peter; Marshall, John; Raine, Keiran; Stebbings, Lucy A.; Futreal, P. Andrew; Stratton, Michael R.; Campbell, Peter J.; Gut, Ivo; López-Guillermo, Armando; Estivill, Xavier; Montserrat, Emili; López-Otín, Carlos; Campo, Elías

2012-01-01

Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution1,2. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes3,4. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer. PMID:21642962

Somatic mutations in histiocytic sarcoma identified by next generation sequencing.

PubMed

Liu, Qingqing; Tomaszewicz, Keith; Hutchinson, Lloyd; Hornick, Jason L; Woda, Bruce; Yu, Hongbo

2016-08-01

Histiocytic sarcoma is a rare malignant neoplasm of presumed hematopoietic origin showing morphologic and immunophenotypic evidence of histiocytic differentiation. Somatic mutation importance in the pathogenesis or disease progression of histiocytic sarcoma was largely unknown. To identify somatic mutations in histiocytic sarcoma, we studied 5 histiocytic sarcomas [3 female and 2 male patients; mean age 54.8 (20-72), anatomic sites include lymph node, uterus, and pleura] and matched normal tissues from each patient as germ line controls. Somatic mutations in 50 "Hotspot" oncogenes and tumor suppressor genes were examined using next generation sequencing. Three (out of five) histiocytic sarcoma cases carried somatic mutations in BRAF. Among them, G464V [variant frequency (VF) of 43.6 %] and G466R (VF of 29.6 %) located at the P loop potentially interfere with the hydrophobic interaction between P and activating loops and ultimately activation of BRAF. Also detected was BRAF somatic mutation N581S (VF of 7.4 %), which was located at the catalytic loop of BRAF kinase domain: its role in modifying kinase activity was unclear. A similar mutational analysis was also performed on nine acute monocytic/monoblastic leukemia cases, which did not identify any BRAF somatic mutations. Our study detected several BRAF mutations in histiocytic sarcomas, which may be important in understanding the tumorigenesis of this rare neoplasm and providing mechanisms for potential therapeutical opportunities.

Disease progression in C9orf72 mutation carriers.

PubMed

Floeter, Mary K; Traynor, Bryan J; Farren, Jennifer; Braun, Laura E; Tierney, Michael; Wiggs, Edythe A; Wu, Tianxia

2017-07-18

To assess changes in 3 clinical measures, the Revised ALS Functional Rating Scale (ALSFRS-R), letter fluency, and Frontal Behavioral Inventory (FBI), over time in C9orf72 mutation carriers (C9+) with varied clinical phenotypes. Thirty-four unrelated participants with mutations in C9orf72 were enrolled in a prospective natural history study. Participants were classified as asymptomatic, amyotrophic lateral sclerosis (ALS), ALS-familial frontotemporal dementia (FTD), or behavioral-variant FTD by clinical diagnostic criteria. Diagnostic cognitive and motor tests were repeated at 6 and 18 months. The ALSFRS-R, letter fluency, and FBI were administered at baseline and follow-up visits at 6, 12, and 18 months. The clinical diagnosis of most patients did not change over the follow-up. ALSFRS-R scores correlated with measures of motor function. Letter fluency correlated with FBI and cognitive tests. ALSFRS-R, letter fluency, and FBI differed among the C9+ diagnostic subgroups at enrollment and worsened over follow-up in symptomatic patients, with different slopes among the subgroups. Most patients survived to the 6-month time point after enrollment. Survival of C9+ patients with ALS and C9+ patients with ALS-FTD declined over the 12- and 18-month follow-up. The pattern of scores of the ALSFRS-R, letter fluency, and FBI distinguished between ALS, ALS-FTD, and FTD presentations of C9orf72 mutation carriers and asymptomatic carriers. Longitudinal changes in these measures occurred with disease progression in a manner consistent with presenting phenotype. © 2017 American Academy of Neurology.

Trafficking Dynamics of PCSK9-Induced LDLR Degradation: Focus on Human PCSK9 Mutations and C-Terminal Domain

PubMed Central

Villeneuve, Louis; Demers, Annie; Mayer, Gaétan

2016-01-01

PCSK9 is a secreted ligand and negative post-translational regulator of low-density lipoprotein receptor (LDLR) in hepatocytes. Gain-of-function (GOF) or loss-of-function (LOF) mutations in PCSK9 are directly correlated with high or low plasma LDL-cholesterol levels, respectively. Therefore, PCSK9 is a prevailing lipid-lowering target to prevent coronary heart diseases and stroke. Herein, we fused monomeric fluorescent proteins to PCSK9 and LDLR to visualize their intra- and extracellular trafficking dynamics by live confocal microscopy. Fluorescence recovery after photobleaching (FRAP) showed that PCSK9 LOF R46L mutant and GOF mutations S127R and D129G, but not the LDLR high-affinity mutant D374Y, significantly accelerate PCSK9 exit from the endoplasmic reticulum (ER). Quantitative analysis of inverse FRAP revealed that only R46L presented a much slower trafficking from the trans-Golgi network (TGN) to the plasma membrane and a lower mobile fraction likely suggesting accumulation or delayed exit at the TGN as an underlying mechanism. While not primarily involved in LDLR binding, PCSK9 C-terminal domain (CTD) was found to be essential to induce LDLR degradation both upon its overexpression in cells or via the extracellular pathway. Our data revealed that PCSK9 CTD is required for the localization of PCSK9 at the TGN and increases its LDLR-mediated endocytosis. Interestingly, intracellular lysosomal targeting of PCSK9-ΔCTD was able to rescue its capacity to induce LDLR degradation emphasizing a role of the CTD in the sorting of PCSK9-LDLR complex towards late endocytic compartments. Finally, we validated our dual fluorescence system as a cell based-assay by preventing PCSK9 internalization using a PCSK9-LDLR blocking antibody, which may be expended to identify protein, peptide or small molecule inhibitors of PCSK9. PMID:27280970

Mutation in the PCSK9 Gene in Omani Arab Subjects with Autosomal Dominant Hypercholesterolemia and its Effect on PCSK9 Protein Structure.

PubMed

Al-Waili, Khalid; Al-Zidi, Ward Al-Muna; Al-Abri, Abdul Rahim; Al-Rasadi, Khalid; Al-Sabti, Hilal Ali; Shah, Karna; Al-Futaisi, Abdullah; Al-Zakwani, Ibrahim; Banerjee, Yajnavalka

2013-01-01

Proprotein convertase subtilisin/kexin type (PCSK9) is a crucial protein in LDL cholesterol (LDL-C) metabolism by virtue of its pivotal role in the degradation of the LDL receptor. Mutations in the PCSK9 gene have previously been found to segregate with autosomal dominant familial hypercholesterolemia (ADFH). In this study, DNA sequencing of the 12 exons of the PCSK9 gene has been performed for two patients with a clinical diagnosis of familial hypercholesterolemia where mutation in the LDL-receptor gene hasn't been excluded. One missense mutation was detected in the exon 9 PCSK9 gene in the two ADFH patients. The patients were found to be heterozygote for Ile474Val (SNP rs562556). Using an array of in silico tools, we have investigated the effect of the above mutation on different structural levels of the PCSK9 protein. Although, the mutation has already been reported in the literature for other populations, to the best of our knowledge this is the first report of a mutation in the PCSK9 gene from the Arab population, including the Omani population.

A novel mutation in SCN9A in a child with congenital insensitivity to pain.

PubMed

Shorer, Zamir; Wajsbrot, Einav; Liran, Tamir-Hostovsky; Levy, Jacov; Parvari, Ruti

2014-01-01

[corrected] Congenital insensitivity to pain (CIP) is a rare condition in which patients have no pain perception and anosmia but are otherwise essentially normal (OMIM 243000). The recent discovery of the genetic defects underlying 3 monogenic pain disorders has provided additional and important insights about some components of human pain. Genetic studies in families demonstrating recessively inherited channelopathy-associated insensitivity to pain have identified nonsense mutations that result in truncation of the voltage-gated sodium channel type IX subunit (SCN9A), a 113.5-kb gene comprising coding 26 exons. Here we describe a patient with CIP with a new mutation in SCN9A not described yet. All exons were sequenced. All 26 coding exons were sequenced and two changes were identified in homozygosity in exon 10: c.1126 A > C causing K376Q and c.1124delG causing p.G375Afs* frame shift. We report a novel, loss-of-function mutation in homozygosity that causes congenital insensitivity to pain and provide a comprehensive clinical description of the patient. This contributes to the clinical and neurophysiological characteristic of the sodium channel Nav1.7 channelopathy and expand our genetic knowledge which might provide more accurate and comprehensive clinical electrophysiological and genetic information. Copyright © 2014 Elsevier Inc. All rights reserved.

Exome capture sequencing identifies a novel mutation in BBS4

PubMed Central

Wang, Hui; Chen, Xianfeng; Dudinsky, Lynn; Patenia, Claire; Chen, Yiyun; Li, Yumei; Wei, Yue; Abboud, Emad B.; Al-Rajhi, Ali A.; Lewis, Richard Alan; Lupski, James R.; Mardon, Graeme; Gibbs, Richard A.; Perkins, Brian D.

2011-01-01

Purpose Leber congenital amaurosis (LCA) is one of the most severe eye dystrophies characterized by severe vision loss at an early stage and accounts for approximately 5% of all retinal dystrophies. The purpose of this study was to identify a novel LCA disease allele or gene and to develop an approach combining genetic mapping with whole exome sequencing. Methods Three patients from King Khaled Eye Specialist Hospital (KKESH205) underwent whole genome single nucleotide polymorphism genotyping, and a single candidate region was identified. Taking advantage of next-generation high-throughput DNA sequencing technologies, whole exome capture sequencing was performed on patient KKESH205#7. Sanger direct sequencing was used during the validation step. The zebrafish model was used to examine the function of the mutant allele. Results A novel missense mutation in Bardet-Biedl syndrome 4 protein (BBS4) was identified in a consanguineous family from Saudi Arabia. This missense mutation in the fifth exon (c.253G>C;p.E85Q) of BBS4 is likely a disease-causing mutation as it segregates with the disease. The mutation is not found in the single nucleotide polymorphism (SNP) database, the 1000 Genomes Project, or matching normal controls. Functional analysis of this mutation in zebrafish indicates that the G253C allele is pathogenic. Coinjection of the G253C allele cannot rescue the mislocalization of rhodopsin in the retina when BBS4 is knocked down by morpholino injection. Immunofluorescence analysis in cell culture shows that this missense mutation in BBS4 does not cause obvious defects in protein expression or pericentriolar localization. Conclusions This mutation likely mainly reduces or abolishes BBS4 function in the retina. Further studies of this allele will provide important insights concerning the pleiotropic nature of BBS4 function. PMID:22219648

Caudal dysgenesis in islet-1 transgenic mice

PubMed Central

Muller, Yunhua Li; Yueh, Yir Gloria; Yaworsky, Paul J.; Salbaum, J. Michael; Kappen, Claudia

2014-01-01

Maternal diabetes during pregnancy is responsible for the occurrence of diabetic embryopathy, a spectrum of birth defects that includes heart abnormalities, neural tube defects, and caudal dysgenesis syndromes. Here, we report that mice transgenic for the homeodomain transcription factor Isl-1 develop profound caudal growth defects that resemble human sacral/caudal agenesis. Isl-1 is normally expressed in the pancreas and is required for pancreas development and endocrine cell differentiation. Aberrant regulation of this pancreatic transcription factor causes increased mesodermal cell death, and the severity of defects is dependent on transgene dosage. Together with the finding that mutation of the pancreatic transcription factor HLXB9 causes sacral agenesis, our results implicate pancreatic transcription factors in the pathogenesis of birth defects associated with diabetes. PMID:12738808

Functional genome-wide siRNA screen identifies KIAA0586 as mutated in Joubert syndrome

PubMed Central

Roosing, Susanne; Hofree, Matan; Kim, Sehyun; Scott, Eric; Copeland, Brett; Romani, Marta; Silhavy, Jennifer L; Rosti, Rasim O; Schroth, Jana; Mazza, Tommaso; Miccinilli, Elide; Zaki, Maha S; Swoboda, Kathryn J; Milisa-Drautz, Joanne; Dobyns, William B; Mikati, Mohamed A; İncecik, Faruk; Azam, Matloob; Borgatti, Renato; Romaniello, Romina; Boustany, Rose-Mary; Clericuzio, Carol L; D'Arrigo, Stefano; Strømme, Petter; Boltshauser, Eugen; Stanzial, Franco; Mirabelli-Badenier, Marisol; Moroni, Isabella; Bertini, Enrico; Emma, Francesco; Steinlin, Maja; Hildebrandt, Friedhelm; Johnson, Colin A; Freilinger, Michael; Vaux, Keith K; Gabriel, Stacey B; Aza-Blanc, Pedro; Heynen-Genel, Susanne; Ideker, Trey; Dynlacht, Brian D; Lee, Ji Eun; Valente, Enza Maria; Kim, Joon; Gleeson, Joseph G

2015-01-01

Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies, including Joubert syndrome (JS), with defective cerebellar vermis development. We performed a high-content genome-wide small interfering RNA (siRNA) screen to identify genes regulating ciliogenesis as candidates for JS. We analyzed results with a supervised-learning approach, using SYSCILIA gold standard, Cildb3.0, a centriole siRNA screen and the GTex project, identifying 591 likely candidates. Intersection of this data with whole exome results from 145 individuals with unexplained JS identified six families with predominantly compound heterozygous mutations in KIAA0586. A c.428del base deletion in 0.1% of the general population was found in trans with a second mutation in an additional set of 9 of 163 unexplained JS patients. KIAA0586 is an orthologue of chick Talpid3, required for ciliogenesis and Sonic hedgehog signaling. Our results uncover a relatively high frequency cause for JS and contribute a list of candidates for future gene discoveries in ciliopathies. DOI: http://dx.doi.org/10.7554/eLife.06602.001 PMID:26026149

Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9.

PubMed

Prykhozhij, Sergey V; Fuller, Charlotte; Steele, Shelby L; Veinotte, Chansey J; Razaghi, Babak; Robitaille, Johane M; McMaster, Christopher R; Shlien, Adam; Malkin, David; Berman, Jason N

2018-06-14

We have optimized point mutation knock-ins into zebrafish genomic sites using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 reagents and single-stranded oligodeoxynucleotides. The efficiency of knock-ins was assessed by a novel application of allele-specific polymerase chain reaction and confirmed by high-throughput sequencing. Anti-sense asymmetric oligo design was found to be the most successful optimization strategy. However, cut site proximity to the mutation and phosphorothioate oligo modifications also greatly improved knock-in efficiency. A previously unrecognized risk of off-target trans knock-ins was identified that we obviated through the development of a workflow for correct knock-in detection. Together these strategies greatly facilitate the study of human genetic diseases in zebrafish, with additional applicability to enhance CRISPR-based approaches in other animal model systems.

Exome-wide Sequencing Shows Low Mutation Rates and Identifies Novel Mutated Genes in Seminomas.

PubMed

Cutcutache, Ioana; Suzuki, Yuka; Tan, Iain Beehuat; Ramgopal, Subhashini; Zhang, Shenli; Ramnarayanan, Kalpana; Gan, Anna; Lee, Heng Hong; Tay, Su Ting; Ooi, Aikseng; Ong, Choon Kiat; Bolthouse, Jonathan T; Lane, Brian R; Anema, John G; Kahnoski, Richard J; Tan, Patrick; Teh, Bin Tean; Rozen, Steven G

2015-07-01

Testicular germ cell tumors are the most common cancer diagnosed in young men, and seminomas are the most common type of these cancers. There have been no exome-wide examinations of genes mutated in seminomas or of overall rates of nonsilent somatic mutations in these tumors. The objective was to analyze somatic mutations in seminomas to determine which genes are affected and to determine rates of nonsilent mutations. Eight seminomas and matched normal samples were surgically obtained from eight patients. DNA was extracted from tissue samples and exome sequenced on massively parallel Illumina DNA sequencers. Single-nucleotide polymorphism chip-based copy number analysis was also performed to assess copy number alterations. The DNA sequencing read data were analyzed to detect somatic mutations including single-nucleotide substitutions and short insertions and deletions. The detected mutations were validated by independent sequencing and further checked for subclonality. The rate of nonsynonymous somatic mutations averaged 0.31 mutations/Mb. We detected nonsilent somatic mutations in 96 genes that were not previously known to be mutated in seminomas, of which some may be driver mutations. Many of the mutations appear to have been present in subclonal populations. In addition, two genes, KIT and KRAS, were affected in two tumors each with mutations that were previously observed in other cancers and are presumably oncogenic. Our study, the first report on exome sequencing of seminomas, detected somatic mutations in 96 new genes, several of which may be targetable drivers. Furthermore, our results show that seminoma mutation rates are five times higher than previously thought, but are nevertheless low compared to other common cancers. Similar low rates are seen in other cancers that also have excellent rates of remission achieved with chemotherapy. We examined the DNA sequences of seminomas, the most common type of testicular germ cell cancer. Our study identified 96

Exome sequencing identifies MAX mutations as a cause of hereditary pheochromocytoma.

PubMed

Comino-Méndez, Iñaki; Gracia-Aznárez, Francisco J; Schiavi, Francesca; Landa, Iñigo; Leandro-García, Luis J; Letón, Rocío; Honrado, Emiliano; Ramos-Medina, Rocío; Caronia, Daniela; Pita, Guillermo; Gómez-Graña, Alvaro; de Cubas, Aguirre A; Inglada-Pérez, Lucía; Maliszewska, Agnieszka; Taschin, Elisa; Bobisse, Sara; Pica, Giuseppe; Loli, Paola; Hernández-Lavado, Rafael; Díaz, José A; Gómez-Morales, Mercedes; González-Neira, Anna; Roncador, Giovanna; Rodríguez-Antona, Cristina; Benítez, Javier; Mannelli, Massimo; Opocher, Giuseppe; Robledo, Mercedes; Cascón, Alberto

2011-06-19

Hereditary pheochromocytoma (PCC) is often caused by germline mutations in one of nine susceptibility genes described to date, but there are familial cases without mutations in these known genes. We sequenced the exomes of three unrelated individuals with hereditary PCC (cases) and identified mutations in MAX, the MYC associated factor X gene. Absence of MAX protein in the tumors and loss of heterozygosity caused by uniparental disomy supported the involvement of MAX alterations in the disease. A follow-up study of a selected series of 59 cases with PCC identified five additional MAX mutations and suggested an association with malignant outcome and preferential paternal transmission of MAX mutations. The involvement of the MYC-MAX-MXD1 network in the development and progression of neural crest cell tumors is further supported by the lack of functional MAX in rat PCC (PC12) cells and by the amplification of MYCN in neuroblastoma and suggests that loss of MAX function is correlated with metastatic potential.

G20210A prothrombin gene mutation identified in patients with venous leg ulcers.

PubMed

Jebeleanu, G; Procopciuc, L

2001-01-01

The G20210A mutation variant of prothrombin gene is the second most frequent mutation identified in patients with deep venous thrombosis, after factor V Leiden. The risk for developing deep venous thrombosis is high in patients identified as heterozygous for G20210A mutation. In order to identify this polymorphism in the gene coding prothrombin, the 345bp fragment in the 3'- untranslated region of the prothrombin gene was amplified using amplification by polymerase chain reaction and enzymatic digestion by HindIII (restriction endonuclease enzyme). The products of amplification and enzymatic's digestion were analized using agarose gel electrophoresis. We investigated 20 patients with venous leg ulcers and we found 2 heterozygous (10%) for G20210A mutation. None of the patients in the control group had G20210A mutation. Our study confirms the presence of G20210A mutation in the Romanian population. Our study also shows the link between venous leg ulcers and this polymorphism in the prothrombin gene.

Monoallelic mutation analysis (MAMA) for identifying germline mutations.

PubMed

Papadopoulos, N; Leach, F S; Kinzler, K W; Vogelstein, B

1995-09-01

Dissection of germline mutations in a sensitive and specific manner presents a continuing challenge. In dominantly inherited diseases, mutations occur in only one allele and are often masked by the normal allele. Here we report the development of a sensitive and specific diagnostic strategy based on somatic cell hybridization termed MAMA (monoallelic mutation analysis). We have demonstrated the utility of this strategy in two different hereditary colorectal cancer syndromes, one caused by a defective tumour suppressor gene on chromosome 5 (familial adenomatous polyposis, FAP) and the other caused by a defective mismatch repair gene on chromosome 2 (hereditary non-polyposis colorectal cancer, HNPCC).

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

Hidden Genetic Variation in LCA9-Associated Congenital Blindness Explained by 5'UTR Mutations and Copy-Number Variations of NMNAT1.

PubMed

Coppieters, Frauke; Todeschini, Anne Laure; Fujimaki, Takuro; Baert, Annelot; De Bruyne, Marieke; Van Cauwenbergh, Caroline; Verdin, Hannah; Bauwens, Miriam; Ongenaert, Maté; Kondo, Mineo; Meire, Françoise; Murakami, Akira; Veitia, Reiner A; Leroy, Bart P; De Baere, Elfride

2015-12-01

Leber congenital amaurosis (LCA) is a severe autosomal-recessive retinal dystrophy leading to congenital blindness. A recently identified LCA gene is NMNAT1, located in the LCA9 locus. Although most mutations in blindness genes are coding variations, there is accumulating evidence for hidden noncoding defects or structural variations (SVs). The starting point of this study was an LCA9-associated consanguineous family in which no coding mutations were found in the LCA9 region. Exploring the untranslated regions of NMNAT1 revealed a novel homozygous 5'UTR variant, c.-70A>T. Moreover, an adjacent 5'UTR variant, c.-69C>T, was identified in a second consanguineous family displaying a similar phenotype. Both 5'UTR variants resulted in decreased NMNAT1 mRNA abundance in patients' lymphocytes, and caused decreased luciferase activity in human retinal pigment epithelial RPE-1 cells. Second, we unraveled pseudohomozygosity of a coding NMNAT1 mutation in two unrelated LCA patients by the identification of two distinct heterozygous partial NMNAT1 deletions. Molecular characterization of the breakpoint junctions revealed a complex Alu-rich genomic architecture. Our study uncovered hidden genetic variation in NMNAT1-associated LCA and emphasized a shift from coding to noncoding regulatory mutations and repeat-mediated SVs in the molecular pathogenesis of heterogeneous recessive disorders such as hereditary blindness. © 2015 The Authors. **Human Mutation published by Wiley Periodicals, Inc.

Identifying mutations in Tunisian families with retinal dystrophy.

PubMed

Habibi, Imen; Chebil, Ahmed; Falfoul, Yosra; Allaman-Pillet, Nathalie; Kort, Fedra; Schorderet, Daniel F; El Matri, Leila

2016-11-22

Retinal dystrophies (RD) are a rare genetic disorder with high genetic heterogeneity. This study aimed at identifying disease-causing variants in fifteen consanguineous Tunisian families. Full ophthalmic examination was performed. Index patients were subjected to IROme analysis or whole exome sequencing followed by homozygosity mapping. All detected variations were confirmed by direct Sanger sequencing. Mutation analysis in our patients revealed two compound heterozygous mutations p.(R91W);(V172D) in RPE65, and five novel homozygous mutations: p.R765C in CNGB1, p.H337R in PDE6B, splice site variant c.1129-2A > G and c.678_681delGAAG in FAM161A and c.1133 + 3_1133 + 6delAAGT in CERKL. The latter mutation impacts pre-mRNA splicing of CERKL. The other changes detected were six previously reported mutations in CNGB3 (p.R203*), ABCA4 (p.W782*), NR2E3 (p.R311Q), RPE65 (p.H182Y), PROM1 (c.1354dupT) and EYS (c.5928-2A > G). Segregation analysis in each family showed that all affected individuals were homozygotes and unaffected individuals were either heterozygote carriers or homozygous wild type allele. These results confirm the involvement of a large number of genes in RD in the Tunisian population.

Mutation screening of the HGD gene identifies a novel alkaptonuria mutation with significant founder effect and high prevalence.

PubMed

Sakthivel, Srinivasan; Zatkova, Andrea; Nemethova, Martina; Surovy, Milan; Kadasi, Ludevit; Saravanan, Madurai P

2014-05-01

Alkaptonuria (AKU) is an autosomal recessive disorder; caused by the mutations in the homogentisate 1, 2-dioxygenase (HGD) gene located on Chromosome 3q13.33. AKU is a rare disorder with an incidence of 1: 250,000 to 1: 1,000,000, but Slovakia and the Dominican Republic have a relatively higher incidence of 1: 19,000. Our study focused on studying the frequency of AKU and identification of HGD gene mutations in nomads. HGD gene sequencing was used to identify the mutations in alkaptonurics. For the past four years, from subjects suspected to be clinically affected, we found 16 positive cases among a randomly selected cohort of 41 Indian nomads (Narikuravar) settled in the specific area of Tamil Nadu, India. HGD gene mutation analysis showed that 11 of these patients carry the same homozygous splicing mutation c.87 + 1G > A; in five cases, this mutation was found to be heterozygous, while the second AKU-causing mutation was not identified in these patients. This result indicates that the founder effect and high degree of consanguineous marriages have contributed to AKU among nomads. Eleven positive samples were homozygous for a novel mutation c.87 + 1G > A, that abolishes an intron 2 donor splice site and most likely causes skipping of exon 2. The prevalence of AKU observed earlier seems to be highly increased in people of nomadic origin. © 2014 John Wiley & Sons Ltd/University College London.

Correction of Hirschsprung-Associated Mutations in Human Induced Pluripotent Stem Cells Via Clustered Regularly Interspaced Short Palindromic Repeats/Cas9, Restores Neural Crest Cell Function.

PubMed

Lai, Frank Pui-Ling; Lau, Sin-Ting; Wong, John Kwong-Leong; Gui, Hongsheng; Wang, Reeson Xu; Zhou, Tingwen; Lai, Wing Hon; Tse, Hung-Fat; Tam, Paul Kwong-Hang; Garcia-Barcelo, Maria-Mercedes; Ngan, Elly Sau-Wai

2017-07-01

Hirschsprung disease is caused by failure of enteric neural crest cells (ENCCs) to fully colonize the bowel, leading to bowel obstruction and megacolon. Heterozygous mutations in the coding region of the RET gene cause a severe form of Hirschsprung disease (total colonic aganglionosis). However, 80% of HSCR patients have short-segment Hirschsprung disease (S-HSCR), which has not been associated with genetic factors. We sought to identify mutations associated with S-HSCR, and used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing system to determine how mutations affect ENCC function. We created induced pluripotent stem cell (iPSC) lines from 1 patient with total colonic aganglionosis (with the G731del mutation in RET) and from 2 patients with S-HSCR (without a RET mutation), as well as RET +/- and RET -/- iPSCs. IMR90-iPSC cells were used as the control cell line. Migration and differentiation capacities of iPSC-derived ENCCs were analyzed in differentiation and migration assays. We searched for mutation(s) associated with S-HSCR by combining genetic and transcriptome data from patient blood- and iPSC-derived ENCCs, respectively. Mutations in the iPSCs were corrected using the CRISPR/Cas9 system. ENCCs derived from all iPSC lines, but not control iPSCs, had defects in migration and neuronal lineage differentiation. RET mutations were associated with differentiation and migration defects of ENCCs in vitro. Genetic and transcriptome analyses associated a mutation in the vinculin gene (VCL M209L) with S-HSCR. CRISPR/Cas9 correction of the RET G731del and VCL M209L mutations in iPSCs restored the differentiation and migration capacities of ENCCs. We identified mutations in VCL associated with S-HSCR. Correction of this mutation in iPSC using CRISPR/Cas9 editing, as well as the RET G731del mutation that causes Hirschsprung disease with total colonic aganglionosis, restored ENCC function. Our study demonstrates how human iPSCs can

Infantile Pain Episodes Associated with Novel Nav1.9 Mutations in Familial Episodic Pain Syndrome in Japanese Families.

PubMed

Okuda, Hiroko; Noguchi, Atsuko; Kobayashi, Hatasu; Kondo, Daiki; Harada, Kouji H; Youssefian, Shohab; Shioi, Hirotomo; Kabata, Risako; Domon, Yuki; Kubota, Kazufumi; Kitano, Yutaka; Takayama, Yasunori; Hitomi, Toshiaki; Ohno, Kousaku; Saito, Yoshiaki; Asano, Takeshi; Tominaga, Makoto; Takahashi, Tsutomu; Koizumi, Akio

2016-01-01

Painful peripheral neuropathy has been correlated with various voltage-gated sodium channel mutations in sensory neurons. Recently Nav1.9, a voltage-gated sodium channel subtype, has been established as a genetic influence for certain peripheral pain syndromes. In this study, we performed a genetic study in six unrelated multigenerational Japanese families with episodic pain syndrome. Affected participants (n = 23) were characterized by infantile recurrent pain episodes with spontaneous mitigation around adolescence. This unique phenotype was inherited in an autosomal-dominant mode. Linkage analysis was performed for two families with 12 affected and nine unaffected members, and a single locus was identified on 3p22 (LOD score 4.32). Exome analysis (n = 14) was performed for affected and unaffected members in these two families and an additional family. Two missense variants were identified: R222H and R222S in SCN11A. Next, we generated a knock-in mouse model harboring one of the mutations (R222S). Behavioral tests (Hargreaves test and cold plate test) using R222S and wild-type C57BL/6 (WT) mice, young (8-9 weeks old; n = 10-12 for each group) and mature (36-38 weeks old; n = 5-6 for each group), showed that R222S mice were significantly (p < 0.05) more hypersensitive to hot and cold stimuli than WT mice. Electrophysiological studies using dorsal root ganglion neurons from 8-9-week-old mice showed no significant difference in resting membrane potential, but input impedance and firing frequency of evoked action potentials were significantly increased in R222S mice compared with WT mice. However, there was no significant difference among Nav1.9 (WT, R222S, and R222H)-overexpressing ND7/23 cell lines. These results suggest that our novel mutation is a gain-of-function mutation that causes infantile familial episodic pain. The mouse model developed here will be useful for drug screening for familial episodic pain syndrome associated with SCN11A mutations.

Novel mutations in the homogentisate 1,2 dioxygenase gene identified in Jordanian patients with alkaptonuria.

PubMed

Al-sbou, Mohammed

2012-06-01

This study was conducted to identify mutations in the homogentisate 1,2 dioxygenase gene (HGD) in alkaptonuria patients among Jordanian population. Blood samples were collected from four alkaptonuria patients, four carriers, and two healthy volunteers. DNA was isolated from peripheral blood. All 14 exons of the HGD gene were amplified using the polymerase chain reaction (PCR) technique. The PCR products were then purified and analyzed by sequencing. Five mutations were identified in our samples. Four of them were novel C1273A, T1046G, 551-552insG, T533G and had not been previously reported, and one mutation T847C has been described before. The types of mutations identified were two missense mutations, one splice site mutation, one frameshift mutation, and one polymorphism. We present the first molecular study of the HGD gene in Jordanian alkaptonuria patients. This study provides valuable information about the molecular basis of alkaptonuria in Jordanian population.

Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets

DOE PAGES

Schulze, Kornelius; Imbeaud, Sandrine; Letouzé, Eric; ...

2015-03-30

Our genomic analyses promise to improve tumor characterization to optimize personalized treatment for patients with hepatocellular carcinoma (HCC). Exome sequencing analysis of 243 liver tumors identified mutational signatures associated with specific risk factors, mainly combined alcohol and tobacco consumption and exposure to aflatoxin B1. We identified 161 putative driver genes associated with 11 recurrently altered pathways. Associations of mutations defined 3 groups of genes related to risk factors and centered on CTNNB1 (alcohol), TP53 (hepatitis B virus, HBV) and AXIN1. These analyses according to tumor stage progression identified TERT promoter mutation as an early event, whereasFGF3, FGF4, FGF19 or CCND1more » amplification and TP53 and CDKN2A alterations appeared at more advanced stages in aggressive tumors. In 28% of the tumors, we identified genetic alterations potentially targetable by US Food and Drug Administration (FDA)–approved drugs. Finally, we identified risk factor–specific mutational signatures and defined the extensive landscape of altered genes and pathways in HCC, which will be useful to design clinical trials for targeted therapy.« less

Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets

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

Schulze, Kornelius; Imbeaud, Sandrine; Letouzé, Eric

Our genomic analyses promise to improve tumor characterization to optimize personalized treatment for patients with hepatocellular carcinoma (HCC). Exome sequencing analysis of 243 liver tumors identified mutational signatures associated with specific risk factors, mainly combined alcohol and tobacco consumption and exposure to aflatoxin B1. We identified 161 putative driver genes associated with 11 recurrently altered pathways. Associations of mutations defined 3 groups of genes related to risk factors and centered on CTNNB1 (alcohol), TP53 (hepatitis B virus, HBV) and AXIN1. These analyses according to tumor stage progression identified TERT promoter mutation as an early event, whereasFGF3, FGF4, FGF19 or CCND1more » amplification and TP53 and CDKN2A alterations appeared at more advanced stages in aggressive tumors. In 28% of the tumors, we identified genetic alterations potentially targetable by US Food and Drug Administration (FDA)–approved drugs. Finally, we identified risk factor–specific mutational signatures and defined the extensive landscape of altered genes and pathways in HCC, which will be useful to design clinical trials for targeted therapy.« less

Three mutations switch H7N9 influenza to human-type receptor specificity.

PubMed

de Vries, Robert P; Peng, Wenjie; Grant, Oliver C; Thompson, Andrew J; Zhu, Xueyong; Bouwman, Kim M; de la Pena, Alba T Torrents; van Breemen, Marielle J; Ambepitiya Wickramasinghe, Iresha N; de Haan, Cornelis A M; Yu, Wenli; McBride, Ryan; Sanders, Rogier W; Woods, Robert J; Verheije, Monique H; Wilson, Ian A; Paulson, James C

2017-06-01

The avian H7N9 influenza outbreak in 2013 resulted from an unprecedented incidence of influenza transmission to humans from infected poultry. The majority of human H7N9 isolates contained a hemagglutinin (HA) mutation (Q226L) that has previously been associated with a switch in receptor specificity from avian-type (NeuAcα2-3Gal) to human-type (NeuAcα2-6Gal), as documented for the avian progenitors of the 1957 (H2N2) and 1968 (H3N2) human influenza pandemic viruses. While this raised concern that the H7N9 virus was adapting to humans, the mutation was not sufficient to switch the receptor specificity of H7N9, and has not resulted in sustained transmission in humans. To determine if the H7 HA was capable of acquiring human-type receptor specificity, we conducted mutation analyses. Remarkably, three amino acid mutations conferred a switch in specificity for human-type receptors that resembled the specificity of the 2009 human H1 pandemic virus, and promoted binding to human trachea epithelial cells.

Three mutations switch H7N9 influenza to human-type receptor specificity

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

de Vries, Robert P.; Peng, Wenjie; Grant, Oliver C.

The avian H7N9 influenza outbreak in 2013 resulted from an unprecedented incidence of influenza transmission to humans from infected poultry. The majority of human H7N9 isolates contained a hemagglutinin (HA) mutation (Q226L) that has previously been associated with a switch in receptor specificity from avian-type (NeuAcα2-3Gal) to human-type (NeuAcα2-6Gal), as documented for the avian progenitors of the 1957 (H2N2) and 1968 (H3N2) human influenza pandemic viruses. While this raised concern that the H7N9 virus was adapting to humans, the mutation was not sufficient to switch the receptor specificity of H7N9, and has not resulted in sustained transmission in humans. Tomore » determine if the H7 HA was capable of acquiring human-type receptor specificity, we conducted mutation analyses. Remarkably, three amino acid mutations conferred a switch in specificity for human-type receptors that resembled the specificity of the 2009 human H1 pandemic virus, and promoted binding to human trachea epithelial cells.« less

VWF mutations and new sequence variations identified in healthy controls are more frequent in the African-American population.

PubMed

Bellissimo, Daniel B; Christopherson, Pamela A; Flood, Veronica H; Gill, Joan Cox; Friedman, Kenneth D; Haberichter, Sandra L; Shapiro, Amy D; Abshire, Thomas C; Leissinger, Cindy; Hoots, W Keith; Lusher, Jeanne M; Ragni, Margaret V; Montgomery, Robert R

2012-03-01

Diagnosis and classification of VWD is aided by molecular analysis of the VWF gene. Because VWF polymorphisms have not been fully characterized, we performed VWF laboratory testing and gene sequencing of 184 healthy controls with a negative bleeding history. The controls included 66 (35.9%) African Americans (AAs). We identified 21 new sequence variations, 13 (62%) of which occurred exclusively in AAs and 2 (G967D, T2666M) that were found in 10%-15% of the AA samples, suggesting they are polymorphisms. We identified 14 sequence variations reported previously as VWF mutations, the majority of which were type 1 mutations. These controls had VWF Ag levels within the normal range, suggesting that these sequence variations might not always reduce plasma VWF levels. Eleven mutations were found in AAs, and the frequency of M740I, H817Q, and R2185Q was 15%-18%. Ten AA controls had the 2N mutation H817Q; 1 was homozygous. The average factor VIII level in this group was 99 IU/dL, suggesting that this variation may confer little or no clinical symptoms. This study emphasizes the importance of sequencing healthy controls to understand ethnic-specific sequence variations so that asymptomatic sequence variations are not misidentified as mutations in other ethnic or racial groups.

Deep mutational scanning identifies sites in influenza nucleoprotein that affect viral inhibition by MxA

PubMed Central

Ashenberg, Orr; Padmakumar, Jai

2017-01-01

The innate-immune restriction factor MxA inhibits influenza replication by targeting the viral nucleoprotein (NP). Human influenza virus is more resistant than avian influenza virus to inhibition by human MxA, and prior work has compared human and avian viral strains to identify amino-acid differences in NP that affect sensitivity to MxA. However, this strategy is limited to identifying sites in NP where mutations that affect MxA sensitivity have fixed during the small number of documented zoonotic transmissions of influenza to humans. Here we use an unbiased deep mutational scanning approach to quantify how all single amino-acid mutations to NP affect MxA sensitivity in the context of replication-competent virus. We both identify new sites in NP where mutations affect MxA resistance and re-identify mutations known to have increased MxA resistance during historical adaptations of influenza to humans. Most of the sites where mutations have the greatest effect are almost completely conserved across all influenza A viruses, and the amino acids at these sites confer relatively high resistance to MxA. These sites cluster in regions of NP that appear to be important for its recognition by MxA. Overall, our work systematically identifies the sites in influenza nucleoprotein where mutations affect sensitivity to MxA. We also demonstrate a powerful new strategy for identifying regions of viral proteins that affect inhibition by host factors. PMID:28346537

Whole-exome sequencing identifies recurrent AKT1 mutations in sclerosing hemangioma of lung

PubMed Central

Jung, Seung-Hyun; Kim, Min Sung; Lee, Sung-Hak; Park, Hyun-Chun; Choi, Hyun Joo; Maeng, Leeso; Min, Ki Ouk; Kim, Jeana; Park, Tae In; Shin, Ok Ran; Kim, Tae-Jung; Xu, Haidong; Lee, Kyo Young; Kim, Tae-Min; Song, Sang Yong; Lee, Charles; Chung, Yeun-Jun; Lee, Sug Hyung

2016-01-01

Pulmonary sclerosing hemangioma (PSH) is a benign tumor with two cell populations (epithelial and stromal cells), for which genomic profiles remain unknown. We conducted exome sequencing of 44 PSHs and identified recurrent somatic mutations of AKT1 (43.2%) and β-catenin (4.5%). We used a second subset of 24 PSHs to confirm the high frequency of AKT1 mutations (overall 31/68, 45.6%; p.E17K, 33.8%) and recurrent β-catenin mutations (overall 3 of 68, 4.4%). Of the PSHs without AKT1 mutations, two exhibited AKT1 copy gain. AKT1 mutations existed in both epithelial and stromal cells. In two separate PSHs from one patient, we observed two different AKT1 mutations, indicating they were not disseminated but independent arising tumors. Because the AKT1 mutations were not found to co-occur with β-catenin mutations (or any other known driver alterations) in any of the PSHs studied, we speculate that this may be the single-most common driver alteration to develop PSHs. Our study revealed genomic differences between PSHs and lung adenocarcinomas, including a high rate of AKT1 mutation in PSHs. These genomic features of PSH identified in the present study provide clues to understanding the biology of PSH and for differential genomic diagnosis of lung tumors. PMID:27601661

Hearing loss caused by a P2RX2 mutation identified in a MELAS family with a coexisting mitochondrial 3243AG mutation

PubMed Central

Moteki, Hideaki; Azaiez, Hela; Booth, Kevin T; Hattori, Mitsuru; Sato, Ai; Sato, Yoshihiko; Motobayashi, Mitsuo; Sloan, Christina M; Kolbe, Diana L; Shearer, A Eliot; Smith, Richard J H; Usami, Shin-ichi

2015-01-01

Objective We present a family with a mitochondrial DNA 3243A>G mutation resulting in MELAS, of which some members have hearing loss where a novel mutation in the P2RX2 gene was identified. Methods One hundred ninety-four (194) Japanese subjects from unrelated families were enrolled in the study. Targeted genomic enrichment and massively parallel sequencing of all known non-syndromic hearing loss genes were performed to identify the genetic causes of hearing loss. Results A novel mutation in the P2RX2 gene, that corresponded to c.601G>A (p.Asp201Tyr) was identified. Two patients carried the mutation, and had severe SNHL, while other members with MELAS (who did not carry the P2RX2 mutation) had normal hearing. Conclusion This is the first case report of a diagnosis of hearing loss caused by P2RX2 mutation in patients with MELAS. A potential explanation is that decreasing ATP production due to MELAS with mitochondrial 3243A>G mutation might suppress activation of P2X2 receptors. We also suggest that hearing loss caused by the P2RX2 mutation might be influenced by the decrease in ATP production due to MELAS, and that nuclear genetic factors may play a modifying role in mitochondrial dysfunction. PMID:25788561

Molecular profiling of appendiceal epithelial tumors using massively parallel sequencing to identify somatic mutations.

PubMed

Liu, Xiaoying; Mody, Kabir; de Abreu, Francine B; Pipas, J Marc; Peterson, Jason D; Gallagher, Torrey L; Suriawinata, Arief A; Ripple, Gregory H; Hourdequin, Kathryn C; Smith, Kerrington D; Barth, Richard J; Colacchio, Thomas A; Tsapakos, Michael J; Zaki, Bassem I; Gardner, Timothy B; Gordon, Stuart R; Amos, Christopher I; Wells, Wendy A; Tsongalis, Gregory J

2014-07-01

Some epithelial neoplasms of the appendix, including low-grade appendiceal mucinous neoplasm and adenocarcinoma, can result in pseudomyxoma peritonei (PMP). Little is known about the mutational spectra of these tumor types and whether mutations may be of clinical significance with respect to therapeutic selection. In this study, we identified somatic mutations using the Ion Torrent AmpliSeq Cancer Hotspot Panel v2. Specimens consisted of 3 nonneoplastic retention cysts/mucocele, 15 low-grade mucinous neoplasms (LAMNs), 8 low-grade/well-differentiated mucinous adenocarcinomas with pseudomyxoma peritonei, and 12 adenocarcinomas with/without goblet cell/signet ring cell features. Barcoded libraries were prepared from up to 10 ng of extracted DNA and multiplexed on single 318 chips for sequencing. Data analysis was performed using Golden Helix SVS. Variants that remained after the analysis pipeline were individually interrogated using the Integrative Genomics Viewer. A single Janus kinase 3 (JAK3) mutation was detected in the mucocele group. Eight mutations were identified in the V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and GNAS complex locus (GNAS) genes among LAMN samples. Additional gene mutations were identified in the AKT1 (v-akt murine thymoma viral oncogene homolog 1), APC (adenomatous polyposis coli), JAK3, MET (met proto-oncogene), phosphatidylinositol-4,5-bisphosphate 3-kinase (PIK3CA), RB1 (retinoblastoma 1), STK11 (serine/threonine kinase 11), and tumor protein p53 (TP53) genes. Among the PMPs, 6 mutations were detected in the KRAS gene and also in the GNAS, TP53, and RB1 genes. Appendiceal cancers showed mutations in the APC, ATM (ataxia telangiectasia mutated), KRAS, IDH1 [isocitrate dehydrogenase 1 (NADP+)], NRAS [neuroblastoma RAS viral (v-ras) oncogene homolog], PIK3CA, SMAD4 (SMAD family member 4), and TP53 genes. Our results suggest molecular heterogeneity among epithelial tumors of the appendix. Next generation sequencing efforts

Biophysical Properties of 9 KCNQ1 Mutations Associated with Long QT Syndrome (LQTS)

PubMed Central

Yang, Tao; Chung, Seo-Kyung; Zhang, Wei; Mullins, Jonathan G.L.; McCulley, Caroline H.; Crawford, Jackie; MacCormick, Judith; Eddy, Carey-Anne; Shelling, Andrew N.; French, John K.; Yang, Ping; Skinner, Jonathan R.; Roden, Dan M.; Rees, Mark I.

2009-01-01

Background Inherited long QT syndrome (LQTS) is characterized by prolonged QT interval on the EKG, syncope and sudden death due to ventricular arrhythmia. Causative mutations occur mostly in cardiac potassium and sodium channel subunit genes. Confidence in mutation pathogenicity is usually reached through family genotype-phenotype tracking, control population studies, molecular modelling and phylogenetic alignments, however, biophysical testing offers a higher degree of validating evidence. Methods and Results By using in-vitro electrophysiological testing of transfected mutant and wild-type LQTS constructs into Chinese Hamster Ovary cells, we investigated the biophysical properties of 9 KCNQ1 missense mutations (A46T, T265I, F269S, A302V, G316E, F339S, R360G, H455Y, and S546L) identified in a New Zealand based LQTS screening programme. We demonstrate through electrophysiology and molecular modeling that seven of the missense mutations have profound pathological dominant negative loss-of-function properties confirming their likely disease-causing nature. This supports the use of these mutations in diagnostic family screening. Two mutations (A46T, T265I) show suggestive evidence of pathogenicity within the experimental limits of biophysical testing, indicating that these variants are disease-causing via delayed or fast activation kinetics. Further investigation of the A46T family has revealed an inconsistent co-segregation of the variant with the clinical phenotype. Conclusions Electrophysiological characterisation should be used to validate LQTS pathogenicity of novel missense channelopathies. When such results are inconclusive, great care should be taken with genetic counselling and screening of such families, and alternative disease causing mechanisms should be considered. PMID:19808498

Genetic Variation at 9p22.2 and Ovarian Cancer Risk for BRCA1 and BRCA2 Mutation Carriers

PubMed Central

Kartsonaki, Christiana; Gayther, Simon A.; Pharoah, Paul D. P.; Sinilnikova, Olga M.; Beesley, Jonathan; Chen, Xiaoqing; McGuffog, Lesley; Healey, Sue; Couch, Fergus J.; Wang, Xianshu; Fredericksen, Zachary; Peterlongo, Paolo; Manoukian, Siranoush; Peissel, Bernard; Zaffaroni, Daniela; Roversi, Gaia; Barile, Monica; Viel, Alessandra; Allavena, Anna; Ottini, Laura; Papi, Laura; Gismondi, Viviana; Capra, Fabio; Radice, Paolo; Greene, Mark H.; Mai, Phuong L.; Andrulis, Irene L.; Glendon, Gord; Ozcelik, Hilmi; Thomassen, Mads; Gerdes, Anne-Marie; Kruse, Torben A.; Cruger, Dorthe; Jensen, Uffe Birk; Caligo, Maria Adelaide; Olsson, Håkan; Kristoffersson, Ulf; Lindblom, Annika; Arver, Brita; Karlsson, Per; Stenmark Askmalm, Marie; Borg, Ake; Neuhausen, Susan L.; Ding, Yuan Chun; Nathanson, Katherine L.; Domchek, Susan M.; Jakubowska, Anna; Lubiński, Jan; Huzarski, Tomasz; Byrski, Tomasz; Gronwald, Jacek; Górski, Bohdan; Cybulski, Cezary; Dębniak, Tadeusz; Osorio, Ana; Durán, Mercedes; Tejada, Maria-Isabel; Benítez, Javier; Hamann, Ute; Rookus, Matti A.; Verhoef, Senno; Tilanus-Linthorst, Madeleine A.; Vreeswijk, Maaike P.; Bodmer, Danielle; Ausems, Margreet G. E. M.; van Os, Theo A.; Asperen, Christi J.; Blok, Marinus J.; Meijers-Heijboer, Hanne E. J.; Peock, Susan; Cook, Margaret; Oliver, Clare; Frost, Debra; Dunning, Alison M.; Evans, D. Gareth; Eeles, Ros; Pichert, Gabriella; Cole, Trevor; Hodgson, Shirley; Brewer, Carole; Morrison, Patrick J.; Porteous, Mary; Kennedy, M. John; Rogers, Mark T.; Side, Lucy E.; Donaldson, Alan; Gregory, Helen; Godwin, Andrew; Stoppa-Lyonnet, Dominique; Moncoutier, Virginie; Castera, Laurent; Mazoyer, Sylvie; Barjhoux, Laure; Bonadona, Valérie; Leroux, Dominique; Faivre, Laurence; Lidereau, Rosette; Nogues, Catherine; Bignon, Yves-Jean; Prieur, Fabienne; Collonge-Rame, Marie-Agnès; Venat-Bouvet, Laurence; Fert-Ferrer, Sandra; Miron, Alex; Buys, Saundra S.; Hopper, John L.; Daly, Mary B.; John, Esther M.; Terry, Mary Beth; Goldgar, David; Hansen, Thomas v. O.; Jønson, Lars; Ejlertsen, Bent; Agnarsson, Bjarni A.; Offit, Kenneth; Kirchhoff, Tomas; Vijai, Joseph; Dutra-Clarke, Ana V. C.; Przybylo, Jennifer A.; Montagna, Marco; Casella, Cinzia; Imyanitov, Evgeny N.; Janavicius, Ramunas; Blanco, Ignacio; Lázaro, Conxi; Moysich, Kirsten B.; Karlan, Beth Y.; Gross, Jenny; Beattie, Mary S.; Schmutzler, Rita; Wappenschmidt, Barbara; Meindl, Alfons; Ruehl, Ina; Fiebig, Britta; Sutter, Christian; Arnold, Norbert; Deissler, Helmut; Varon-Mateeva, Raymonda; Kast, Karin; Niederacher, Dieter; Gadzicki, Dorothea; Caldes, Trinidad; de la Hoya, Miguel; Nevanlinna, Heli; Aittomäki, Kristiina; Simard, Jacques; Soucy, Penny; Spurdle, Amanda B.; Holland, Helene; Chenevix-Trench, Georgia; Easton, Douglas F.; Antoniou, Antonis C.

2011-01-01

Background Germline mutations in the BRCA1 and BRCA2 genes are associated with increased risks of breast and ovarian cancers. Although several common variants have been associated with breast cancer susceptibility in mutation carriers, none have been associated with ovarian cancer susceptibility. A genome-wide association study recently identified an association between the rare allele of the single-nucleotide polymorphism (SNP) rs3814113 (ie, the C allele) at 9p22.2 and decreased risk of ovarian cancer for women in the general population. We evaluated the association of this SNP with ovarian cancer risk among BRCA1 or BRCA2 mutation carriers by use of data from the Consortium of Investigators of Modifiers of BRCA1/2. Methods We genotyped rs3814113 in 10 029 BRCA1 mutation carriers and 5837 BRCA2 mutation carriers. Associations with ovarian and breast cancer were assessed with a retrospective likelihood approach. All statistical tests were two-sided. Results The minor allele of rs3814113 was associated with a reduced risk of ovarian cancer among BRCA1 mutation carriers (per-allele hazard ratio of ovarian cancer = 0.78, 95% confidence interval = 0.72 to 0.85; P = 4.8 × 10-9) and BRCA2 mutation carriers (hazard ratio of ovarian cancer = 0.78, 95% confidence interval = 0.67 to 0.90; P = 5.5 × 10-4). This SNP was not associated with breast cancer risk among either BRCA1 or BRCA2 mutation carriers. BRCA1 mutation carriers with the TT genotype at SNP rs3814113 were predicted to have an ovarian cancer risk to age 80 years of 48%, and those with the CC genotype were predicted to have a risk of 33%. Conclusion Common genetic variation at the 9p22.2 locus was associated with decreased risk of ovarian cancer for carriers of a BRCA1 or BRCA2 mutation. PMID:21169536

The CDC Hemophilia B mutation project mutation list: a new online resource.

PubMed

Li, Tengguo; Miller, Connie H; Payne, Amanda B; Craig Hooper, W

2013-11-01

Hemophilia B (HB) is caused by mutations in the human gene F9. The mutation type plays a pivotal role in genetic counseling and prediction of inhibitor development. To help the HB community understand the molecular etiology of HB, we have developed a listing of all F9 mutations that are reported to cause HB based on the literature and existing databases. The Centers for Disease Control and Prevention (CDC) Hemophilia B Mutation Project (CHBMP) mutation list is compiled in an easily accessible format of Microsoft Excel and contains 1083 unique mutations that are reported to cause HB. Each mutation is identified using Human Genome Variation Society (HGVS) nomenclature standards. The mutation types and the predicted changes in amino acids, if applicable, are also provided. Related information including the location of mutation, severity of HB, the presence of inhibitor, and original publication reference are listed as well. Therefore, our mutation list provides an easily accessible resource for genetic counselors and HB researchers to predict inhibitors. The CHBMP mutation list is freely accessible at http://www.cdc.gov/hemophiliamutations.

PCSK 9 gain-of-function mutations (R496W and D374Y) and clinical cardiovascular characteristics in a cohort of Turkish patients with familial hypercholesterolemia.

PubMed

Kaya, Esra; Kayıkçıoğlu, Meral; Tetik Vardarlı, Aslı; Eroğlu, Zuhal; Payzın, Serdar; Can, Levent

2017-10-01

The molecular basis of the mutations in the PCSK9 gene that produces familial hypercholesterolemia (FH) in the Turkish population is unknown. This study was conducted to determine the presence of four different PCSK9 gain-of-function (GOF) mutations (F216L, R496W, S127R, and D374Y) in a group of patients with FH. A total of 80 consecutive patients with FH (mean age: 56±11 years; mean maximum LDL cholesterol: 251±76 mg/dL) were included in the study. Patients with FH were diagnosed according to the Dutch Lipid Clinic Network criteria based on serum cholesterol levels, personal and family histories of cardiovascular disease, tendon xanthomas, and genetic analysis. To identify F216L, R496W, S127R, and D374Y mutations of the PCSK9 gene, high-resolution melting analysis was performed on isolated DNAs. Of the 80 patients, there were 11 patients (13.8%) with PCSK9 GOF mutations. Detected mutations were D374Y mutation in four (5.0%) patients and R496W in seven patients (8.7%). Only one patient was homozygous for R496W mutation. The other two GOF mutations (S127R and F216 variants) were not detected. There was no significant difference with regard to demographic characteristics and CV disease risk factors and clinical course of the disease between the PCSK9 mutation-positive and PCSK9 mutation-negative groups. This is the first study from a Turkish FH cohort, revealing a higher frequency (approximately 14%) of two PCSK9 GOF mutations (D374Y and R496W) and a different disease course compared to the world literature.

Targeted mutations and MD simulations of a methanol-stable lipase YLIP9 from Yarrowia lipolytica MSR80 to develop a biodiesel enzyme.

PubMed

Syal, Poonam; Verma, Ved Vrat; Gupta, Rani

2017-11-01

Biodiesel, an environment friendly alternative for fuels, contains methyl esters of long-chain fatty acids. Our group has reported a methanol-stable YLIP9 from Yarrowia lipolytica MSR80 that shows poor catalysis of long-chain fatty acids. To shift its substrate specificity, residues within lid and binding pocket were identified for sequential mutations using YLIP2 as the template. Of the two point mutations (Glu116Leu and Ser119Val) introduced in the lid, the former mutation (YLIP9L1) increased the catalytic rate by ∼2-fold without any change in substrate specificity. In this mutant, six binding pocket residues (Bp2-Bp7) were further mutated to obtain six double mutants. YLIP9L1Bp3 showed significant shift in substrate specificity towards long-chain pNPesters with 11-fold increase in catalytic efficiency than YLIP9. Double mutations also led to increased thermostability and lowered activation energy of YLIP9L1Bp3 thereby shifting its optimum temperature from 60°C to 50°C. In silico molecular dynamics simulations revealed improved lid flexibility and increased catalytic triad volume in YLIP9L1Bp3. The enzyme YLIP9L1Bp3 was methanol-stable having selectivity for long-chain fatty acids with improved catalytic efficiency. Its application as a biodiesel enzyme was validated by transesterification of palm oil in presence of methanol, where it showed 8-fold increase in conversion of oil to methyl esters. Copyright © 2017 Elsevier B.V. All rights reserved.

Targeted next generation sequencing identifies novel NOTCH3 gene mutations in CADASIL diagnostics patients.

PubMed

Maksemous, Neven; Smith, Robert A; Haupt, Larisa M; Griffiths, Lyn R

2016-11-24

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic, hereditary, small vessel disease of the brain causing stroke and vascular dementia in adults. CADASIL has previously been shown to be caused by varying mutations in the NOTCH3 gene. The disorder is often misdiagnosed due to its significant clinical heterogeneic manifestation with familial hemiplegic migraine and several ataxia disorders as well as the location of the currently identified causative mutations. The aim of this study was to develop a new, comprehensive and efficient single assay strategy for complete molecular diagnosis of NOTCH3 mutations through the use of a custom next-generation sequencing (NGS) panel for improved routine clinical molecular diagnostic testing. Our custom NGS panel identified nine genetic variants in NOTCH3 (p.D139V, p.C183R, p.R332C, p.Y465C, p.C597W, p.R607H, p.E813E, p.C977G and p.Y1106C). Six mutations were stereotypical CADASIL mutations leading to an odd number of cysteine residues in one of the 34 NOTCH3 gene epidermal growth factor (EGF)-like repeats, including three new typical cysteine mutations identified in exon 11 (p.C597W; c.1791C>G); exon 18 (p.C977G; c.2929T>G) and exon 20 (p.Y1106C; c.3317A>G). Interestingly, a novel missense mutation in the CACNA1A gene was also identified in one CADASIL patient. All variants identified (novel and known) were further investigated using in silico bioinformatic analyses and confirmed through Sanger sequencing. NGS provides an improved and effective methodology for the diagnosis of CADASIL. The NGS approach reduced time and cost for comprehensive genetic diagnosis, placing genetic diagnostic testing within reach of more patients.

Systematic analysis of mutation distribution in three dimensional protein structures identifies cancer driver genes.

PubMed

Fujimoto, Akihiro; Okada, Yukinori; Boroevich, Keith A; Tsunoda, Tatsuhiko; Taniguchi, Hiroaki; Nakagawa, Hidewaki

2016-05-26

Protein tertiary structure determines molecular function, interaction, and stability of the protein, therefore distribution of mutation in the tertiary structure can facilitate the identification of new driver genes in cancer. To analyze mutation distribution in protein tertiary structures, we applied a novel three dimensional permutation test to the mutation positions. We analyzed somatic mutation datasets of 21 types of cancers obtained from exome sequencing conducted by the TCGA project. Of the 3,622 genes that had ≥3 mutations in the regions with tertiary structure data, 106 genes showed significant skew in mutation distribution. Known tumor suppressors and oncogenes were significantly enriched in these identified cancer gene sets. Physical distances between mutations in known oncogenes were significantly smaller than those of tumor suppressors. Twenty-three genes were detected in multiple cancers. Candidate genes with significant skew of the 3D mutation distribution included kinases (MAPK1, EPHA5, ERBB3, and ERBB4), an apoptosis related gene (APP), an RNA splicing factor (SF1), a miRNA processing factor (DICER1), an E3 ubiquitin ligase (CUL1) and transcription factors (KLF5 and EEF1B2). Our study suggests that systematic analysis of mutation distribution in the tertiary protein structure can help identify cancer driver genes.

Systematic analysis of mutation distribution in three dimensional protein structures identifies cancer driver genes

PubMed Central

Fujimoto, Akihiro; Okada, Yukinori; Boroevich, Keith A.; Tsunoda, Tatsuhiko; Taniguchi, Hiroaki; Nakagawa, Hidewaki

2016-01-01

Protein tertiary structure determines molecular function, interaction, and stability of the protein, therefore distribution of mutation in the tertiary structure can facilitate the identification of new driver genes in cancer. To analyze mutation distribution in protein tertiary structures, we applied a novel three dimensional permutation test to the mutation positions. We analyzed somatic mutation datasets of 21 types of cancers obtained from exome sequencing conducted by the TCGA project. Of the 3,622 genes that had ≥3 mutations in the regions with tertiary structure data, 106 genes showed significant skew in mutation distribution. Known tumor suppressors and oncogenes were significantly enriched in these identified cancer gene sets. Physical distances between mutations in known oncogenes were significantly smaller than those of tumor suppressors. Twenty-three genes were detected in multiple cancers. Candidate genes with significant skew of the 3D mutation distribution included kinases (MAPK1, EPHA5, ERBB3, and ERBB4), an apoptosis related gene (APP), an RNA splicing factor (SF1), a miRNA processing factor (DICER1), an E3 ubiquitin ligase (CUL1) and transcription factors (KLF5 and EEF1B2). Our study suggests that systematic analysis of mutation distribution in the tertiary protein structure can help identify cancer driver genes. PMID:27225414

A novel mutation R190H in the AT-hook 1 domain of MeCP2 identified in an atypical Rett syndrome.

PubMed

Zhou, Xiao; Liao, Yuangao; Xu, Miaojing; Ji, Zhong; Xu, Yunqi; Zhou, Liang; Wei, Xiaoming; Hu, Peiqian; Han, Peng; Yang, Fanghan; Pan, Suyue; Hu, Yafang

2017-10-10

Mutations in Methyl-CpG binding protein 2 ( MECP2 ) have been identified as the disease-causing mutations in Rett Syndrome (RTT). However, no mutation in the AT-hook 1 domain of MECP2 has been reported in RTT yet. The function of AT-hook 1 domain of MECP2 has not been described either. The clinical and radiological features of a girl with progressive hyperactivity and loss of acquired linguistic and motor functions were presented. Next generation sequencing was used to screen the causative gene. Effect of the mutant protein on histone 3 methylation was assessed in vitro experiment. The patient was diagnosed with an atypical RTT at the age of nine. Magnetic resonance imaging revealed a loss of whole-brain volume and abnormal myelination. Genetic analysis identified a de novo novel missense mutation of MECP2 (NM_004992, c.570G->A, p.Arg190His). This mutation is located in the AT-hook 1 domain of MeCP2 protein. Overexpression of the mutant MeCP2 in cultured neuroblastoma cells SH-SY5Y revealed increased level of dimethylated histone 3 lysine 9, a transcriptional repressor marker. A novel missense mutation in AT-hook 1 domain of MeCP2 was identified in a patient with atypical RTT. Clinical data and in vitro experiment result imply that R190H mutation in AT-hook1 may cause dysfunction of MeCP2 and be a pathogenic variant.

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii.

PubMed

Shen, Bang; Powell, Robin H; Behnke, Michael S

2017-06-22

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.

A homozygous CARD9 mutation in a family with susceptibility to fungal infections.

PubMed

Glocker, Erik-Oliver; Hennigs, Andre; Nabavi, Mohammad; Schäffer, Alejandro A; Woellner, Cristina; Salzer, Ulrich; Pfeifer, Dietmar; Veelken, Hendrik; Warnatz, Klaus; Tahami, Fariba; Jamal, Sarah; Manguiat, Annabelle; Rezaei, Nima; Amirzargar, Ali Akbar; Plebani, Alessandro; Hannesschläger, Nicole; Gross, Olaf; Ruland, Jürgen; Grimbacher, Bodo

2009-10-29

Chronic mucocutaneous candidiasis may be manifested as a primary immunodeficiency characterized by persistent or recurrent infections of the mucosa or the skin with candida species. Most cases are sporadic, but both autosomal dominant inheritance and autosomal recessive inheritance have been described. We performed genetic studies in 36 members of a large, consanguineous five-generation family, in which 4 members had recurrent fungal infections and an additional 3 members died during adolescence, 2 after invasive infection of the brain with candida species. All 36 family members were enrolled in the study, and 22 had blood samples taken for DNA analysis. Homozygosity mapping was used to locate the mutated gene. In the 4 affected family members (patients) and the 18 unaffected members we sequenced CARD9, the gene encoding the caspase recruitment domain-containing protein 9, carried out T-cell phenotyping, and performed functional studies, with the use of either leukocytes from the patients or a reconstituted murine model of the genetic defect. We found linkage (lod score, 3.6) to a genomic interval on chromosome 9q, including CARD9. All four patients had a homozygous point mutation in CARD9, resulting in a premature termination codon (Q295X). Healthy family members had wild-type expression of the CARD9 protein; the four patients lacked wild-type expression, which was associated with low numbers of Th17 cells (helper T cells producing interleukin-17). Functional studies based on genetic reconstitution of myeloid cells from Card9(-/-) mice showed that the Q295X mutation impairs innate signaling from the antifungal pattern-recognition receptor dectin-1. An autosomal recessive form of susceptibility to chronic mucocutaneous candidiasis is associated with homozygous mutations in CARD9. 2009 Massachusetts Medical Society

Whole Exome Sequencing Identifies de Novo Mutations in GATA6 Associated with Congenital Diaphragmatic Hernia

PubMed Central

Yu, Lan; Bennett, James T.; Wynn, Julia; Carvill, Gemma L.; Cheung, Yee Him; Shen, Yufeng; Mychaliska, George B.; Azarow, Kenneth S.; Crombleholme, Timothy M.; Chung, Dai H.; Potoka, Douglas; Warner, Brad W.; Bucher, Brian; Lim, Foong-Yen; Pietsch, John; Stolar, Charles; Aspelund, Gudrun; Arkovitz, Marc S.; Mefford, Heather; Chung, Wendy K.

2014-01-01

Background Congenital diaphragmatic hernia (CDH) is a common birth defect affecting 1 in 3,000 births. It is characterized by herniation of abdominal viscera through an incompletely formed diaphragm. Although chromosomal anomalies and mutations in several genes have been implicated, the cause for most patients is unknown. Methods We used whole exome sequencing in two families with CDH and congenital heart disease, and identified mutations in GATA6 in both. Results In the first family, we identified a de novo missense mutation (c.1366C>T, p.R456C) in a sporadic CDH patient with tetralogy of Fallot. In the second, a nonsense mutation (c.712G>T, p.G238*) was identified in two siblings with CDH and a large ventricular septal defect. The G238* mutation was inherited from their mother, who was clinically affected with congenital absence of the pericardium, patent ductus arteriosus, and intestinal malrotation. Deep sequencing of blood and saliva derived DNA from the mother suggested somatic mosaicism as an explanation for her milder phenotype, with only approximately 15% mutant alleles. To determine the frequency of GATA6 mutations in CDH, we sequenced the gene in 378 patients with CDH. We identified one additional de novo mutation (c.1071delG, p.V358Cfs34*). Conclusions Mutations in GATA6 have been previously associated with pancreatic agenesis and congenital heart disease. We conclude that, in addition to the heart and the pancreas, GATA6 is involved in development of two additional organs, the diaphragm and the pericardium. In addition we have shown that de novo mutations can contribute to the development of CDH, a common birth defect. PMID:24385578

Human Nonsyndromic Hereditary Deafness DFNA17 Is Due to a Mutation in Nonmuscle Myosin MYH9

PubMed Central

Lalwani, Anil K.; Goldstein, Jayne A.; Kelley, Michael J.; Luxford, William; Castelein, Caley M.; Mhatre, Anand N.

2000-01-01

The authors had previously mapped a new locus—DFNA17, for nonsyndromic hereditary hearing impairment—to chromosome 22q12.2-q13.3. DFNA17 spans a 17- to 23-cM region, and MYH9, a nonmuscle–myosin heavy-chain gene, is located within the linked region. Because of the importance of myosins in hearing, MYH9 was tested as a candidate gene for DFNA17. Expression of MYH9 in the rat cochlea was confirmed using reverse transcriptase–PCR and immunohistochemistry. MYH9 was immunolocalized in the organ of Corti, the subcentral region of the spiral ligament, and the Reissner membrane. Sequence analysis of MYH9 in a family with DFNA17 identified, at nucleotide 2114, a G→A transposition that cosegregated with the inherited autosomal dominant hearing impairment. This missense mutation changes codon 705 from an invariant arginine (R) to histidine (H), R705H, within a highly conserved SH1 linker region. Previous studies have shown that modification of amino acid residues within the SH1 helix causes dysfunction of the ATPase activity of the motor domain in myosin II. Both the precise role of MYH9 in the cochlea and the mechanism by which the R705H mutation leads to the DFNA17 phenotype (progressive hearing impairment and cochleosaccular degeneration) remain to be elucidated. PMID:11023810

CRISPR/Cas9-mediated ASXL1 mutations in U937 cells disrupt myeloid differentiation

PubMed Central

Wu, Zhi-Jie; Zhao, Xin; Banaszak, Lauren G.; Gutierrez-Rodrigues, Fernanda; Keyvanfar, Keyvan; Gao, Shou-Guo; Raffo, Diego Quinones; Kajigaya, Sachiko; Young, Neal S.

2018-01-01

Additional sex combs-like 1 (ASXL1) is a well-known tumor suppressor gene and epigenetic modifier. ASXL1 mutations are frequent in myeloid malignances; these mutations are risk factors for the development of myelodysplasia and also appear as small clones during normal aging. ASXL1 appears to act as an epigenetic regulator of cell survival and myeloid differentiation; however, the molecular mechanisms underlying the malignant transformation of cells with ASXL1 mutations are not well defined. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome editing, heterozygous and homozygous ASXL1 mutations were introduced into human U937 leukemic cells. Comparable cell growth and cell cycle progression were observed between wild-type (WT) and ASXL1-mutated U937 cells. Drug-induced cytotoxicity, as measured by growth inhibition and apoptosis in the presence of the cell-cycle active agent 5-fluorouracil, was variable among the mutated clones but was not significantly different from WT cells. In addition, ASXL1-mutated cells exhibited defects in monocyte/macrophage differentiation. Transcriptome analysis revealed that ASXL1 mutations altered differentiation of U937 cells by disturbing genes involved in myeloid differentiation, including cytochrome B-245 β chain and C-type lectin domain family 5, member A. Dysregulation of numerous gene sets associated with cell death and survival were also observed in ASXL1-mutated cells. These data provide evidence regarding the underlying molecular mechanisms induced by mutated ASXL1 in leukemogenesis. PMID:29532865

Association of KIT exon 9 mutations with nongastric primary site and aggressive behavior: KIT mutation analysis and clinical correlates of 120 gastrointestinal stromal tumors.

PubMed

Antonescu, Cristina R; Sommer, Gunhild; Sarran, Lisa; Tschernyavsky, Sylvia J; Riedel, Elyn; Woodruff, James M; Robson, Mark; Maki, Robert; Brennan, Murray F; Ladanyi, Marc; DeMatteo, Ronald P; Besmer, Peter

2003-08-15

Activating mutations of the KIT juxtamembrane region are the most common genetic events in gastrointestinal stromal tumors (GISTs) and have been noted as independent prognostic factors. The impact of KIT mutation in other regions, such as the extracellular or kinase domains, is not well-defined and fewer than 30 cases have been published to date. One hundred twenty GISTs, confirmed by KIT immunoreactivity, were evaluated for the presence of KIT exon 9, 11, 13, and 17 mutations. The relation between the presence/type of KIT mutation and clinicopathological factors was analyzed using Fisher's exact test and log-rank test. Forty-four % of the tumors were located in the stomach, 47% in the small bowel, 6% in the rectum, and 3% in the retroperitoneum. Overall, KIT mutations were detected in 78% of patients as follows: 67% in exon 11, 11% in exon 9, and none in exon 13 or 17. The types of KIT exon 11 mutations were heterogeneous and clustered in the classic "hot spot" at the 5' end of exon 11. Seven % of cases showed internal tandem duplications (ITD) at the 3' end of exon 11, in a region that we designate as a second hot spot for KIT mutations. Interestingly, these cases were associated with: female predominance, stomach location, occurrence in older patients, and favorable outcome. There were significant associations between exon 9 mutations and large tumor size (P < 0.001) and extragastric location (P = 0.02). Ten of these 13 patients with more than 1-year follow-up have developed recurrent disease. Most KIT-expressing GISTs show KIT mutations that are preferentially located within the classic hot spot of exon 11. In addition, we found an association between a second hot spot at the 3'end of exon 11, characterized by ITDs, and a subgroup of clinically indolent gastric GISTs in older females. KIT exon 9 mutations seem to define a distinct subset of GISTs, located predominantly in the small bowel and associated with an unfavorable clinical course.

Targeted next generation sequencing identifies functionally deleterious germline mutations in novel genes in early-onset/familial prostate cancer.

PubMed

Paulo, Paula; Maia, Sofia; Pinto, Carla; Pinto, Pedro; Monteiro, Augusta; Peixoto, Ana; Teixeira, Manuel R

2018-04-01

Considering that mutations in known prostate cancer (PrCa) predisposition genes, including those responsible for hereditary breast/ovarian cancer and Lynch syndromes, explain less than 5% of early-onset/familial PrCa, we have sequenced 94 genes associated with cancer predisposition using next generation sequencing (NGS) in a series of 121 PrCa patients. We found monoallelic truncating/functionally deleterious mutations in seven genes, including ATM and CHEK2, which have previously been associated with PrCa predisposition, and five new candidate PrCa associated genes involved in cancer predisposing recessive disorders, namely RAD51C, FANCD2, FANCI, CEP57 and RECQL4. Furthermore, using in silico pathogenicity prediction of missense variants among 18 genes associated with breast/ovarian cancer and/or Lynch syndrome, followed by KASP genotyping in 710 healthy controls, we identified "likely pathogenic" missense variants in ATM, BRIP1, CHEK2 and TP53. In conclusion, this study has identified putative PrCa predisposing germline mutations in 14.9% of early-onset/familial PrCa patients. Further data will be necessary to confirm the genetic heterogeneity of inherited PrCa predisposition hinted in this study.

Targeted next-generation sequencing analysis identifies novel mutations in families with severe familial exudative vitreoretinopathy.

PubMed

Huang, Xiao-Yan; Zhuang, Hong; Wu, Ji-Hong; Li, Jian-Kang; Hu, Fang-Yuan; Zheng, Yu; Tellier, Laurent Christian Asker M; Zhang, Sheng-Hai; Gao, Feng-Juan; Zhang, Jian-Guo; Xu, Ge-Zhi

2017-01-01

Familial exudative vitreoretinopathy (FEVR) is a genetically and clinically heterogeneous disease, characterized by failure of vascular development of the peripheral retina. The symptoms of FEVR vary widely among patients in the same family, and even between the two eyes of a given patient. This study was designed to identify the genetic defect in a patient cohort of ten Chinese families with a definitive diagnosis of FEVR. To identify the causative gene, next-generation sequencing (NGS)-based target capture sequencing was performed. Segregation analysis of the candidate variant was performed in additional family members by using Sanger sequencing and quantitative real-time PCR (QPCR). Of the cohort of ten FEVR families, six pathogenic variants were identified, including four novel and two known heterozygous mutations. Of the variants identified, four were missense variants, and two were novel heterozygous deletion mutations [ LRP5 , c.4053 DelC (p.Ile1351IlefsX88); TSPAN12 , EX8Del]. The two novel heterozygous deletion mutations were not observed in the control subjects and could give rise to a relatively severe FEVR phenotype, which could be explained by the protein function prediction. We identified two novel heterozygous deletion mutations [ LRP5 , c.4053 DelC (p.Ile1351IlefsX88); TSPAN12 , EX8Del] using targeted NGS as a causative mutation for FEVR. These genetic deletion variations exhibit a severe form of FEVR, with tractional retinal detachments compared with other known point mutations. The data further enrich the mutation spectrum of FEVR and enhance our understanding of genotype-phenotype correlations to provide useful information for disease diagnosis, prognosis, and effective genetic counseling.

Targeted next-generation sequencing analysis identifies novel mutations in families with severe familial exudative vitreoretinopathy

PubMed Central

Huang, Xiao-Yan; Zhuang, Hong; Wu, Ji-Hong; Li, Jian-Kang; Hu, Fang-Yuan; Zheng, Yu; Tellier, Laurent Christian Asker M.; Zhang, Sheng-Hai; Gao, Feng-Juan; Zhang, Jian-Guo

2017-01-01

Purpose Familial exudative vitreoretinopathy (FEVR) is a genetically and clinically heterogeneous disease, characterized by failure of vascular development of the peripheral retina. The symptoms of FEVR vary widely among patients in the same family, and even between the two eyes of a given patient. This study was designed to identify the genetic defect in a patient cohort of ten Chinese families with a definitive diagnosis of FEVR. Methods To identify the causative gene, next-generation sequencing (NGS)-based target capture sequencing was performed. Segregation analysis of the candidate variant was performed in additional family members by using Sanger sequencing and quantitative real-time PCR (QPCR). Results Of the cohort of ten FEVR families, six pathogenic variants were identified, including four novel and two known heterozygous mutations. Of the variants identified, four were missense variants, and two were novel heterozygous deletion mutations [LRP5, c.4053 DelC (p.Ile1351IlefsX88); TSPAN12, EX8Del]. The two novel heterozygous deletion mutations were not observed in the control subjects and could give rise to a relatively severe FEVR phenotype, which could be explained by the protein function prediction. Conclusions We identified two novel heterozygous deletion mutations [LRP5, c.4053 DelC (p.Ile1351IlefsX88); TSPAN12, EX8Del] using targeted NGS as a causative mutation for FEVR. These genetic deletion variations exhibit a severe form of FEVR, with tractional retinal detachments compared with other known point mutations. The data further enrich the mutation spectrum of FEVR and enhance our understanding of genotype–phenotype correlations to provide useful information for disease diagnosis, prognosis, and effective genetic counseling. PMID:28867931

A Mismatch EndoNuclease Array-Based Methodology (MENA) for Identifying Known SNPs or Novel Point Mutations.

PubMed

Comeron, Josep M; Reed, Jordan; Christie, Matthew; Jacobs, Julia S; Dierdorff, Jason; Eberl, Daniel F; Manak, J Robert

2016-04-05

Accurate and rapid identification or confirmation of single nucleotide polymorphisms (SNPs), point mutations and other human genomic variation facilitates understanding the genetic basis of disease. We have developed a new methodology (called MENA (Mismatch EndoNuclease Array)) pairing DNA mismatch endonuclease enzymology with tiling microarray hybridization in order to genotype both known point mutations (such as SNPs) as well as identify previously undiscovered point mutations and small indels. We show that our assay can rapidly genotype known SNPs in a human genomic DNA sample with 99% accuracy, in addition to identifying novel point mutations and small indels with a false discovery rate as low as 10%. Our technology provides a platform for a variety of applications, including: (1) genotyping known SNPs as well as confirming newly discovered SNPs from whole genome sequencing analyses; (2) identifying novel point mutations and indels in any genomic region from any organism for which genome sequence information is available; and (3) screening panels of genes associated with particular diseases and disorders in patient samples to identify causative mutations. As a proof of principle for using MENA to discover novel mutations, we report identification of a novel allele of the beethoven (btv) gene in Drosophila, which encodes a ciliary cytoplasmic dynein motor protein important for auditory mechanosensation.

Computational engineering of cellulase Cel9A-68 functional motions through mutations in its linker region.

PubMed

Costa, M G S; Silva, Y F; Batista, P R

2018-03-14

Microbial cellulosic degradation by cellulases has become a complementary approach for biofuel production. However, its efficiency is hindered by the recalcitrance of cellulose fibres. In this context, computational protein design methods may offer an efficient way to obtain variants with improved enzymatic activity. Cel9A-68 is a cellulase from Thermobifida fusca that is still active at high temperatures. In a previous work, we described a collective bending motion, which governs the overall cellulase dynamics. This movement promotes the approximation of its CBM and CD structural domains (that are connected by a flexible linker). We have identified two residues (G460 and P461) located at the linker that act as a hinge point. Herein, we applied a new level of protein design, focusing on the modulation of this collective motion to obtain cellulase variants with enhanced functional dynamics. We probed whether specific linker mutations would affect Cel9A-68 dynamics through computational simulations. We assumed that P461G and G460+ (with an extra glycine) constructs would present enhanced interdomain motions, while the G460P mutant would be rigid. From our results, the P461G mutation resulted in a broader exploration of the conformational space, as confirmed by clustering and free energy analyses. The WT enzyme was the most rigid system. However, G460P and P460+ explored distinct conformational states described by opposite directions of low-frequency normal modes; they sampled preferentially closed and open conformations, respectively. Overall, we highlight two significant findings: (i) all mutants explored larger conformational spaces than the WT; (ii) the selection of distinct conformational populations was intimately associated with the mutation considered. Thus, the engineering of Cel9A-68 motions through linker mutations may constitute an efficient way to improve cellulase activity, facilitating the disruption of cellulose fibres.

Screening for ATM Mutations in an African-American Population to Identify a Predictor of Breast Cancer Susceptibility

DTIC Science & Technology

2006-07-01

ATM genetic variant identified affects radiosensitivity and levels of the protein encoded by the ATM gene for each mutation examined. 15. SUBJECT...women without breast cancer. An additional objective is to determine the functional impact upon the protein encoded by the ATM gene for each mutation ...each ATM variant identified affects radiosensitivity and levels of the protein encoded by the ATM gene for mutations identified. Body STATEMENT

CTNNB1 (beta-catenin) mutation identifies low grade, early stage endometrial cancer patients at increased risk of recurrence.

PubMed

Kurnit, Katherine C; Kim, Grace N; Fellman, Bryan M; Urbauer, Diana L; Mills, Gordon B; Zhang, Wei; Broaddus, Russell R

2017-07-01

Although the majority of low grade, early stage endometrial cancer patients will have good survival outcomes with surgery alone, those patients who do recur tend to do poorly. Optimal identification of the subset of patients who are at high risk of recurrence and would benefit from adjuvant treatment has been difficult. The purpose of this study was to evaluate the impact of somatic tumor mutation on survival outcomes in this patient population. For this study, low grade was defined as endometrioid FIGO grades 1 or 2, while early stage was defined as endometrioid stages I or II (disease confined to the uterus). Next-generation sequencing was performed using panels comprised of 46-200 genes. Recurrence-free and overall survival was compared across gene mutational status in both univariate and multivariate analyses. In all, 342 patients were identified, 245 of which had endometrioid histology. For grades 1-2, stages I-II endometrioid endometrial cancer patients, age (HR 1.07, 95% CI 1.03-1.10), CTNNB1 mutation (HR 5.97, 95% CI 2.69-13.21), and TP53 mutation (HR 4.07, 95% CI 1.57-10.54) were associated with worse recurrence-free survival on multivariate analysis. When considering endometrioid tumors of all grades and stages, CTNNB1 mutant tumors were associated with significantly higher rates of grades 1-2 disease, lower rates of deep myometrial invasion, and lower rates of lymphatic/vascular space invasion. When both TP53 and CTNNB1 mutations were considered, presence of either TP53 mutation or CTNNB1 mutation remained a statistically significant predictor of recurrence-free survival on multivariate analysis and was associated with a more precise confidence interval (HR 4.69, 95% CI 2.38-9.24). Thus, mutational analysis of a 2 gene panel of CTNNB1 and TP53 can help to identify a subset of low grade, early stage endometrial cancer patients who are at high risk of recurrence.

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

PubMed

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

2017-01-01

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

Exome Sequencing Identifies Biallelic MSH3 Germline Mutations as a Recessive Subtype of Colorectal Adenomatous Polyposis.

PubMed

Adam, Ronja; Spier, Isabel; Zhao, Bixiao; Kloth, Michael; Marquez, Jonathan; Hinrichsen, Inga; Kirfel, Jutta; Tafazzoli, Aylar; Horpaopan, Sukanya; Uhlhaas, Siegfried; Stienen, Dietlinde; Friedrichs, Nicolaus; Altmüller, Janine; Laner, Andreas; Holzapfel, Stefanie; Peters, Sophia; Kayser, Katrin; Thiele, Holger; Holinski-Feder, Elke; Marra, Giancarlo; Kristiansen, Glen; Nöthen, Markus M; Büttner, Reinhard; Möslein, Gabriela; Betz, Regina C; Brieger, Angela; Lifton, Richard P; Aretz, Stefan

2016-08-04

In ∼30% of families affected by colorectal adenomatous polyposis, no germline mutations have been identified in the previously implicated genes APC, MUTYH, POLE, POLD1, and NTHL1, although a hereditary etiology is likely. To uncover further genes with high-penetrance causative mutations, we performed exome sequencing of leukocyte DNA from 102 unrelated individuals with unexplained adenomatous polyposis. We identified two unrelated individuals with differing compound-heterozygous loss-of-function (LoF) germline mutations in the mismatch-repair gene MSH3. The impact of the MSH3 mutations (c.1148delA, c.2319-1G>A, c.2760delC, and c.3001-2A>C) was indicated at the RNA and protein levels. Analysis of the diseased individuals' tumor tissue demonstrated high microsatellite instability of di- and tetranucleotides (EMAST), and immunohistochemical staining illustrated a complete loss of nuclear MSH3 in normal and tumor tissue, confirming the LoF effect and causal relevance of the mutations. The pedigrees, genotypes, and frequency of MSH3 mutations in the general population are consistent with an autosomal-recessive mode of inheritance. Both index persons have an affected sibling carrying the same mutations. The tumor spectrum in these four persons comprised colorectal and duodenal adenomas, colorectal cancer, gastric cancer, and an early-onset astrocytoma. Additionally, we detected one unrelated individual with biallelic PMS2 germline mutations, representing constitutional mismatch-repair deficiency. Potentially causative variants in 14 more candidate genes identified in 26 other individuals require further workup. In the present study, we identified biallelic germline MSH3 mutations in individuals with a suspected hereditary tumor syndrome. Our data suggest that MSH3 mutations represent an additional recessive subtype of colorectal adenomatous polyposis. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Recurrent TERT promoter mutations identified in a large-scale study of multiple tumor types are associated with increased TERT expression and telomerase activation

PubMed Central

Huang, Dong-Sheng; Wang, Zhaohui; He, Xu-Jun; Diplas, Bill H.; Yang, Rui; Killela, Patrick J.; Liang, Junbo; Meng, Qun; Ye, Zai-Yuan; Wang, Wei; Jiang, Xiao-Ting; Xu, Li; He, Xiang-Lei; Zhao, Zhong-Sheng; Xu, Wen-Juan; Wang, Hui-Ju; Ma, Ying-Yu; Xia, Ying-Jie; Li, Li; Zhang, Ru-Xuan; Jin, Tao; Zhao, Zhong-Kuo; Xu, Ji; Yu, Sheng; Wu, Fang; Wang, Si-Zhen; Jiao, Yu-Chen; Yan, Hai; Tao, Hou-Quan

2015-01-01

Background Several somatic mutation hotspots were recently identified in the TERT promoter region in human cancers. Large scale studies of these mutations in multiple tumor types are limited, in particular in Asian populations. This study aimed to: analyze TERT promoter mutations in multiple tumor types in a large Chinese patient cohort, investigate novel tumor types and assess the functional significance of the mutations. Methods TERT promoter mutation status was assessed by Sanger sequencing for 13 different tumor types and 799 tumor tissues from Chinese cancer patients. Thymic epithelial tumors, gastrointestinal leiomyoma, and gastric schwannoma were included, for which the TERT promoter has not been previously sequenced. Functional studies included TERT expression by RT-qPCR, telomerase activity by the TRAP assay, and promoter activity by the luciferase reporter assay. Results TERT promoter mutations were highly frequent in glioblastoma (83.9%), urothelial carcinoma (64.5%), oligodendroglioma (70.0%), medulloblastoma (33.3%), and hepatocellular carcinoma (31.4%). C228T and C250T were the most common mutations. In urothelial carcinoma, several novel rare mutations were identified. TERT promoter mutations were absent in GIST, thymic epithelial tumors, gastrointestinal leiomyoma, gastric schwannoma, cholangiocarcinoma, gastric and pancreatic cancer. TERT promoter mutations highly correlated with upregulated TERT mRNA expression and telomerase activity in adult gliomas. These mutations differentially enhanced the transcriptional activity of the TERT core promoter. Conclusions TERT promoter mutations are frequent in multiple tumor types and have similar distributions in Chinese cancer patients. The functional significance of these mutations reflect the importance to telomere maintenance and hence tumorigenesis, making them potential therapeutic targets. PMID:25843513

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

PubMed

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

2016-12-01

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

Mutations in the newly identified RAX regulatory sequence are not a frequent cause of micro/anophthalmia.

PubMed

Chassaing, Nicolas; Vigouroux, Adeline; Calvas, Patrick

2009-06-01

Microphthalmia and anophthalmia are at the severe end of the spectrum of abnormalities in ocular development. A few genes (SOX2, OTX2, RAX, and CHX10) have been implicated in isolated micro/anophthalmia, but causative mutations of these genes explain less than a quarter of these developmental defects. A specifically conserved SOX2/OTX2-mediated RAX expression regulatory sequence has recently been identified. We postulated that mutations in this sequence could lead to micro/anophthalmia, and thus we performed molecular screening of this regulatory element in patients suffering from micro/anophthalmia. Fifty-one patients suffering from nonsyndromic microphthalmia (n = 40) or anophthalmia (n = 11) were included in this study after negative molecular screening for SOX2, OTX2, RAX, and CHX10 mutations. Mutation screening of the RAX regulatory sequence was performed by direct sequencing for these patients. No mutations were identified in the highly conserved RAX regulatory sequence in any of the 51 patients. Mutations in the newly identified RAX regulatory sequence do not represent a frequent cause of nonsyndromic micro/anophthalmia.

A novel dysfunctional germline P53 mutation identified in a family with Li-Fraumeni syndrome.

PubMed

Ji, Min; Wang, Lin; Shao, Yuguo; Cao, Wei; Xu, Ting; Chen, Shujie; Wang, Zhiwei; He, Qi; Yang, Kuo

2018-01-01

Li-Fraumeni Syndrome (LFS), which is a rare dominantly inherited cancer predisposition syndrome, is associated with germline P53 mutations. Mutations of the tumor suppressor protein P53 are associated with more than 50% of human cancers; however, almost 30% of P53 mutations occur rarely and this has raised questions about their significance. It therefore appeared of particular interest that we identified a novel mutation in a patient suffering from breast cancer and fulfilling the diagnostic criteria of LFS. In this study, a patient with remarkable family history developed breast cancer and was diagnosed with LFS. By performing next-generation sequencing on the patient and subsequent verification by Sanger sequencing among other family members, a new germ-line P53 replication error, a trinucleotide repeat mutation in the coding region, was identified in two generations of this Li-Fraumeni family.

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.

Characterization of an FTLD-PDB family with the coexistence of SQSTM1 mutation and hexanucleotide (G₄C₂) repeat expansion in C9orf72 gene.

PubMed

Almeida, Maria Rosário; Letra, Liliana; Pires, Paula; Santos, Ana; Rebelo, Olinda; Guerreiro, Rita; van der Zee, Julie; Van Broeckhoven, Christine; Santana, Isabel

2016-04-01

The C9orf72 expansion is considered a major genetic cause of familial frontotemporal dementia (FTD) in several patients' cohorts. Interestingly, C9orf72 expansion carriers, present also abundant neuronal p62-positive inclusions. Although p62/SQSTM1 mutations were initially associated with Paget disease of bone (PDB), they have been also identified in FTD. We describe an FTD-PDB family in which the proband presented with behavioral FTD phenotype and concomitant Paget disease. The molecular genetic analysis revealed the co-occurrence of 2 mutations; the pathogenic C9orf72 expansion and p.P392L heterozygous missense mutation in SQSTM1 gene. Amongst the 6 family members analyzed, the p.P392L SQSTM1 mutation segregated as expected with PDB, whereas the C9orf72 expansion segregated with frontal cognitive impairment or dementia in all but one carrier. The coexistence of these conditions could be underestimated since neither patients with FTD nor patients with PDB undergo bone scintigraphy or cognitive assessment, respectively. The number of cases with double mutations could also be over looked as the molecular strategy adopted in most laboratories ends with the identification of one pathogenic mutation in one of the known causative genes. Therefore, we advocate for further clinical and molecular evaluation in suspect cases. Copyright © 2016 Elsevier Inc. All rights reserved.

Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus.

PubMed

Merkle, Florian T; Neuhausser, Werner M; Santos, David; Valen, Eivind; Gagnon, James A; Maas, Kristi; Sandoe, Jackson; Schier, Alexander F; Eggan, Kevin

2015-05-12

The CRISPR-Cas9 system has the potential to revolutionize genome editing in human pluripotent stem cells (hPSCs), but its advantages and pitfalls are still poorly understood. We systematically tested the ability of CRISPR-Cas9 to mediate reporter gene knockin at 16 distinct genomic sites in hPSCs. We observed efficient gene targeting but found that targeted clones carried an unexpectedly high frequency of insertion and deletion (indel) mutations at both alleles of the targeted gene. These indels were induced by Cas9 nuclease, as well as Cas9-D10A single or dual nickases, and often disrupted gene function. To overcome this problem, we designed strategies to physically destroy or separate CRISPR target sites at the targeted allele and developed a bioinformatic pipeline to identify and eliminate clones harboring deleterious indels at the other allele. This two-pronged approach enables the reliable generation of knockin hPSC reporter cell lines free of unwanted mutations at the targeted locus. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

PubMed Central

Shen, Bang; Powell, Robin H.; Behnke, Michael S.

2017-01-01

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites. PMID:28671645

Coding Sequence Mutations Identified in MYH7, TNNT2, SCN5A, CSRP3, LBD3, and TCAP from 313 Patients with Familial or Idiopathic Dilated Cardiomyopathy

PubMed Central

Hershberger, Ray E.; Parks, Sharie B.; Kushner, Jessica D.; Li, Duanxiang; Ludwigsen, Susan; Jakobs, Petra; Nauman, Deirdre; Burgess, Donna; Partain, Julie; Litt, Michael

2008-01-01

Abstract Background: More than 20 genes have been reported to cause idiopathic and familial dilated cardiomyopathy (IDC/FDC), but the frequency of genetic causation remains poorly understood. Methods and Results: Blood samples were collected and DNA prepared from 313 patients, 183 with FDC and 130 with IDC. Genomic DNA underwent bidirectional sequencing of six genes, and mutation carriers were followed up by evaluation of additional family members. We identified in 36 probands, 31 unique protein‐altering variants (11.5% overall) that were not identified in 253 control subjects (506 chromosomes). These included 13 probands (4.2%) with 12 β‐myosin heavy chain (MYH7) mutations, nine probands (2.9%) with six different cardiac troponin T (TNNT2) mutations, eight probands (2.6%) carrying seven different cardiac sodium channel (SCN5A) mutations, three probands (1.0%) with three titin‐cap or telethonin (TCAP) mutations, three probands (1.0%) with two LIM domain binding 3 (LDB3) mutations, and one proband (0.3%) with a muscle LIM protein (CSRP3) mutation. Four nucleotide changes did not segregate with phentoype and/or did not alter a conserved amino acid and were therefore considered unlikely to be disease‐causing. Mutations in 11 probands were assessed as likely disease‐causing, and in 21 probands were considered possibly disease‐causing. These 32 probands included 14 of the 130 with IDC (10.8%) and 18 of the 183 with FDC (9.8%) Conclusions: Mutations of these six genes each account for a small fraction of the genetic cause of FDC/IDC. The frequency of possible or likely disease‐causing mutations in these genes is similar for IDC and FDC. PMID:19412328

Prevailing PA Mutation K356R in Avian Influenza H9N2 Virus Increases Mammalian Replication and Pathogenicity.

PubMed

Xu, Guanlong; Zhang, Xuxiao; Gao, Weihua; Wang, Chenxi; Wang, Jinliang; Sun, Honglei; Sun, Yipeng; Guo, Lu; Zhang, Rui; Chang, Kin-Chow; Liu, Jinhua; Pu, Juan

2016-09-15

Adaptation of the viral polymerase complex comprising PB1, PB2, and PA is necessary for efficient influenza A virus replication in new host species. We found that PA mutation K356R (PA-K356R) has become predominant since 2014 in avian H9N2 viruses in China as with seasonal human H1N1 viruses. The same mutation is also found in most human isolates of emergent avian H7N9 and H10N8 viruses whose six internal gene segments are derived from the H9N2 virus. We further demonstrated the mammalian adaptive functionality of the PA-K356R mutation. Avian H9N2 virus with the PA-K356R mutation in human A549 cells showed increased nuclear accumulation of PA and increased viral polymerase activity that resulted in elevated levels of viral transcription and virus output. The same mutant virus in mice also enhanced virus replication and caused lethal infection. In addition, combined mutation of PA-K356R and PB2-E627K, a well-known mammalian adaptive marker, in the H9N2 virus showed further cooperative increases in virus production and severity of infection in vitro and in vivo In summary, PA-K356R behaves as a novel mammalian tropism mutation, which, along with other mutations such as PB2-E627K, might render avian H9N2 viruses adapted for human infection. Mutations of the polymerase complex (PB1, PB2, and PA) of influenza A virus are necessary for viral adaptation to new hosts. This study reports a novel and predominant mammalian adaptive mutation, PA-K356R, in avian H9N2 viruses and human isolates of emergent H7N9 and H10N8 viruses. We found that PA-356R in H9N2 viruses causes significant increases in virus replication and severity of infection in human cells and mice and that PA-K356R cooperates with the PB2-E627K mutation, a well-characterized human adaptive marker, to exacerbate mammalian infection in vitro and in vivo Therefore, the PA-K356R mutation is a significant adaptation in H9N2 viruses and related H7N9 and H10N8 reassortants toward human infectivity. Copyright © 2016

Prevailing PA Mutation K356R in Avian Influenza H9N2 Virus Increases Mammalian Replication and Pathogenicity

PubMed Central

Xu, Guanlong; Zhang, Xuxiao; Gao, Weihua; Wang, Chenxi; Wang, Jinliang; Sun, Honglei; Sun, Yipeng; Guo, Lu; Zhang, Rui; Chang, Kin-Chow; Liu, Jinhua

2016-01-01

ABSTRACT Adaptation of the viral polymerase complex comprising PB1, PB2, and PA is necessary for efficient influenza A virus replication in new host species. We found that PA mutation K356R (PA-K356R) has become predominant since 2014 in avian H9N2 viruses in China as with seasonal human H1N1 viruses. The same mutation is also found in most human isolates of emergent avian H7N9 and H10N8 viruses whose six internal gene segments are derived from the H9N2 virus. We further demonstrated the mammalian adaptive functionality of the PA-K356R mutation. Avian H9N2 virus with the PA-K356R mutation in human A549 cells showed increased nuclear accumulation of PA and increased viral polymerase activity that resulted in elevated levels of viral transcription and virus output. The same mutant virus in mice also enhanced virus replication and caused lethal infection. In addition, combined mutation of PA-K356R and PB2-E627K, a well-known mammalian adaptive marker, in the H9N2 virus showed further cooperative increases in virus production and severity of infection in vitro and in vivo. In summary, PA-K356R behaves as a novel mammalian tropism mutation, which, along with other mutations such as PB2-E627K, might render avian H9N2 viruses adapted for human infection. IMPORTANCE Mutations of the polymerase complex (PB1, PB2, and PA) of influenza A virus are necessary for viral adaptation to new hosts. This study reports a novel and predominant mammalian adaptive mutation, PA-K356R, in avian H9N2 viruses and human isolates of emergent H7N9 and H10N8 viruses. We found that PA-356R in H9N2 viruses causes significant increases in virus replication and severity of infection in human cells and mice and that PA-K356R cooperates with the PB2-E627K mutation, a well-characterized human adaptive marker, to exacerbate mammalian infection in vitro and in vivo. Therefore, the PA-K356R mutation is a significant adaptation in H9N2 viruses and related H7N9 and H10N8 reassortants toward human

Two novel mutations in the homogentisate-1,2-dioxygenase gene identified in Chinese Han Child with Alkaptonuria.

PubMed

Li, Hongying; Zhang, Kaihui; Xu, Qun; Ma, Lixia; Lv, Xin; Sun, Ruopeng

2015-03-01

Alkaptonuria (AKU) is an autosomal recessive disorder of tyrosine metabolism, which is caused by a defect in the enzyme homogentisate 1,2-dioxygenase (HGD) with subsequent accumulation of homogentisic acid. Presently, more than 100 HGD mutations have been identified as the cause of the inborn error of metabolism across different populations worldwide. However, the HGD mutation is very rarely reported in Asia, especially China. In this study, we present mutational analyses of HGD gene in one Chinese Han child with AKU, which had been identified by gas chromatography-mass spectrometry detection of organic acids in urine samples. PCR and DNA sequencing of the entire coding region as well as exon-intron boundaries of HGD have been performed. Two novel mutations were identified in the HGD gene in this AKU case, a frameshift mutation of c.115delG in exon 3 and the splicing mutation of IVS5+3 A>C, a donor splice site of the exon 5 and exon-intron junction. The identification of these mutations in this study further expands the spectrum of known HGD gene mutations and contributes to prenatal molecular diagnosis of AKU.

Identification of mutations in the MYO9A gene in patients with congenital myasthenic syndrome

PubMed Central

O’Connor, Emily; Töpf, Ana; Müller, Juliane S.; Cox, Daniel; Evangelista, Teresinha; Colomer, Jaume; Abicht, Angela; Senderek, Jan; Hasselmann, Oswald; Yaramis, Ahmet; Laval, Steven H.

2016-01-01

Abstract Congenital myasthenic syndromes are a group of rare and genetically heterogenous disorders resulting from defects in the structure and function of the neuromuscular junction. Patients with congenital myasthenic syndrome exhibit fatigable muscle weakness with a variety of accompanying phenotypes depending on the protein affected. A cohort of patients with a clinical diagnosis of congenital myasthenic syndrome that lacked a genetic diagnosis underwent whole exome sequencing in order to identify genetic causation. Missense biallelic mutations in the MYO9A gene, encoding an unconventional myosin, were identified in two unrelated families. Depletion of MYO9A in NSC-34 cells revealed a direct effect of MYO9A on neuronal branching and axon guidance. Morpholino-mediated knockdown of the two MYO9A orthologues in zebrafish, myo9aa/ab, demonstrated a requirement for MYO9A in the formation of the neuromuscular junction during development. The morphants displayed shortened and abnormally branched motor axons, lack of movement within the chorion and abnormal swimming in response to tactile stimulation. We therefore conclude that MYO9A deficiency may affect the presynaptic motor axon, manifesting in congenital myasthenic syndrome. These results highlight the involvement of unconventional myosins in motor axon functionality, as well as the need to look outside traditional neuromuscular junction-specific proteins for further congenital myasthenic syndrome candidate genes. PMID:27259756

Defining the association of TMEM106B variants among frontotemporal lobar degeneration patients with GRN mutations and C9orf72 repeat expansions.

PubMed

Lattante, Serena; Le Ber, Isabelle; Galimberti, Daniela; Serpente, Maria; Rivaud-Péchoux, Sophie; Camuzat, Agnès; Clot, Fabienne; Fenoglio, Chiara; Scarpini, Elio; Brice, Alexis; Kabashi, Edor

2014-11-01

TMEM106B was identified as a risk factor for frontotemporal lobar degeneration (FTD) with TAR DNA-binding protein 43 kDa inclusions. It has been reported that variants in this gene are genetic modifiers of the disease and that this association is stronger in patients carrying a GRN mutation or a pathogenic expansion in chromosome 9 open reading frame 72 (C9orf72) gene. Here, we investigated the contribution of TMEM106B polymorphisms in cohorts of FTD and FTD with amyotrophic lateral sclerosis patients from France and Italy. Patients carrying the C9orf72 expansion (n = 145) and patients with GRN mutations (n = 76) were compared with a group of FTD patients (n = 384) negative for mutations and to a group of healthy controls (n = 552). In our cohorts, the presence of the C9orf72 expansion did not correlate with TMEM106B genotypes but the association was very strong in individuals with pathogenic GRN mutations (p = 9.54 × 10(-6)). Our data suggest that TMEM106B genotypes differ in FTD patient cohorts and strengthen the protective role of TMEM106B in GRN carriers. Further studies are needed to determine whether TMEM106B polymorphisms are associated with other genetic causes for FTD, including C9orf72 repeat expansions. Copyright © 2014 Elsevier Inc. All rights reserved.

TP53, PIK3CA, FBXW7 and KRAS Mutations in Esophageal Cancer Identified by Targeted Sequencing.

PubMed

Zheng, Huili; Wang, Yan; Tang, Chuanning; Jones, Lindsey; Ye, Hua; Zhang, Guangchun; Cao, Weihai; Li, Jingwen; Liu, Lifeng; Liu, Zhencong; Zhang, Chao; Lou, Feng; Liu, Zhiyuan; Li, Yangyang; Shi, Zhenfen; Zhang, Jingbo; Zhang, Dandan; Sun, Hong; Dong, Haichao; Dong, Zhishou; Guo, Baishuai; Yan, H E; Lu, Qingyu; Huang, Xue; Chen, Si-Yi

2016-01-01

Esophageal cancer (EC) is a common malignancy with significant morbidity and mortality. As individual cancers exhibit unique mutation patterns, identifying and characterizing gene mutations in EC that may serve as biomarkers might help predict patient outcome and guide treatment. Traditionally, personalized cancer DNA sequencing was impractical and expensive. Recent technological advancements have made targeted DNA sequencing more cost- and time-effective with reliable results. This technology may be useful for clinicians to direct patient treatment. The Ion PGM and AmpliSeq Cancer Panel was used to identify mutations at 737 hotspot loci of 45 cancer-related genes in 64 EC samples from Chinese patients. Frequent mutations were found in TP53 and less frequent mutations in PIK3CA, FBXW7 and KRAS. These results demonstrate that targeted sequencing can reliably identify mutations in individual tumors that make this technology a possibility for clinical use. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

GNAq mutations are not identified in papillary thyroid carcinomas and hyperfunctioning thyroid nodules.

PubMed

Cassol, Clarissa A; Guo, Miao; Ezzat, Shereen; Asa, Sylvia L

2010-12-01

Activating mutations of GNAq protein in a hotspot at codon 209 have been recently described in uveal melanomas. Since these neoplasms share with thyroid carcinomas a high frequency of MAP kinase pathway-activating mutations, we hypothesized whether GNAq mutations could also play a role in the development of thyroid carcinomas. Additionally, activating mutations of another subtype of G protein (GNAS1) are frequently found in hyperfunctioning thyroid adenomas, making it plausible that GNAq-activating mutations could also be found in some of these nodules. To investigate thyroid papillary carcinomas and thyroid hyperfunctioning nodules for GNAq mutations in exon 5, codon 209, a total of 32 RET/PTC, BRAF, and RAS negative thyroid papillary carcinomas and 13 hyperfunctioning thyroid nodules were evaluated. No mutations were identified. Although plausible, GNAq mutations seem not to play an important role in the development of thyroid follicular neoplasms, either benign hyperfunctioning nodules or malignant papillary carcinomas. Our results are in accordance with the literature, in which no GNAq hotspot mutations were found in thyroid papillary carcinomas, as well as in an extensive panel of other tumors. The molecular basis for MAP-kinase pathway activation in RET-PTC/BRAF/RAS negative thyroid carcinomas remains to be determined.

Remarkable stabilization of a psychrotrophic RNase HI by a combination of thermostabilizing mutations identified by the suppressor mutation method.

PubMed

Tadokoro, Takashi; Matsushita, Kyoko; Abe, Yumi; Rohman, Muhammad Saifur; Koga, Yuichi; Takano, Kazufumi; Kanaya, Shigenori

2008-08-05

Ribonuclease HI from the psychrotrophic bacterium Shewanella oneidensis MR-1 (So-RNase HI) is much less stable than Escherichia coli RNase HI (Ec-RNase HI) by 22.4 degrees C in T m and 12.5 kJ mol (-1) in Delta G(H 2O), despite their high degrees of structural and functional similarity. To examine whether the stability of So-RNase HI increases to a level similar to that of Ec-RNase HI via introduction of several mutations, the mutations that stabilize So-RNase HI were identified by the suppressor mutation method and combined. So-RNase HI and its variant with a C-terminal four-residue truncation (154-RNase HI) complemented the RNase H-dependent temperature-sensitive (ts) growth phenotype of E. coli strain MIC3001, while 153-RNase HI with a five-residue truncation could not. Analyses of the activity and stability of these truncated proteins suggest that 153-RNase HI is nonfunctional in vivo because of a great decrease in stability. Random mutagenesis of 153-RNase HI using error-prone PCR, followed by screening for the revertants, allowed us to identify six single suppressor mutations that make 153-RNase HI functional in vivo. Four of them markedly increased the stability of the wild-type protein by 3.6-6.7 degrees C in T m and 1.7-5.2 kJ mol (-1) in Delta G(H 2O). The effects of these mutations were nearly additive, and combination of these mutations increased protein stability by 18.7 degrees C in T m and 12.2 kJ mol (-1) in Delta G(H 2O). These results suggest that several residues are not optimal for the stability of So-RNase HI, and their replacement with other residues strikingly increases it to a level similar to that of the mesophilic counterpart.

Ultra-sensitive Sequencing Identifies High Prevalence of Clonal Hematopoiesis-Associated Mutations throughout Adult Life.

PubMed

Acuna-Hidalgo, Rocio; Sengul, Hilal; Steehouwer, Marloes; van de Vorst, Maartje; Vermeulen, Sita H; Kiemeney, Lambertus A L M; Veltman, Joris A; Gilissen, Christian; Hoischen, Alexander

2017-07-06

Clonal hematopoiesis results from somatic mutations in hematopoietic stem cells, which give an advantage to mutant cells, driving their clonal expansion and potentially leading to leukemia. The acquisition of clonal hematopoiesis-driver mutations (CHDMs) occurs with normal aging and these mutations have been detected in more than 10% of individuals ≥65 years. We aimed to examine the prevalence and characteristics of CHDMs throughout adult life. We developed a targeted re-sequencing assay combining high-throughput with ultra-high sensitivity based on single-molecule molecular inversion probes (smMIPs). Using smMIPs, we screened more than 100 loci for CHDMs in more than 2,000 blood DNA samples from population controls between 20 and 69 years of age. Loci screened included 40 regions known to drive clonal hematopoiesis when mutated and 64 novel candidate loci. We identified 224 somatic mutations throughout our cohort, of which 216 were coding mutations in known driver genes (DNMT3A, JAK2, GNAS, TET2, and ASXL1), including 196 point mutations and 20 indels. Our assay's improved sensitivity allowed us to detect mutations with variant allele frequencies as low as 0.001. CHDMs were identified in more than 20% of individuals 60 to 69 years of age and in 3% of individuals 20 to 29 years of age, approximately double the previously reported prevalence despite screening a limited set of loci. Our findings support the occurrence of clonal hematopoiesis-associated mutations as a widespread mechanism linked with aging, suggesting that mosaicism as a result of clonal evolution of cells harboring somatic mutations is a universal mechanism occurring at all ages in healthy humans. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Recurrent TERT promoter mutations identified in a large-scale study of multiple tumour types are associated with increased TERT expression and telomerase activation.

PubMed

Huang, Dong-Sheng; Wang, Zhaohui; He, Xu-Jun; Diplas, Bill H; Yang, Rui; Killela, Patrick J; Meng, Qun; Ye, Zai-Yuan; Wang, Wei; Jiang, Xiao-Ting; Xu, Li; He, Xiang-Lei; Zhao, Zhong-Sheng; Xu, Wen-Juan; Wang, Hui-Ju; Ma, Ying-Yu; Xia, Ying-Jie; Li, Li; Zhang, Ru-Xuan; Jin, Tao; Zhao, Zhong-Kuo; Xu, Ji; Yu, Sheng; Wu, Fang; Liang, Junbo; Wang, Sizhen; Jiao, Yuchen; Yan, Hai; Tao, Hou-Quan

2015-05-01

Several somatic mutation hotspots were recently identified in the telomerase reverse transcriptase (TERT) promoter region in human cancers. Large scale studies of these mutations in multiple tumour types are limited, in particular in Asian populations. This study aimed to: analyse TERT promoter mutations in multiple tumour types in a large Chinese patient cohort, investigate novel tumour types and assess the functional significance of the mutations. TERT promoter mutation status was assessed by Sanger sequencing for 13 different tumour types and 799 tumour tissues from Chinese cancer patients. Thymic epithelial tumours, gastrointestinal leiomyoma, and gastric schwannoma were included, for which the TERT promoter has not been previously sequenced. Functional studies included TERT expression by reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR), telomerase activity by the telomeric repeat amplification protocol (TRAP) assay and promoter activity by the luciferase reporter assay. TERT promoter mutations were highly frequent in glioblastoma (83.9%), urothelial carcinoma (64.5%), oligodendroglioma (70.0%), medulloblastoma (33.3%) and hepatocellular carcinoma (31.4%). C228T and C250T were the most common mutations. In urothelial carcinoma, several novel rare mutations were identified. TERT promoter mutations were absent in gastrointestinal stromal tumour (GIST), thymic epithelial tumours, gastrointestinal leiomyoma, gastric schwannoma, cholangiocarcinoma, gastric and pancreatic cancer. TERT promoter mutations highly correlated with upregulated TERT mRNA expression and telomerase activity in adult gliomas. These mutations differentially enhanced the transcriptional activity of the TERT core promoter. TERT promoter mutations are frequent in multiple tumour types and have similar distributions in Chinese cancer patients. The functional significance of these mutations reflect the importance to telomere maintenance and hence tumourigenesis, making them

CRISPR/Cas9/sgRNA-mediated targeted gene modification confirms the cause-effect relationship between gyrA mutation and quinolone resistance in Escherichia coli.

PubMed

Qiu, Haixiang; Gong, Jiansen; Butaye, Patrick; Lu, Guangwu; Huang, Ke; Zhu, Guoqiang; Zhang, Jilei; Hathcock, Terri; Cheng, Darong; Wang, Chengming

2018-05-14

Quinolones are broad-spectrum antibiotics that have been used for decades in treating bacterial infections in humans and animals, and subsequently bacterial resistance to these agents has increased. While studies indicated the relationship between gyrA mutations and bacterial resistance to quinolones, CRISPR/Cas9 was used in this study to investigate causal role of gyrA mutation in the quinolone resistance. In this study, 818 clinical Escherichia coli isolates were analyzed for gyrA mutations and their resistance to quinolones. The CRISPR/Cas9 system was used to generate gyrA mutations in quinolone-susceptible E. coli ATCC 25922, and quinolone-resistant clinical E. coli. The antimicrobial resistance prevalence rate in E. coli against nalidixic acid, ciprofloxacin and enrofloxacin was 77.1% (631/818), 51.1% (418/818) and 49.8% (407/818), respectively. The gyrA mutations were identified in nucleotide positions 248, 255, 259, 260, 261, 273 and 300, and mutations at positions 248 and 259 resulting in amino acid changes at positions 83 and 87 were associated with quinolone resistance. Double-site amino acid mutations increase resistance to quinolones. The gyrA mutations causing changes at amino acids 83 and 87 reversed the features of quinolone resistance in ATCC and clinical strains, verifying the causal role of gyrA mutation in the quinolone resistance of E. coli.

Whole-exome sequencing identifies novel homozygous mutation in NPAS2 in family with nonobstructive azoospermia.

PubMed

Ramasamy, Ranjith; Bakırcıoğlu, M Emre; Cengiz, Cenk; Karaca, Ender; Scovell, Jason; Jhangiani, Shalini N; Akdemir, Zeynep C; Bainbridge, Matthew; Yu, Yao; Huff, Chad; Gibbs, Richard A; Lupski, James R; Lamb, Dolores J

2015-08-01

To investigate the genetic cause of nonobstructive azoospermia (NOA) in a consanguineous Turkish family through homozygosity mapping followed by targeted exon/whole-exome sequencing to identify genetic variations. Whole-exome sequencing (WES). Research laboratory. Two siblings in a consanguineous family with NOA. Validating all variants passing filter criteria with Sanger sequencing to confirm familial segregation and absence in the control population. Discovery of a mutation that could potentially cause NOA. A novel nonsynonymous mutation in the neuronal PAS-2 domain (NPAS2) was identified in a consanguineous family from Turkey. This mutation in exon 14 (chr2: 101592000 C>G) of NPAS2 is likely a disease-causing mutation as it is predicted to be damaging, it is a novel variant, and it segregates with the disease. Family segregation of the variants showed the presence of the homozygous mutation in the three brothers with NOA and a heterozygous mutation in the mother as well as one brother and one sister who were both fertile. The mutation is not found in the single-nucleotide polymorphism database, the 1000 Genomes Project, the Baylor College of Medicine cohort of 500 Turkish patients (not a population-specific polymorphism), or the matching 50 fertile controls. With the use of WES we identified a novel homozygous mutation in NPAS2 as a likely disease-causing variant in a Turkish family diagnosed with NOA. Our data reinforce the clinical role of WES in the molecular diagnosis of highly heterogeneous genetic diseases for which conventional genetic approaches have previously failed to find a molecular diagnosis. Copyright © 2015 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Exome Sequencing Identifies Mitochondrial Alanyl-tRNA Synthetase Mutations in Infantile Mitochondrial Cardiomyopathy

PubMed Central

Götz, Alexandra; Tyynismaa, Henna; Euro, Liliya; Ellonen, Pekka; Hyötyläinen, Tuulia; Ojala, Tiina; Hämäläinen, Riikka H.; Tommiska, Johanna; Raivio, Taneli; Oresic, Matej; Karikoski, Riitta; Tammela, Outi; Simola, Kalle O.J.; Paetau, Anders; Tyni, Tiina; Suomalainen, Anu

2011-01-01

Infantile cardiomyopathies are devastating fatal disorders of the neonatal period or the first year of life. Mitochondrial dysfunction is a common cause of this group of diseases, but the underlying gene defects have been characterized in only a minority of cases, because tissue specificity of the manifestation hampers functional cloning and the heterogeneity of causative factors hinders collection of informative family materials. We sequenced the exome of a patient who died at the age of 10 months of hypertrophic mitochondrial cardiomyopathy with combined cardiac respiratory chain complex I and IV deficiency. Rigorous data analysis allowed us to identify a homozygous missense mutation in AARS2, which we showed to encode the mitochondrial alanyl-tRNA synthetase (mtAlaRS). Two siblings from another family, both of whom died perinatally of hypertrophic cardiomyopathy, had the same mutation, compound heterozygous with another missense mutation. Protein structure modeling of mtAlaRS suggested that one of the mutations affected a unique tRNA recognition site in the editing domain, leading to incorrect tRNA aminoacylation, whereas the second mutation severely disturbed the catalytic function, preventing tRNA aminoacylation. We show here that mutations in AARS2 cause perinatal or infantile cardiomyopathy with near-total combined mitochondrial respiratory chain deficiency in the heart. Our results indicate that exome sequencing is a powerful tool for identifying mutations in single patients and allows recognition of the genetic background in single-gene disorders of variable clinical manifestation and tissue-specific disease. Furthermore, we show that mitochondrial disorders extend to prenatal life and are an important cause of early infantile cardiac failure. PMID:21549344

Genetic mutations in human rectal cancers detected by targeted sequencing.

PubMed

Bai, Jun; Gao, Jinglong; Mao, Zhijun; Wang, Jianhua; Li, Jianhui; Li, Wensheng; Lei, Yu; Li, Shuaishuai; Wu, Zhuo; Tang, Chuanning; Jones, Lindsey; Ye, Hua; Lou, Feng; Liu, Zhiyuan; Dong, Zhishou; Guo, Baishuai; Huang, Xue F; Chen, Si-Yi; Zhang, Enke

2015-10-01

Colorectal cancer (CRC) is widespread with significant mortality. Both inherited and sporadic mutations in various signaling pathways influence the development and progression of the cancer. Identifying genetic mutations in CRC is important for optimal patient treatment and many approaches currently exist to uncover these mutations, including next-generation sequencing (NGS) and commercially available kits. In the present study, we used a semiconductor-based targeted DNA-sequencing approach to sequence and identify genetic mutations in 91 human rectal cancer samples. Analysis revealed frequent mutations in KRAS (58.2%), TP53 (28.6%), APC (16.5%), FBXW7 (9.9%) and PIK3CA (9.9%), and additional mutations in BRAF, CTNNB1, ERBB2 and SMAD4 were also detected at lesser frequencies. Thirty-eight samples (41.8%) also contained two or more mutations, with common combination mutations occurring between KRAS and TP53 (42.1%), and KRAS and APC (31.6%). DNA sequencing for individual cancers is of clinical importance for targeted drug therapy and the advantages of such targeted gene sequencing over other NGS platforms or commercially available kits in sensitivity, cost and time effectiveness may aid clinicians in treating CRC patients in the near future.

Molecular Testing of 163 Patients with Morquio A (Mucopolysaccharidosis IVA) Identifies 39 Novel GALNS Mutations

PubMed Central

Morrone, A; Tylee, K.L.; Al-Sayed, M; Brusius-Facchin, A.C.; Caciotti, A.; Church, H.J.; Coll, M.J.; Davidson, K.; Fietz, M.J.; Gort, L.; Hegde, M.; Kubaski, F.; Lacerda, L.; Laranjeira, F.; Leistner-Segal, S.; Mooney, S.; Pajares, S.; Pollard, L.; Riberio, I.; Wang, R.Y.; Miller, N.

2014-01-01

Morquio A (Mucopolysaccharidosis IVA; MPS IVA) is an autosomal recessive lysosomal storage disorder caused by partial or total deficiency of the enzyme galactosamine-6-sulfate sulfatase (GALNS; also known as N-acetylgalactosamine-6-sulfate sulfatase) encoded by the GALNS gene. Patients who inherit two mutated GALNS gene alleles produce protein with decreased ability to degrade the glycosaminoglycans (GAGs) keratan sulfate and chondroitin 6-sulfate, thereby causing GAG accumulation within lysosomes and consequently pleiotropic disease. GALNS mutations occur throughout the gene and many mutations are identified only in single patients or families, causing difficulties both in mutation detection and interpretation. In this study, molecular analysis of 163 patients with Morquio A identified 99 unique mutations in the GALNS gene believed to negatively impact GALNS protein function, of which 39 are previously unpublished, together with 26 single-nucleotide polymorphisms. Recommendations for the molecular testing of patients, clear reporting of sequence findings, and interpretation of sequencing data are provided. PMID:24726177

Comprehensive mutation profiling of mucinous gastric carcinoma.

PubMed

Rokutan, Hirofumi; Hosoda, Fumie; Hama, Natsuko; Nakamura, Hiromi; Totoki, Yasushi; Furukawa, Eisaku; Arakawa, Erika; Ohashi, Shoko; Urushidate, Tomoko; Satoh, Hironori; Shimizu, Hiroko; Igarashi, Keiko; Yachida, Shinichi; Katai, Hitoshi; Taniguchi, Hirokazu; Fukayama, Masashi; Shibata, Tatsuhiro

2016-10-01

Mucinous gastric carcinoma (MGC) is a unique subtype of gastric cancer with a poor survival outcome. Comprehensive molecular profiles and putative therapeutic targets of MGC remain undetermined. We subjected 16 tumour-normal tissue pairs to whole-exome sequencing (WES) and an expanded set of 52 tumour-normal tissue pairs to subsequent targeted sequencing. The latter focused on 114 genes identified by WES. Twenty-two histologically differentiated MGCs (D-MGCs) and 46 undifferentiated MGCs (U-MGCs) were analysed. Chromatin modifier genes, including ARID1A (21%), MLL2 (19%), MLL3 (15%), and KDM6A (7%), were frequently mutated (47%) in MGC. We also identified mutations in potential therapeutic target genes, including MTOR (9%), BRCA2 (9%), BRCA1 (7%), and ERBB3 (6%). RHOA mutation was detected only in 4% of U-MGCs and in no D-MGCs. MYH9 was recurrently (13%) mutated in MGC, with all these being of the U-MGC subtype (p = 0.023). Three U-MGCs harboured MYH9 nonsense mutations. MYH9 knockdown enhanced cell migration and induced intracytoplasmic mucin and cellular elongation. BCOR mutation was associated with improved survival. In U-MGCs, the MLH1 expression status and combined mutation status (TP53/BCL11B or TP53/MLL2) were prognostic factors. A comparative analysis of driver genes revealed that the mutation profile of D-MGC was similar to that of intestinal-type gastric cancer, whereas U-MGC was a distinct entity, harbouring a different mutational profile to intestinal- and diffuse-type gastric cancers. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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.

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.

An undergraduate laboratory class using CRISPR/Cas9 technology to mutate drosophila genes.

PubMed

Adame, Vanesa; Chapapas, Holly; Cisneros, Marilyn; Deaton, Carol; Deichmann, Sophia; Gadek, Chauncey; Lovato, TyAnna L; Chechenova, Maria B; Guerin, Paul; Cripps, Richard M

2016-05-06

CRISPR/Cas9 genome editing technology is used in the manipulation of genome sequences and gene expression. Because of the ease and rapidity with which genes can be mutated using CRISPR/Cas9, we sought to determine if a single-semester undergraduate class could be successfully taught, wherein students isolate mutants for specific genes using CRISPR/Cas9. Six students were each assigned a single Drosophila gene, for which no mutants currently exist. Each student designed and created plasmids to encode single guide RNAs that target their selected gene; injected the plasmids into Cas9-expressing embryos, in order to delete the selected gene; carried out a three-generation cross to test for germline transmission of a mutated allele and generate a stable stock of the mutant; and characterized the mutant alleles by PCR and sequencing. Three genes out of six were successfully mutated. Pre- and post- survey evaluations of the students in the class revealed that student attitudes towards their research competencies increased, although the changes were not statistically significant. We conclude that it is feasible to develop a laboratory genome editing class, to provide effective laboratory training to undergraduate students, and to generate mutant lines for use by the broader scientific community. © 2016 by The International Union of Biochemistry and Molecular Biology, 44:263-275, 2016. © 2016 The International Union of Biochemistry and Molecular Biology.

SLC3A1 and SLC7A9 mutations in autosomal recessive or dominant canine cystinuria: a new classification system.

PubMed

Brons, A-K; Henthorn, P S; Raj, K; Fitzgerald, C A; Liu, J; Sewell, A C; Giger, U

2013-01-01

Cystinuria, one of the first recognized inborn errors of metabolism, has been reported in many dog breeds. To determine urinary cystine concentrations, inheritance, and mutations in the SLC3A1 and SLC7A9 genes associated with cystinuria in 3 breeds. Mixed and purebred Labrador Retrievers (n = 6), Australian Cattle Dogs (6), Miniature Pinschers (4), and 1 mixed breed dog with cystine urolithiasis, relatives and control dogs. Urinary cystinuria and aminoaciduria was assessed and exons of the SLC3A1 and SLC7A9 genes were sequenced from genomic DNA. In each breed, male and female dogs, independent of neuter status, were found to form calculi. A frameshift mutation in SLC3A1 (c.350delG) resulting in a premature stop codon was identified in autosomal-recessive (AR) cystinuria in Labrador Retrievers and mixed breed dogs. A 6 bp deletion (c.1095_1100del) removing 2 threonines in SLC3A1 was found in autosomal-dominant (AD) cystinuria with a more severe phenotype in homozygous than in heterozygous Australian Cattle Dogs. A missense mutation in SLC7A9 (c.964G>A) was discovered in AD cystinuria in Miniature Pinschers with only heterozygous affected dogs observed to date. Breed-specific DNA tests were developed, but the prevalence of each mutation remains unknown. These studies describe the first AD inheritance and the first putative SLC7A9 mutation to cause cystinuria in dogs and expand our understanding of this phenotypically and genetically heterogeneous disease, leading to a new classification system for canine cystinuria and better therapeutic management and genetic control in these breeds. Copyright © 2013 by the American College of Veterinary Internal Medicine.

Identification of mutations in the MYO9A gene in patients with congenital myasthenic syndrome.

PubMed

O'Connor, Emily; Töpf, Ana; Müller, Juliane S; Cox, Daniel; Evangelista, Teresinha; Colomer, Jaume; Abicht, Angela; Senderek, Jan; Hasselmann, Oswald; Yaramis, Ahmet; Laval, Steven H; Lochmüller, Hanns

2016-08-01

Congenital myasthenic syndromes are a group of rare and genetically heterogenous disorders resulting from defects in the structure and function of the neuromuscular junction. Patients with congenital myasthenic syndrome exhibit fatigable muscle weakness with a variety of accompanying phenotypes depending on the protein affected. A cohort of patients with a clinical diagnosis of congenital myasthenic syndrome that lacked a genetic diagnosis underwent whole exome sequencing in order to identify genetic causation. Missense biallelic mutations in the MYO9A gene, encoding an unconventional myosin, were identified in two unrelated families. Depletion of MYO9A in NSC-34 cells revealed a direct effect of MYO9A on neuronal branching and axon guidance. Morpholino-mediated knockdown of the two MYO9A orthologues in zebrafish, myo9aa/ab, demonstrated a requirement for MYO9A in the formation of the neuromuscular junction during development. The morphants displayed shortened and abnormally branched motor axons, lack of movement within the chorion and abnormal swimming in response to tactile stimulation. We therefore conclude that MYO9A deficiency may affect the presynaptic motor axon, manifesting in congenital myasthenic syndrome. These results highlight the involvement of unconventional myosins in motor axon functionality, as well as the need to look outside traditional neuromuscular junction-specific proteins for further congenital myasthenic syndrome candidate genes. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain.

Detection of EGFR mutations with mutation-specific antibodies in stage IV non-small-cell lung cancer

PubMed Central

2010-01-01

Background Immunohistochemistry (IHC) with mutation-specific antibodies may be an ancillary method of detecting EGFR mutations in lung cancer patients. Methods EGFR mutation status was analyzed by DNA assays, and compared with IHC results in five non-small-cell lung cancer (NSCLC) cell lines and tumor samples from 78 stage IV NSCLC patients. Results IHC correctly identified del 19 in the H1650 and PC9 cell lines, L858R in H1975, and wild-type EGFR in H460 and A549, as well as wild-type EGFR in tumor samples from 22 patients. IHC with the mAb against EGFR with del 19 was highly positive for the protein in all 17 patients with a 15-bp (ELREA) deletion in exon 19, whereas in patients with other deletions, IHC was weakly positive in 3 cases and negative in 9 cases. IHC with the mAb against the L858R mutation showed high positivity for the protein in 25/27 (93%) patients with exon 21 EGFR mutations (all with L858R) but did not identify the L861Q mutation in the remaining two patients. Conclusions IHC with mutation-specific mAbs against EGFR is a promising method for detecting EGFR mutations in NSCLC patients. However these mAbs should be validated with additional studies to clarify their possible role in routine clinical practice for screening EGFR mutations in NSCLC patients. PMID:21167064

New VMD2 gene mutations identified in patients affected by Best vitelliform macular dystrophy

PubMed Central

Marchant, D; Yu, K; Bigot, K; Roche, O; Germain, A; Bonneau, D; Drouin‐Garraud, V; Schorderet, D F; Munier, F; Schmidt, D; Neindre, P Le; Marsac, C; Menasche, M; Dufier, J L; Fischmeister, R; Hartzell, C; Abitbol, M

2007-01-01

Purpose The mutations responsible for Best vitelliform macular dystrophy (BVMD) are found in a gene called VMD2. The VMD2 gene encodes a transmembrane protein named bestrophin‐1 (hBest1) which is a Ca2+‐sensitive chloride channel. This study was performed to identify disease‐specific mutations in 27 patients with BVMD. Because this disease is characterised by an alteration in Cl− channel function, patch clamp analysis was used to test the hypothesis that one of the VMD2 mutated variants causes the disease. Methods Direct sequencing analysis of the 11 VMD2 exons was performed to detect new abnormal sequences. The mutant of hBest1 was expressed in HEK‐293 cells and the associated Cl− current was examined using whole‐cell patch clamp analysis. Results Six new VMD2 mutations were identified, located exclusively in exons four, six and eight. One of these mutations (Q293H) was particularly severe. Patch clamp analysis of human embryonic kidney cells expressing the Q293H mutant showed that this mutant channel is non‐functional. Furthermore, the Q293H mutant inhibited the function of wild‐type bestrophin‐1 channels in a dominant negative manner. Conclusions This study provides further support for the idea that mutations in VMD2 are a necessary factor for Best disease. However, because variable expressivity of VMD2 was observed in a family with the Q293H mutation, it is also clear that a disease‐linked mutation in VMD2 is not sufficient to produce BVMD. The finding that the Q293H mutant does not form functional channels in the membrane could be explained either by disruption of channel conductance or gating mechanisms or by improper trafficking of the protein to the plasma membrane. PMID:17287362

[Two novel pathogenic mutations of GAN gene identified in a patient with giant axonal neuropathy].

PubMed

Wang, Juan; Ma, Qingwen; Cai, Qin; Liu, Yanna; Wang, Wei; Ren, Zhaorui

2016-06-01

To explore the disease-causing mutations in a patient suspected for giant axonal neuropathy(GAN). Target sequence capture sequencing was used to screen potential mutations in genomic DNA extracted from peripheral blood sample of the patient. Sanger sequencing was applied to confirm the detected mutation. The mutation was verified among 400 GAN alleles from 200 healthy individuals by Sanger sequencing. The function of the mutations was predicted by bioinformatics analysis. The patient was identified as a compound heterozygote carrying two novel pathogenic GAN mutations, i.e., c.778G>T (p.Glu260Ter) and c.277G>A (p.Gly93Arg). Sanger sequencing confirmed that the c.778G>T (p.Glu260Ter) mutation was inherited from his father, while c.277G>A (p.Gly93Arg) was inherited from his mother. The same mutations was not found in the 200 healthy individuals. Bioinformatics analysis predicted that the two mutations probably caused functional abnormality of gigaxonin. Two novel GAN mutations were detected in a patient with GAN. Both mutations are pathogenic and can cause abnormalities of gigaxonin structure and function, leading to pathogenesis of GAN. The results may also offer valuable information for similar diseases.

Correction of nonsense BMPR2 and SMAD9 mutations by ataluren in pulmonary arterial hypertension.

PubMed

Drake, Kylie M; Dunmore, Benjamin J; McNelly, Lauren N; Morrell, Nicholas W; Aldred, Micheala A

2013-09-01

Heritable pulmonary arterial hypertension (HPAH) is a serious lung vascular disease caused by heterozygous mutations in the bone morphogenetic protein (BMP) pathway genes, BMPR2 and SMAD9. One noncanonical function of BMP signaling regulates biogenesis of a subset of microRNAs. We have previously shown that this function is abrogated in patients with HPAH, making it a highly sensitive readout of BMP pathway integrity. Ataluren (PTC124) is an investigational drug that permits ribosomal readthrough of premature stop codons, resulting in a full-length protein. It exhibits oral bioavailability and limited toxicity in human trials. Here, we tested ataluren in lung- or blood-derived cells from patients with HPAH with nonsense mutations in BMPR2 (n = 6) or SMAD9 (n = 1). Ataluren significantly increased BMP-mediated microRNA processing in six of the seven cases. Moreover, rescue was achieved even for mutations exhibiting significant nonsense-mediated mRNA decay. Response to ataluren was dose dependent, and complete correction was achieved at therapeutic doses currently used in clinical trials for cystic fibrosis. BMP receptor (BMPR)-II protein levels were normalized and ligand-dependent phosphorylation of downstream target Smads was increased. Furthermore, the usually hyperproliferative phenotype of pulmonary artery endothelial and smooth muscle cells was reversed by ataluren. These results indicate that ataluren can effectively suppress a high proportion of BMPR2 and SMAD9 nonsense mutations and correct BMP signaling in vitro. Approximately 29% of all HPAH mutations are nonsense point mutations. In light of this, we propose ataluren as a potential new personalized therapy for this significant subgroup of patients with PAH.

Mutations in the Kinase Domain of the HER2/ERBB2 Gene Identified in a Wide Variety of Human Cancers.

PubMed

Wen, Wenhsiang; Chen, Wangjuh Sting; Xiao, Nick; Bender, Ryan; Ghazalpour, Anatole; Tan, Zheng; Swensen, Jeffrey; Millis, Sherri Z; Basu, Gargi; Gatalica, Zoran; Press, Michael F

2015-09-01

The HER2 (official name ERBB2) gene encodes a membrane receptor in the epidermal growth factor receptor family amplified and overexpressed in adenocarcinoma. Activating mutations also occur in several cancers. We report mutation analyses of the HER2 kinase domain in 7497 histologically diverse cancers. Forty-five genes, including the kinase domain of HER2 with HER2 IHC and dual in situ hybridization, were analyzed in tumors from 7497 patients with cancer, including 850 breast, 770 colorectal, 910 non-small cell lung, 823 uterine or cervical, 1372 ovarian, and 297 pancreatic cancers, as well as 323 melanomas and 2152 other solid tumors. Sixty-nine HER2 kinase domain mutations were identified in tumors from 68 patients (approximately 1% of all cases, ranging from absent in sarcomas to 4% in urothelial cancers), which included previously published activating mutations and 13 novel mutations. Fourteen cases with coexisting HER2 mutation and amplification and/or overexpression were identified. Fifty-two of 68 patients had additional mutations in other analyzed genes, whereas 16 patients (23%) had HER2 mutations identified as the sole driver mutation. HER2 mutations coexisted with HER2 gene amplification and overexpression and with mutations in other functionally important genes. HER2 mutations were identified as the only driver mutation in a significant proportion of solid cancers. Evaluation of anti-HER2 therapies in nonamplified, HER2-mutated cancers is warranted. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Exome sequencing identifies a DNAJB6 mutation in a family with dominantly-inherited limb-girdle muscular dystrophy.

PubMed

Couthouis, Julien; Raphael, Alya R; Siskind, Carly; Findlay, Andrew R; Buenrostro, Jason D; Greenleaf, William J; Vogel, Hannes; Day, John W; Flanigan, Kevin M; Gitler, Aaron D

2014-05-01

Limb-girdle muscular dystrophy primarily affects the muscles of the hips and shoulders (the "limb-girdle" muscles), although it is a heterogeneous disorder that can present with varying symptoms. There is currently no cure. We sought to identify the genetic basis of limb-girdle muscular dystrophy type 1 in an American family of Northern European descent using exome sequencing. Exome sequencing was performed on DNA samples from two affected siblings and one unaffected sibling and resulted in the identification of eleven candidate mutations that co-segregated with the disease. Notably, this list included a previously reported mutation in DNAJB6, p.Phe89Ile, which was recently identified as a cause of limb-girdle muscular dystrophy type 1D. Additional family members were Sanger sequenced and the mutation in DNAJB6 was only found in affected individuals. Subsequent haplotype analysis indicated that this DNAJB6 p.Phe89Ile mutation likely arose independently of the previously reported mutation. Since other published mutations are located close by in the G/F domain of DNAJB6, this suggests that the area may represent a mutational hotspot. Exome sequencing provided an unbiased and effective method for identifying the genetic etiology of limb-girdle muscular dystrophy type 1 in a previously genetically uncharacterized family. This work further confirms the causative role of DNAJB6 mutations in limb-girdle muscular dystrophy type 1D. Copyright © 2014 Elsevier B.V. All rights reserved.

Efficient Generation of Gene-Modified Pigs Harboring Precise Orthologous Human Mutation via CRISPR/Cas9-Induced Homology-Directed Repair in Zygotes.

PubMed

Zhou, Xiaoyang; Wang, Lulu; Du, Yinan; Xie, Fei; Li, Liang; Liu, Yu; Liu, Chuanhong; Wang, Shiqiang; Zhang, Shibing; Huang, Xingxu; Wang, Yong; Wei, Hong

2016-01-01

Precise genetic mutation of model animals is highly valuable for functional investigation of human mutations. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)-induced homology-directed repair (HDR) is usually used for precise genetic mutation, being limited by the relatively low efficiency compared with that of non-homologous end joining (NHEJ). Although inhibition of NHEJ was shown to enhance HDR-derived mutation, in this work, without inhibition of NHEJ, we first generated gene-modified pigs harboring precise orthologous human mutation (Sox10 c.A325>T) via CRISPR/Cas9-induced HDR in zygotes using single-strand oligo DNA (ssODN) as template with an efficiency as high as 80%, indicating that pig zygotes exhibited high activities of HDR relative to NHEJ and were highly amendable to genetic mutation via CIRSPR/Cas9-induced HDR. Besides, we found a higher concentration of ssODN remarkably reduced HDR-derived mutation in pig zygotes, suggesting a possible balance for optimal HDR-derived mutation in zygotes between the excessive accessibility to HDR templates and the activities of HDR relative to NHEJ which appeared to be negatively correlated to ssODN concentration. In addition, the HDR-derived mutation, as well as those from NHEJ, extensively integrated into various tissues including gonad of founder pig without detected off-targeting, suggesting CRISPR/Cas9-induced HDR in zygotes is a reliable approach for precise genetic mutation in pigs. © 2015 WILEY PERIODICALS, INC.

Microfluidic screening and whole-genome sequencing identifies mutations associated with improved protein secretion by yeast.

PubMed

Huang, Mingtao; Bai, Yunpeng; Sjostrom, Staffan L; Hallström, Björn M; Liu, Zihe; Petranovic, Dina; Uhlén, Mathias; Joensson, Haakan N; Andersson-Svahn, Helene; Nielsen, Jens

2015-08-25

There is an increasing demand for biotech-based production of recombinant proteins for use as pharmaceuticals in the food and feed industry and in industrial applications. Yeast Saccharomyces cerevisiae is among preferred cell factories for recombinant protein production, and there is increasing interest in improving its protein secretion capacity. Due to the complexity of the secretory machinery in eukaryotic cells, it is difficult to apply rational engineering for construction of improved strains. Here we used high-throughput microfluidics for the screening of yeast libraries, generated by UV mutagenesis. Several screening and sorting rounds resulted in the selection of eight yeast clones with significantly improved secretion of recombinant α-amylase. Efficient secretion was genetically stable in the selected clones. We performed whole-genome sequencing of the eight clones and identified 330 mutations in total. Gene ontology analysis of mutated genes revealed many biological processes, including some that have not been identified before in the context of protein secretion. Mutated genes identified in this study can be potentially used for reverse metabolic engineering, with the objective to construct efficient cell factories for protein secretion. The combined use of microfluidics screening and whole-genome sequencing to map the mutations associated with the improved phenotype can easily be adapted for other products and cell types to identify novel engineering targets, and this approach could broadly facilitate design of novel cell factories.

Genomic characterization of biliary tract cancers identifies driver genes and predisposing mutations.

PubMed

Wardell, Christopher P; Fujita, Masashi; Yamada, Toru; Simbolo, Michele; Fassan, Matteo; Karlic, Rosa; Polak, Paz; Kim, Jaegil; Hatanaka, Yutaka; Maejima, Kazuhiro; Lawlor, Rita T; Nakanishi, Yoshitsugu; Mitsuhashi, Tomoko; Fujimoto, Akihiro; Furuta, Mayuko; Ruzzenente, Andrea; Conci, Simone; Oosawa, Ayako; Sasaki-Oku, Aya; Nakano, Kaoru; Tanaka, Hiroko; Yamamoto, Yujiro; Michiaki, Kubo; Kawakami, Yoshiiku; Aikata, Hiroshi; Ueno, Masaki; Hayami, Shinya; Gotoh, Kunihito; Ariizumi, Shun-Ichi; Yamamoto, Masakazu; Yamaue, Hiroki; Chayama, Kazuaki; Miyano, Satoru; Getz, Gad; Scarpa, Aldo; Hirano, Satoshi; Nakamura, Toru; Nakagawa, Hidewaki

2018-05-01

Biliary tract cancers (BTCs) are clinically and pathologically heterogeneous and respond poorly to treatment. Genomic profiling can offer a clearer understanding of their carcinogenesis, classification and treatment strategy. We performed large-scale genome sequencing analyses on BTCs to investigate their somatic and germline driver events and characterize their genomic landscape. We analyzed 412 BTC samples from Japanese and Italian populations, 107 by whole-exome sequencing (WES), 39 by whole-genome sequencing (WGS), and a further 266 samples by targeted sequencing. The subtypes were 136 intrahepatic cholangiocarcinomas (ICCs), 101 distal cholangiocarcinomas (DCCs), 109 peri-hilar type cholangiocarcinomas (PHCs), and 66 gallbladder or cystic duct cancers (GBCs/CDCs). We identified somatic alterations and searched for driver genes in BTCs, finding pathogenic germline variants of cancer-predisposing genes. We predicted cell-of-origin for BTCs by combining somatic mutation patterns and epigenetic features. We identified 32 significantly and commonly mutated genes including TP53, KRAS, SMAD4, NF1, ARID1A, PBRM1, and ATR, some of which negatively affected patient prognosis. A novel deletion of MUC17 at 7q22.1 affected patient prognosis. Cell-of-origin predictions using WGS and epigenetic features suggest hepatocyte-origin of hepatitis-related ICCs. Deleterious germline mutations of cancer-predisposing genes such as BRCA1, BRCA2, RAD51D, MLH1, or MSH2 were detected in 11% (16/146) of BTC patients. BTCs have distinct genetic features including somatic events and germline predisposition. These findings could be useful to establish treatment and diagnostic strategies for BTCs based on genetic information. We here analyzed genomic features of 412 BTC samples from Japanese and Italian populations. A total of 32 significantly and commonly mutated genes were identified, some of which negatively affected patient prognosis, including a novel deletion of MUC17 at 7q22.1. Cell

A point mutation in the polymerase protein PB2 allows a reassortant H9N2 influenza isolate of wild-bird origin to replicate in human cells.

USGS Publications Warehouse

Hussein, Islam T.M.; Ma, Eric J.; Meixell, Brandt W.; Hill, Nichola J.; Lindberg, Mark S.; Albrecht , Randy A.; Bahl, Justin; Runstadler, Jonathan A.

2016-01-01

H9N2 influenza A viruses are on the list of potentially pandemic subtypes. Therefore, it is important to understand how genomic reassortment and genetic polymorphisms affect phenotypes of H9N2 viruses circulating in the wild bird reservoir. A comparative genetic analysis of North American H9N2 isolates of wild bird origin identified a naturally occurring reassortant virus containing gene segments derived from both North American and Eurasian lineage ancestors. The PB2 segment of this virus encodes 10 amino acid changes that distinguish it from other H9 strains circulating in North America. G590S, one of the 10 amino acid substitutions observed, was present in ~ 12% of H9 viruses worldwide. This mutation combined with R591 has been reported as a marker of pathogenicity for human pandemic 2009 H1N1 viruses. Screening by polymerase reporter assay of all the natural polymorphisms at these two positions identified G590/K591 and S590/K591 as the most active, with the highest polymerase activity recorded for the SK polymorphism. Rescued viruses containing these two polymorphic combinations replicated more efficiently in MDCK cells and they were the only ones tested that were capable of establishing productive infection in NHBE cells. A global analysis of all PB2 sequences identified the K591 signature in six viral HA/NA subtypes isolated from several hosts in seven geographic locations. Interestingly, introducing the K591 mutation into the PB2 of a human-adapted H3N2 virus did not affect its polymerase activity. Our findings demonstrate that a single point mutation in the PB2 of a low pathogenic H9N2 isolate could have a significant effect on viral phenotype and increase its propensity to infect mammals. However, this effect is not universal, warranting caution in interpreting point mutations without considering protein sequence context.

A point mutation in the polymerase protein PB2 allows a reassortant H9N2 influenza isolate of wild-bird origin to replicate in human cells.

PubMed

Hussein, Islam T M; Ma, Eric J; Hill, Nichola J; Meixell, Brandt W; Lindberg, Mark; Albrecht, Randy A; Bahl, Justin; Runstadler, Jonathan A

2016-07-01

H9N2 influenza A viruses are on the list of potentially pandemic subtypes. Therefore, it is important to understand how genomic reassortment and genetic polymorphisms affect phenotypes of H9N2 viruses circulating in the wild bird reservoir. A comparative genetic analysis of North American H9N2 isolates of wild bird origin identified a naturally occurring reassortant virus containing gene segments derived from both North American and Eurasian lineage ancestors. The PB2 segment of this virus encodes 10 amino acid changes that distinguish it from other H9 strains circulating in North America. G590S, one of the 10 amino acid substitutions observed, was present in ~12% of H9 viruses worldwide. This mutation combined with R591 has been reported as a marker of pathogenicity for human pandemic 2009 H1N1 viruses. Screening by polymerase reporter assay of all the natural polymorphisms at these two positions identified G590/K591 and S590/K591 as the most active, with the highest polymerase activity recorded for the SK polymorphism. Rescued viruses containing these two polymorphic combinations replicated more efficiently in MDCK cells and they were the only ones tested that were capable of establishing productive infection in NHBE cells. A global analysis of all PB2 sequences identified the K591 signature in six viral HA/NA subtypes isolated from several hosts in seven geographic locations. Interestingly, introducing the K591 mutation into the PB2 of a human-adapted H3N2 virus did not affect its polymerase activity. Our findings demonstrate that a single point mutation in the PB2 of a low pathogenic H9N2 isolate could have a significant effect on viral phenotype and increase its propensity to infect mammals. However, this effect is not universal, warranting caution in interpreting point mutations without considering protein sequence context. Copyright © 2016 Elsevier B.V. All rights reserved.

An Undergraduate Laboratory Class Using CRISPR/Cas9 Technology to Mutate Drosophila Genes

ERIC Educational Resources Information Center

Adame, Vanesa; Chapapas, Holly; Cisneros, Marilyn; Deaton, Carol; Deichmann, Sophia; Gadek, Chauncey; Lovato, TyAnna L.; Chechenova, Maria B.; Guerin, Paul; Cripps, Richard M.

2016-01-01

CRISPR/Cas9 genome editing technology is used in the manipulation of genome sequences and gene expression. Because of the ease and rapidity with which genes can be mutated using CRISPR/Cas9, we sought to determine if a single-semester undergraduate class could be successfully taught, wherein students isolate mutants for specific genes using…

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.

Infertility due to congenital absence of vas deferens in mainly caused by variable exon 9 skipping of the CFTR gene in heterozygous males for cystic fibrosis mutations

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

Chillon, M.; Casals, T.; Nunes, V.

1994-09-01

About 65% or the individuals with congenital bilateral absence of the vas deferens (CBAVD) have mutations in at least one of the CFTR alleles. We have studied the phenotypic effects of the CFTR gene intron 8 polyT tract 5T allele in 90 CBAVD subjects and in parents of CF patients. This group was compared with normal individuals, and with fathers and mothers of CF patients. Allele 5T was significantly associated with CBAVD (19.6%) when compared to the general population (5.2%) ({chi}{sup 2} = 33.3%; p<<0.0001). It was represented poorly in fathers of CF patients (1.3%). Mutations were identified in onemore » (60%) or both CFTR alleles (8.9%) of CBAVD patients. Heterozygosity for the 5T allele was strongly associated with heterozygosity for CF mutations ({chi}{sup 2} = 10.9; p<0.0004). The strong correlation between allele 5T and CBAVD, together with the low frequency of this allele in fathers of CF patients, demonstrates that variable {Delta}exon 9 produces infertility in males if associated with a CF mutation on the other chromosome. The 30% of CBAVD cases with only one CFTR mutation and without a 5T-allele may be due to other molecular mechanisms involving CFTR, distinct from {Delta}exon 9. Since there is a relatively high proportion of CBAVD without CF mutations (25%), other gene(s), distinct from CFTR, may have a role in the CBAVD phenotype.« less

New mutations in non-syndromic primary ovarian insufficiency patients identified via whole-exome sequencing.

PubMed

Patiño, Liliana Catherine; Beau, Isabelle; Carlosama, Carolina; Buitrago, July Constanza; González, Ronald; Suárez, Carlos Fernando; Patarroyo, Manuel Alfonso; Delemer, Brigitte; Young, Jacques; Binart, Nadine; Laissue, Paul

2017-07-01

Is it possible to identify new mutations potentially associated with non-syndromic primary ovarian insufficiency (POI) via whole-exome sequencing (WES)? WES is an efficient tool to study genetic causes of POI as we have identified new mutations, some of which lead to protein destablization potentially contributing to the disease etiology. POI is a frequently occurring complex pathology leading to infertility. Mutations in only few candidate genes, mainly identified by Sanger sequencing, have been definitively related to the pathogenesis of the disease. This is a retrospective cohort study performed on 69 women affected by POI. WES and an innovative bioinformatics analysis were used on non-synonymous sequence variants in a subset of 420 selected POI candidate genes. Mutations in BMPR1B and GREM1 were modeled by using fragment molecular orbital analysis. Fifty-five coding variants in 49 genes potentially related to POI were identified in 33 out of 69 patients (48%). These genes participate in key biological processes in the ovary, such as meiosis, follicular development, granulosa cell differentiation/proliferation and ovulation. The presence of at least two mutations in distinct genes in 42% of the patients argued in favor of a polygenic nature of POI. It is possible that regulatory regions, not analyzed in the present study, carry further variants related to POI. WES and the in silico analyses presented here represent an efficient approach for mapping variants associated with POI etiology. Sequence variants presented here represents potential future genetic biomarkers. This study was supported by the Universidad del Rosario and Colciencias (Grants CS/CIGGUR-ABN062-2016 and 672-2014). Colciencias supported Liliana Catherine Patiño´s work (Fellowship: 617, 2013). The authors declare no conflict of interest. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For

ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts

PubMed Central

Valletta, Simona; Dolatshad, Hamid; Bartenstein, Matthias; Yip, Bon Ham; Bello, Erica; Gordon, Shanisha; Yu, Yiting; Shaw, Jacqueline; Roy, Swagata; Scifo, Laura; Schuh, Anna; Pellagatti, Andrea; Fulga, Tudor A.; Verma, Amit; Boultwood, Jacqueline

2015-01-01

Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas9-mediated ASXL1 homozygous correction resulted in protein re-expression with restored normal function, including down-regulation of Polycomb repressive complex 2 target genes. Significantly reduced cell growth and increased myeloid differentiation were observed in ASXL1 mutation-corrected cells, providing new insights into the role of ASXL1 in human myeloid cell differentiation. Mice xenografted with mutation-corrected KBM5 cells showed significantly longer survival than uncorrected xenografts. These results show that the sole correction of a driver mutation in leukemia cells increases survival in vivo in mice. This study provides proof-of-concept for driver gene mutation correction via CRISPR/Cas9 technology in human leukemia cells and presents a strategy to illuminate the impact of oncogenic mutations on cellular function and survival. PMID:26623729

ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts.

PubMed

Valletta, Simona; Dolatshad, Hamid; Bartenstein, Matthias; Yip, Bon Ham; Bello, Erica; Gordon, Shanisha; Yu, Yiting; Shaw, Jacqueline; Roy, Swagata; Scifo, Laura; Schuh, Anna; Pellagatti, Andrea; Fulga, Tudor A; Verma, Amit; Boultwood, Jacqueline

2015-12-29

Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas9-mediated ASXL1 homozygous correction resulted in protein re-expression with restored normal function, including down-regulation of Polycomb repressive complex 2 target genes. Significantly reduced cell growth and increased myeloid differentiation were observed in ASXL1 mutation-corrected cells, providing new insights into the role of ASXL1 in human myeloid cell differentiation. Mice xenografted with mutation-corrected KBM5 cells showed significantly longer survival than uncorrected xenografts. These results show that the sole correction of a driver mutation in leukemia cells increases survival in vivo in mice. This study provides proof-of-concept for driver gene mutation correction via CRISPR/Cas9 technology in human leukemia cells and presents a strategy to illuminate the impact of oncogenic mutations on cellular function and survival.

Comparative analysis of primary versus relapse/refractory DLBCL identifies shifts in mutation spectrum.

PubMed

Greenawalt, Danielle M; Liang, Winnie S; Saif, Sakina; Johnson, Justin; Todorov, Petar; Dulak, Austin; Enriquez, Daniel; Halperin, Rebecca; Ahmed, Ambar; Saveliev, Vladislav; Carpten, John; Craig, David; Barrett, J Carl; Dougherty, Brian; Zinda, Michael; Fawell, Stephen; Dry, Jonathan R; Byth, Kate

2017-11-21

Current understanding of the mutation spectrum of relapsed/refractory (RR) tumors is limited. We performed whole exome sequencing (WES) on 47 diffuse large B cell lymphoma (DLBCL) tumors that persisted after R-CHOP treatment, 8 matched to primary biopsies. We compared genomic alterations from the RR cohort against two treatment-naïve DLBCL cohorts (n=112). While the overall number and types of mutations did not differ significantly, we identified frequency changes in DLBCL driver genes. The overall frequency of MYD88 mutant samples increased (12% to 19%), but we noted a decrease in p.L265P (8% to 4%) and increase in p.S219C mutations (2% to 6%). CARD11 p.D230N, PIM1 p.K115N and CD79B p.Y196C mutations were not observed in the RR cohort, although these mutations were prominent in the primary DLBCL samples. We observed an increase in BCL2 mutations (21% to 38% of samples), BCL2 amplifications (3% to 6% of samples) and CREBBP mutations (31% to 42% of samples) in the RR cohort, supported by acquisition of mutations in these genes in relapsed compared to diagnostic biopsies from the same patient. These increases may reflect the genetic characteristics of R-CHOP RR tumors expected to be enriched for during clinical trial enrollment. These findings hold significance for a number of emerging targeted therapies aligned to genetic targets and biomarkers in DLBCL, reinforcing the importance of time-of-treatment biomarker screening during DLBCL therapy selection.

Comparative analysis of primary versus relapse/refractory DLBCL identifies shifts in mutation spectrum

PubMed Central

Greenawalt, Danielle M.; Liang, Winnie S.; Saif, Sakina; Johnson, Justin; Todorov, Petar; Dulak, Austin; Enriquez, Daniel; Halperin, Rebecca; Ahmed, Ambar; Saveliev, Vladislav; Carpten, John; Craig, David; Barrett, J. Carl; Dougherty, Brian; Zinda, Michael; Fawell, Stephen; Dry, Jonathan R.; Byth, Kate

2017-01-01

Current understanding of the mutation spectrum of relapsed/refractory (RR) tumors is limited. We performed whole exome sequencing (WES) on 47 diffuse large B cell lymphoma (DLBCL) tumors that persisted after R-CHOP treatment, 8 matched to primary biopsies. We compared genomic alterations from the RR cohort against two treatment-naïve DLBCL cohorts (n=112). While the overall number and types of mutations did not differ significantly, we identified frequency changes in DLBCL driver genes. The overall frequency of MYD88 mutant samples increased (12% to 19%), but we noted a decrease in p.L265P (8% to 4%) and increase in p.S219C mutations (2% to 6%). CARD11 p.D230N, PIM1 p.K115N and CD79B p.Y196C mutations were not observed in the RR cohort, although these mutations were prominent in the primary DLBCL samples. We observed an increase in BCL2 mutations (21% to 38% of samples), BCL2 amplifications (3% to 6% of samples) and CREBBP mutations (31% to 42% of samples) in the RR cohort, supported by acquisition of mutations in these genes in relapsed compared to diagnostic biopsies from the same patient. These increases may reflect the genetic characteristics of R-CHOP RR tumors expected to be enriched for during clinical trial enrollment. These findings hold significance for a number of emerging targeted therapies aligned to genetic targets and biomarkers in DLBCL, reinforcing the importance of time-of-treatment biomarker screening during DLBCL therapy selection. PMID:29245897

Exome sequencing identifies complex I NDUFV2 mutations as a novel cause of Leigh syndrome.

PubMed

Cameron, Jessie M; MacKay, Nevena; Feigenbaum, Annette; Tarnopolsky, Mark; Blaser, Susan; Robinson, Brian H; Schulze, Andreas

2015-09-01

Two siblings with hypertrophic cardiomyopathy and brain atrophy were diagnosed with Complex I deficiency based on low enzyme activity in muscle and high lactate/pyruvate ratio in fibroblasts. Whole exome sequencing results of fibroblast gDNA from one sibling was narrowed down to 190 SNPs or In/Dels in 185 candidate genes by selecting non-synonymous coding sequence base pair changes that were not present in the SNP database. Two compound heterozygous mutations were identified in both siblings in NDUFV2, encoding the 24 kDa subunit of Complex I. The intronic mutation (c.IVS2 + 1delGTAA) is disease causing and has been reported before. The other mutation is novel (c.669_670insG, p.Ser224Valfs*3) and predicted to cause a pathogenic frameshift in the protein. Subsequent investigation of 10 probands with complex I deficiency from different families revealed homozygosity for the intronic c.IVS2 + 1delGTAA mutation in a second, consanguineous family. In this family three of five siblings were affected. Interestingly, they presented with Leigh syndrome but no cardiac involvement. The same genotype had been reported previously in a two families but presenting with hypertrophic cardiomyopathy, trunk hypotonia and encephalopathy. We have identified NDUFV2 mutations in two families with Complex I deficiency, including a novel mutation. The diagnosis of Leigh syndrome expands the clinical phenotypes associated with the c.IVS2 + 1delGTAA mutation in this gene. Copyright © 2015 European Paediatric Neurology Society. Published by Elsevier Ltd. All rights reserved.

A G to C transversion at the last nucleotide of exon 25 of the MYH9 gene results in a missense mutation rather than in a splicing defect.

PubMed

Vettore, Silvia; De Rocco, Daniela; Gerber, Bernhard; Scandellari, Raffaella; Bianco, Anna Monica; Balduini, Carlo L; Pecci, Alessandro; Fabris, Fabrizio; Savoia, Anna

2010-01-01

MYH9-related disease (MYH9-RD) is a rare autosomal dominant disorder caused by mutations in MYH9, the gene encoding the heavy chain of non-muscle myosin IIA. Patients present with congenital macrothrombocytopenia and inclusion bodies in neutrophils and might develop sensorineural deafness, presenile cataract, and/or progressive nephropathy leading to end-stage renal failure. In two families with macrothrombocytopenia we identified a novel c.3485G > C mutation in the last nucleotide of exon 25. Bioinformatic tools for splice site prediction and minigene functional test predicted splicing anomalies of exon 25. However, analysis of RNA purified from patient's peripheral blood did not allowed us to detect any anomalies, suggesting that RNA processing is correct at least in this tissue. Therefore, we concluded that c.3485G > C leads to a novel missense mutation (p.Arg1162Thr) of myosin-9, which resulted to be slightly degraded in patient platelets. A precise definition of the effect of mutations is fundamental to improve our knowledge into the pathogenetic mechanisms responsible for the disease. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

Pathogenic mutations in TULP1 responsible for retinitis pigmentosa identified in consanguineous familial cases

PubMed Central

Ullah, Inayat; Kabir, Firoz; Iqbal, Muhammad; Gottsch, Clare Brooks S.; Naeem, Muhammad Asif; Assir, Muhammad Zaman; Khan, Shaheen N.; Akram, Javed; Riazuddin, Sheikh; Ayyagari, Radha; Hejtmancik, J. Fielding

2016-01-01

Purpose To identify pathogenic mutations responsible for autosomal recessive retinitis pigmentosa (arRP) in consanguineous familial cases. Methods Seven large familial cases with multiple individuals diagnosed with retinitis pigmentosa were included in the study. Affected individuals in these families underwent ophthalmic examinations to document the symptoms and confirm the initial diagnosis. Blood samples were collected from all participating members, and genomic DNA was extracted. An exclusion analysis with microsatellite markers spanning the TULP1 locus on chromosome 6p was performed, and two-point logarithm of odds (LOD) scores were calculated. All coding exons along with the exon–intron boundaries of TULP1 were sequenced bidirectionally. We constructed a single nucleotide polymorphism (SNP) haplotype for the four familial cases harboring the K489R allele and estimated the likelihood of a founder effect. Results The ophthalmic examinations of the affected individuals in these familial cases were suggestive of RP. Exclusion analyses confirmed linkage to chromosome 6p harboring TULP1 with positive two-point LOD scores. Subsequent Sanger sequencing identified the single base pair substitution in exon14, c.1466A>G (p.K489R), in four families. Additionally, we identified a two-base deletion in exon 4, c.286_287delGA (p.E96Gfs77*); a homozygous splice site variant in intron 14, c.1495+4A>C; and a novel missense variation in exon 15, c.1561C>T (p.P521S). All mutations segregated with the disease phenotype in the respective families and were absent in ethnically matched control chromosomes. Haplotype analysis suggested (p<10−6) that affected individuals inherited the causal mutation from a common ancestor. Conclusions Pathogenic mutations in TULP1 are responsible for the RP phenotype in seven familial cases with a common ancestral mutation responsible for the disease phenotype in four of the seven families. PMID:27440997

Expression Profile Analysis of Zinc Transporters (ZIP4, ZIP9, ZIP11, ZnT9) in Gliomas and their Correlation with IDH1 Mutation Status.

PubMed

Kang, Xing; Chen, Rong; Zhang, Jie; Li, Gang; Dai, Peng-Gao; Chen, Chao; Wang, Hui-Juan

2015-01-01

Zinc transporters have been considered as essential regulators in many cancers; however, their mechanisms remain unknown, especially in gliomas. Isocitrate dehydrogenase 1(IDH1) mutation is crucial to glioma. This study aimed to investigate whether zinc transporters are correlated with glioma grade and IDH1 mutation status. IDH1 mutation status and mRNA expression of four zinc transporters (ZIP4, ZIP9, ZIP11, and ZnT9) were determined by subjecting a panel of 74 glioma tissue samples to quantitative real-time PCR and pyrosequencing. The correlations between the expression levels of these zinc transporter genes and the grade of glioma, as well as IDH1 mutation status, were investigated. Among the four zinc transporter genes, high ZIP4 expression and low ZIP11 expression were significantly associated with higher grade (grades III and IV) tumors compared with lower grade (grades I and II) counterparts (p<0.0001). However, only ZIP11 exhibited weak correlation with IDH1 mutation status (p=0.045). Samples with mutations in IDH1 displayed higher ZIP11 expression than those without IDH1 mutations. This finding indicated that zinc transporters may interact with IDH1 mutation by direct modulation or action in some shared pathways or genes to promote the development of glioma. Zinc transporters may play an important role in glioma. ZIP4 and ZIP11 are promising molecular diagnostic markers and novel therapeutic targets. Nevertheless, the detailed biological function of zinc transporters and the mechanism of the potential interaction between ZIP11 and IDH1 mutation in gliomagenesis should be further investigated.

Fabry Disease: prevalence of affected males and heterozygotes with pathogenic GLA mutations identified by screening renal, cardiac and stroke clinics, 1995-2017.

PubMed

Doheny, Dana; Srinivasan, Ram; Pagant, Silvere; Chen, Brenden; Yasuda, Makiko; Desnick, Robert J

2018-04-01

Fabry Disease (FD), an X linked lysosomal storage disease due to pathogenic α-galactosidase A ( GLA ) mutations, results in two major subtypes, the early-onset Type 1 'Classic' and the Type 2 'Later-Onset' phenotypes. To identify previously unrecognised patients, investigators screened cardiac, renal and stroke clinics by enzyme assays. However, some screening studies did not perform confirmatory GLA mutation analyses, and many included recently recognised 'benign/likely-benign' variants, thereby inflating prevalence estimates. Online databases were searched for all FD screening studies in high-risk clinics (1995-2017). Studies reporting GLA mutations were re-analysed for pathogenic mutations, sex and phenotype. Phenotype-specific and sex-specific prevalence rates were determined. Of 67 studies, 63 that screened 51363patients (33943M and 17420F) and provided GLA mutations were reanalysed for disease-causing mutations. Of reported GLA mutations, benign variants occurred in 47.9% of males and 74.1% of females. The following were the revised prevalence estimates: among 36820 (23954M and 12866F) haemodialysis screenees, 0.21% males and 0.15% females; among 3074 (2031M and 1043F) renal transplant screenees, 0.25% males and no females; among 5491 (4054M and 1437F) cardiac screenees, 0.94% males and 0.90% females; and among 5978 (3904M and 2074F) stroke screenees, 0.13% males and 0.14% females. Among male and female screenees with pathogenic mutations, the type 1 Classic phenotype was predominant (~60%), except more male cardiac patients (75%) had type 2 Later-Onset phenotype. Compared with previous findings, reanalysis of 63 studies increased the screenee numbers (~3.4-fold), eliminated 20 benign/likely benign variants, and provided more accurate sex-specific and phenotype-specific prevalence estimates, ranging from ~0.13% of stroke to ~0.9% of cardiac male or female screenees. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article

Potential ligand-binding residues in rat olfactory receptors identified by correlated mutation analysis

NASA Technical Reports Server (NTRS)

Singer, M. S.; Oliveira, L.; Vriend, G.; Shepherd, G. M.

1995-01-01

A family of G-protein-coupled receptors is believed to mediate the recognition of odor molecules. In order to identify potential ligand-binding residues, we have applied correlated mutation analysis to receptor sequences from the rat. This method identifies pairs of sequence positions where residues remain conserved or mutate in tandem, thereby suggesting structural or functional importance. The analysis supported molecular modeling studies in suggesting several residues in positions that were consistent with ligand-binding function. Two of these positions, dominated by histidine residues, may play important roles in ligand binding and could confer broad specificity to mammalian odor receptors. The presence of positive (overdominant) selection at some of the identified positions provides additional evidence for roles in ligand binding. Higher-order groups of correlated residues were also observed. Each group may interact with an individual ligand determinant, and combinations of these groups may provide a multi-dimensional mechanism for receptor diversity.

Frameshift mutational target gene analysis identifies similarities and differences in constitutional mismatch repair-deficiency and Lynch syndrome.

PubMed

Maletzki, Claudia; Huehns, Maja; Bauer, Ingrid; Ripperger, Tim; Mork, Maureen M; Vilar, Eduardo; Klöcking, Sabine; Zettl, Heike; Prall, Friedrich; Linnebacher, Michael

2017-07-01

Mismatch-repair deficient (MMR-D) malignancies include Lynch Syndrome (LS), which is secondary to germline mutations in one of the MMR genes, and the rare childhood-form of constitutional mismatch repair-deficiency (CMMR-D); caused by bi-allelic MMR gene mutations. A hallmark of LS-associated cancers is microsatellite instability (MSI), characterized by coding frameshift mutations (cFSM) in target genes. By contrast, tumors arising in CMMR-D patients are thought to display a somatic mutation pattern differing from LS. This study has the main goal to identify cFSM in MSI target genes relevant in CMMR-D and to compare the spectrum of common somatic mutations, including alterations in DNA polymerases POLE and D1 between LS and CMMR-D. CMMR-D-associated tumors harbored more somatic mutations compared to LS cases, especially in the TP53 gene and in POLE and POLD1, where novel mutations were additionally identified. Strikingly, MSI in classical mononucleotide markers BAT40 and CAT25 was frequent in CMMR-D cases. MSI-target gene analysis revealed mutations in CMMR-D-associated tumors, some of them known to be frequently hit in LS, such as RNaseT2, HT001, and TGFβR2. Our results imply a general role for these cFSM as potential new drivers of MMR-D tumorigenesis. © 2017 Wiley Periodicals, Inc.

Evolutionary Analysis Predicts Sensitive Positions of MMP20 and Validates Newly- and Previously-Identified MMP20 Mutations Causing Amelogenesis Imperfecta

PubMed Central

Gasse, Barbara; Prasad, Megana; Delgado, Sidney; Huckert, Mathilde; Kawczynski, Marzena; Garret-Bernardin, Annelyse; Lopez-Cazaux, Serena; Bailleul-Forestier, Isabelle; Manière, Marie-Cécile; Stoetzel, Corinne; Bloch-Zupan, Agnès; Sire, Jean-Yves

2017-01-01

Amelogenesis imperfecta (AI) designates a group of genetic diseases characterized by a large range of enamel disorders causing important social and health problems. These defects can result from mutations in enamel matrix proteins or protease encoding genes. A range of mutations in the enamel cleavage enzyme matrix metalloproteinase-20 gene (MMP20) produce enamel defects of varying severity. To address how various alterations produce a range of AI phenotypes, we performed a targeted analysis to find MMP20 mutations in French patients diagnosed with non-syndromic AI. Genomic DNA was isolated from saliva and MMP20 exons and exon-intron boundaries sequenced. We identified several homozygous or heterozygous mutations, putatively involved in the AI phenotypes. To validate missense mutations and predict sensitive positions in the MMP20 sequence, we evolutionarily compared 75 sequences extracted from the public databases using the Datamonkey webserver. These sequences were representative of mammalian lineages, covering more than 150 million years of evolution. This analysis allowed us to find 324 sensitive positions (out of the 483 MMP20 residues), pinpoint functionally important domains, and build an evolutionary chart of important conserved MMP20 regions. This is an efficient tool to identify new- and previously-identified mutations. We thus identified six functional MMP20 mutations in unrelated families, finding two novel mutated sites. The genotypes and phenotypes of these six mutations are described and compared. To date, 13 MMP20 mutations causing AI have been reported, making these genotypes and associated hypomature enamel phenotypes the most frequent in AI. PMID:28659819

Evolutionary Analysis Predicts Sensitive Positions of MMP20 and Validates Newly- and Previously-Identified MMP20 Mutations Causing Amelogenesis Imperfecta.

PubMed

Gasse, Barbara; Prasad, Megana; Delgado, Sidney; Huckert, Mathilde; Kawczynski, Marzena; Garret-Bernardin, Annelyse; Lopez-Cazaux, Serena; Bailleul-Forestier, Isabelle; Manière, Marie-Cécile; Stoetzel, Corinne; Bloch-Zupan, Agnès; Sire, Jean-Yves

2017-01-01

Amelogenesis imperfecta (AI) designates a group of genetic diseases characterized by a large range of enamel disorders causing important social and health problems. These defects can result from mutations in enamel matrix proteins or protease encoding genes. A range of mutations in the enamel cleavage enzyme matrix metalloproteinase-20 gene ( MMP20 ) produce enamel defects of varying severity. To address how various alterations produce a range of AI phenotypes, we performed a targeted analysis to find MMP20 mutations in French patients diagnosed with non-syndromic AI. Genomic DNA was isolated from saliva and MMP20 exons and exon-intron boundaries sequenced. We identified several homozygous or heterozygous mutations, putatively involved in the AI phenotypes. To validate missense mutations and predict sensitive positions in the MMP20 sequence, we evolutionarily compared 75 sequences extracted from the public databases using the Datamonkey webserver. These sequences were representative of mammalian lineages, covering more than 150 million years of evolution. This analysis allowed us to find 324 sensitive positions (out of the 483 MMP20 residues), pinpoint functionally important domains, and build an evolutionary chart of important conserved MMP20 regions. This is an efficient tool to identify new- and previously-identified mutations. We thus identified six functional MMP20 mutations in unrelated families, finding two novel mutated sites. The genotypes and phenotypes of these six mutations are described and compared. To date, 13 MMP20 mutations causing AI have been reported, making these genotypes and associated hypomature enamel phenotypes the most frequent in AI.

Sequencing ASMT identifies rare mutations in Chinese Han patients with autism.

PubMed

Wang, Lifang; Li, Jun; Ruan, Yanyan; Lu, Tianlan; Liu, Chenxing; Jia, Meixiang; Yue, Weihua; Liu, Jing; Bourgeron, Thomas; Zhang, Dai

2013-01-01

Melatonin is involved in the regulation of circadian and seasonal rhythms and immune function. Prior research reported low melatonin levels in autism spectrum disorders (ASD). ASMT located in pseudo-autosomal region 1 encodes the last enzyme of the melatonin biosynthesis pathway. A previous study reported an association between ASD and single nucleotide polymorphisms (SNPs) rs4446909 and rs5989681 located in the promoter of ASMT. Furthermore, rare deleterious mutations were identified in a subset of patients. To investigate the association between ASMT and autism, we sequenced all ASMT exons and its neighboring region in 398 Chinese Han individuals with autism and 437 healthy controls. Although our study did not detect significant differences of genotypic distribution and allele frequencies of the common SNPs in ASMT between patients with autism and healthy controls, we identified new rare coding mutations of ASMT. Among these rare variants, 4 were exclusively detected in patients with autism including a stop mutation (p.R115W, p.V166I, p.V179G, and p.W257X). These four coding variants were observed in 6 of 398 (1.51%) patients with autism and none in 437 controls (Chi-Square test, Continuity Correction p = 0.032, two-sided). Functional prediction of impact of amino acid showed that p.R115W might affect protein function. These results indicate that ASMT might be a susceptibility gene for autism. Further studies in larger samples are needed to better understand the degree of variation in this gene as well as to understand the biochemical and clinical impacts of ASMT/melatonin deficiency.

Identification of Mediterranean mutation in Egyptian favism patients.

PubMed

Osman, H G; Zahran, F M; El-Sokkary, A M A; El-Said, A; Sabry, A M

2014-10-01

Identify and screen the G6PD Mediterranean mutation in favism patients by applying a Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS-PCR). A total of 114 unrelated Egyptians patients were included in the present study; their ages ranged between (2-9) years with male to female ratio 4.5:1. G6PD activity was determined qualitatively from red cell hemolysate during attack. The G6PD Mediterranean mutation in patients has been identified by ARMS-PCR. G6PD deficiency was detected in 87.7%, (n=100). The frequency of G6PD Mediterranean mutation was (94.7%), (n=108). The association between G6PD deficiency and Mediterranean mutation was a highly significant. Glucose-6-phosphate dehydrogenase Mediterranean mutation is one of the most common mutations causing G6PD deficiency among Egyptian children with favism.

In Southern Africa, Brown Oculocutaneous Albinism (BOCA) Maps to the OCA2 Locus on Chromosome 15q: P-Gene Mutations Identified

PubMed Central

Manga, Prashiela; Kromberg, Jennifer G. R.; Turner, Angela; Jenkins, Trefor; Ramsay, Michele

2001-01-01

In southern Africa, brown oculocutaneous albinism (BOCA) is a distinct pigmentation phenotype. In at least two cases, it has occurred in the same families as tyrosinase-positive oculocutaneous albinism (OCA2), suggesting that it may be allelic, despite the fact that this phenotype was attributed to mutations in the TYRP1 gene in an American individual of mixed ancestry. Linkage analysis in five families mapped the BOCA locus to the same region as the OCA2 locus (maximum LOD 3.07; θ=0 using a six-marker haplotype). Mutation analysis of the human homologue of the mouse pink-eyed dilution gene (P), in 10 unrelated individuals with BOCA revealed that 9 had one copy of the 2.7-kb deletion. No other mutations were identified. Additional haplotype studies, based on closely linked markers (telomere to centromere: D15S1048, D15S1019, D15S1533, P-gene 2.7-kb deletion, D15S219, and D15S156) revealed several BOCA-associated P haplotypes. These could be divided into two core haplotypes, suggesting that a limited number of P-gene mutations give rise to this phenotype. PMID:11179026

Common variants at 12p11, 12q24, 9p21, 9q31.2 and in ZNF365 are associated with breast cancer risk for BRCA1 and/or BRCA2 mutation carriers

PubMed Central

2012-01-01

Introduction Several common alleles have been shown to be associated with breast and/or ovarian cancer risk for BRCA1 and BRCA2 mutation carriers. Recent genome-wide association studies of breast cancer have identified eight additional breast cancer susceptibility loci: rs1011970 (9p21, CDKN2A/B), rs10995190 (ZNF365), rs704010 (ZMIZ1), rs2380205 (10p15), rs614367 (11q13), rs1292011 (12q24), rs10771399 (12p11 near PTHLH) and rs865686 (9q31.2). Methods To evaluate whether these single nucleotide polymorphisms (SNPs) are associated with breast cancer risk for BRCA1 and BRCA2 carriers, we genotyped these SNPs in 12,599 BRCA1 and 7,132 BRCA2 mutation carriers and analysed the associations with breast cancer risk within a retrospective likelihood framework. Results Only SNP rs10771399 near PTHLH was associated with breast cancer risk for BRCA1 mutation carriers (per-allele hazard ratio (HR) = 0.87, 95% CI: 0.81 to 0.94, P-trend = 3 × 10-4). The association was restricted to mutations proven or predicted to lead to absence of protein expression (HR = 0.82, 95% CI: 0.74 to 0.90, P-trend = 3.1 × 10-5, P-difference = 0.03). Four SNPs were associated with the risk of breast cancer for BRCA2 mutation carriers: rs10995190, P-trend = 0.015; rs1011970, P-trend = 0.048; rs865686, 2df-P = 0.007; rs1292011 2df-P = 0.03. rs10771399 (PTHLH) was predominantly associated with estrogen receptor (ER)-negative breast cancer for BRCA1 mutation carriers (HR = 0.81, 95% CI: 0.74 to 0.90, P-trend = 4 × 10-5) and there was marginal evidence of association with ER-negative breast cancer for BRCA2 mutation carriers (HR = 0.78, 95% CI: 0.62 to 1.00, P-trend = 0.049). Conclusions The present findings, in combination with previously identified modifiers of risk, will ultimately lead to more accurate risk prediction and an improved understanding of the disease etiology in BRCA1 and BRCA2 mutation carriers. PMID:22348646

LNDriver: identifying driver genes by integrating mutation and expression data based on gene-gene interaction network.

PubMed

Wei, Pi-Jing; Zhang, Di; Xia, Junfeng; Zheng, Chun-Hou

2016-12-23

Cancer is a complex disease which is characterized by the accumulation of genetic alterations during the patient's lifetime. With the development of the next-generation sequencing technology, multiple omics data, such as cancer genomic, epigenomic and transcriptomic data etc., can be measured from each individual. Correspondingly, one of the key challenges is to pinpoint functional driver mutations or pathways, which contributes to tumorigenesis, from millions of functional neutral passenger mutations. In this paper, in order to identify driver genes effectively, we applied a generalized additive model to mutation profiles to filter genes with long length and constructed a new gene-gene interaction network. Then we integrated the mutation data and expression data into the gene-gene interaction network. Lastly, greedy algorithm was used to prioritize candidate driver genes from the integrated data. We named the proposed method Length-Net-Driver (LNDriver). Experiments on three TCGA datasets, i.e., head and neck squamous cell carcinoma, kidney renal clear cell carcinoma and thyroid carcinoma, demonstrated that the proposed method was effective. Also, it can identify not only frequently mutated drivers, but also rare candidate driver genes.

Clinical and functional characterization of TNNT2 mutations identified in patients with dilated cardiomyopathy

PubMed Central

Hershberger, Ray E.; Pinto, Jose Renato; Parks, Sharie B.; Kushner, Jessica D.; Li, Duanxiang; Ludwigsen, Susan; Cowan, Jason; Morales, Ana; Parvatiyar, Michelle S.; Potter, James D.

2009-01-01

Background A key issue for cardiovascular genetic medicine is ascertaining if a putative mutation indeed causes dilated cardiomyopathy (DCM). This is critically important as genetic DCM, usually presenting with advanced, life-threatening disease, may be preventable with early intervention in relatives known to carry the mutation. Methods and Results We recently undertook bidirectional resequencing of TNNT2, the cardiac troponin T gene, in 313 probands with DCM. We identified six TNNT2 protein-altering variants in nine probands, all who had early onset, aggressive disease. Additional family members of mutation carriers were then studied when available. Four of the nine probands had DCM without a family history, and five had familial DCM. Only one mutation (Lys210del) could be attributed as definitively causative from prior reports. Four of the five missense mutations were novel (Arg134Gly, Arg151Cys, Arg159Gln, Arg205Trp), and one was previously reported with hypertrophic cardiomyopathy (Glu244Asp). Based on the clinical, pedigree and molecular genetic data these five mutations were considered possibly or likely disease causing. To further clarify their potential pathophysiologic impact, we undertook functional studies of these mutations in cardiac myocytes reconstituted with mutant troponin T proteins. We observed decreased Ca2+ sensitivity of force development, a hallmark of DCM, in support of the conclusion that these mutations are disease-causing. Conclusions We conclude that the combination of clinical, pedigree, molecular genetic and functional data strengthen the interpretation of TNNT2 mutations in DCM. PMID:20031601

A novel APOC2 gene mutation identified in a Chinese patient with severe hypertriglyceridemia and recurrent pancreatitis.

PubMed

Jiang, Jingjing; Wang, Yuhui; Ling, Yan; Kayoumu, Abudurexiti; Liu, George; Gao, Xin

2016-01-16

The severe forms of hypertriglyceridemia are usually caused by genetic defects. In this study, we described a Chinese female with severe hypertriglyceridemia caused by a novel homozygous mutation in the APOC2 gene. Lipid profiles of the pedigree were studied in detail. LPL and HL activity were also measured. The coding regions of 5 candidate genes (namely LPL, APOC2, APOA5, LMF1, and GPIHBP1) were sequenced using genomic DNA from peripheral leucocytes. The ApoE gene was also genotyped. Serum triglyceride level was extremely high in the proband, compared with other family members. Plasma LPL activity was also significantly reduced in the proband. Serum ApoCII was very low in the proband as well as in the heterozygous mutation carriers. A novel mutation (c.86A > CC) was identified on exon 3 [corrected] of the APOC2 gene, which converted the Asp [corrected] codon at position 29 into Ala, followed by a termination codon (TGA). This study presented the first case of ApoCII deficiency in the Chinese population, with a novel mutation c.86A > CC in the APOC2 gene identified. Serum ApoCII protein might be a useful screening test for identifying mutation carriers.

Two females with mutations in USP9X highlight the variable expressivity of the intellectual disability syndrome.

PubMed

Au, P Y B; Huang, L; Broley, S; Gallagher, L; Creede, E; Lahey, D; Ordorica, S; Mina, K; Boycott, K M; Baynam, G; Dyment, D A

2017-07-01

The genetic causes of intellectual disability (ID) are heterogeneous and include both chromosomal and monogenic etiologies. The X-chromosome is known to contain many ID-related genes and males show a marked predominance for intellectual disability. Here we report two females with syndromic intellectual disability. The first individual was relatively mild in her presentation with mild-moderate intellectual disability, hydronephrosis and altered pigmentation along the lines of Blaschko without additional congenital anomalies. A second female presented shortly after birth with dysmorphic facial features, post-axial polydactyly and, on follow-up assessment, demonstrated moderate intellectual disability. Chromosomal studies for Individual 1 identified an X-chromosome deletion due to a de novo pericentric inversion; the inversion breakpoint was associated with deletion of the 5'UTR of the USP9X, a gene which has been implicated in a syndromic intellectual disability affecting females. The second individual had a de novo frameshift mutation detected by whole-exome sequencing that was predicted to be deleterious, NM_001039590.2 (USP9X): c.4104_4105del (p.(Arg1368Serfs*2)). Haploinsufficiency of USP9X in females has been associated with ID and congenital malformations that include heart defects, scoliosis, dental abnormalities, anal atresia, polydactyly, Dandy Walker malformation and hypoplastic corpus callosum. The extent of the congenital malformations observed in Individual 1 was less striking than Individual 2 and other individuals previously reported in the literature, and suggests that USP9X mutations in females can have a wider spectrum of presentation than previously appreciated. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

An integrative somatic mutation analysis to identify pathways linked with survival outcomes across 19 cancer types

PubMed Central

Park, Sunho; Kim, Seung-Jun; Yu, Donghyeon; Peña-Llopis, Samuel; Gao, Jianjiong; Park, Jin Suk; Chen, Beibei; Norris, Jessie; Wang, Xinlei; Chen, Min; Kim, Minsoo; Yong, Jeongsik; Wardak, Zabi; Choe, Kevin; Story, Michael; Starr, Timothy; Cheong, Jae-Ho; Hwang, Tae Hyun

2016-01-01

Motivation: Identification of altered pathways that are clinically relevant across human cancers is a key challenge in cancer genomics. Precise identification and understanding of these altered pathways may provide novel insights into patient stratification, therapeutic strategies and the development of new drugs. However, a challenge remains in accurately identifying pathways altered by somatic mutations across human cancers, due to the diverse mutation spectrum. We developed an innovative approach to integrate somatic mutation data with gene networks and pathways, in order to identify pathways altered by somatic mutations across cancers. Results: We applied our approach to The Cancer Genome Atlas (TCGA) dataset of somatic mutations in 4790 cancer patients with 19 different types of tumors. Our analysis identified cancer-type-specific altered pathways enriched with known cancer-relevant genes and targets of currently available drugs. To investigate the clinical significance of these altered pathways, we performed consensus clustering for patient stratification using member genes in the altered pathways coupled with gene expression datasets from 4870 patients from TCGA, and multiple independent cohorts confirmed that the altered pathways could be used to stratify patients into subgroups with significantly different clinical outcomes. Of particular significance, certain patient subpopulations with poor prognosis were identified because they had specific altered pathways for which there are available targeted therapies. These findings could be used to tailor and intensify therapy in these patients, for whom current therapy is suboptimal. Availability and implementation: The code is available at: http://www.taehyunlab.org. Contact: jhcheong@yuhs.ac or taehyun.hwang@utsouthwestern.edu or taehyun.cs@gmail.com Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26635139

Exome sequencing identifies titin mutations causing hereditary myopathy with early respiratory failure (HMERF) in families of diverse ethnic origins.

PubMed

Toro, Camilo; Olivé, Montse; Dalakas, Marinos C; Sivakumar, Kumaraswami; Bilbao, Juan M; Tyndel, Felix; Vidal, Noemí; Farrero, Eva; Sambuughin, Nyamkhishig; Goldfarb, Lev G

2013-03-20

Hereditary myopathy with early respiratory failure (HMERF) was described in several North European families and recently linked to a titin gene (TTN) mutation. We independently studied HMERF-like diseases with the purpose to identify the cause, refine diagnostic criteria, and estimate the frequency of this disease among myopathy patients of various ethnic origins. Whole exome sequencing analysis was carried out in a large U.S. family that included seven members suffering from skeletal muscle weakness and respiratory failure. Subsequent mutation screening was performed in further 45 unrelated probands with similar phenotypes. Studies included muscle strength evaluation, nerve conduction studies and concentric needle EMG, respiratory function test, cardiologic examination, and muscle biopsy. A novel TTN p.Gly30150Asp mutation was identified in the highly conserved A-band of titin that co-segregated with the disease in the U.S. family. Screening of 45 probands initially diagnosed as myofibrillar myopathy (MFM) but excluded based on molecular screening for the known MFM genes led to the identification of a previously reported TTN p.Cys30071Arg mutation in one patient. This same mutation was also identified in a patient with suspected HMERF. The p.Gly30150Asp and p.Cys30071Arg mutations are localized to a side chain of fibronectin type III element A150 of the 10th C-zone super-repeat of titin. Missense mutations in TTN are the cause of HMERF in families of diverse origins. A comparison of phenotypic features of HMERF caused by the three known TTN mutations in various populations allowed to emphasize distinct clinical/pathological features that can serve as the basis for diagnosis. The newly identified p.Gly30150Asp and the p.Cys30071Arg mutation are localized to a side chain of fibronectin type III element A150 of the 10th C-zone super-repeat of titin.

Phenotypes of Recessive Pediatric Cataract in a Cohort of Children with Identified Homozygous Gene Mutations (An American Ophthalmological Society Thesis)

PubMed Central

Khan, Arif O.; Aldahmesh, Mohammed A.; Alkuraya, Fowzan S.

2015-01-01

Purpose: To assess for phenotype-genotype correlations in families with recessive pediatric cataract and identified gene mutations. Methods: Retrospective review (2004 through 2013) of 26 Saudi Arabian apparently nonsyndromic pediatric cataract families referred to one of the authors (A.O.K.) and for which recessive gene mutations were identified. Results: Fifteen different homozygous recessive gene mutations were identified in the 26 consanguineous families; two genes and five families are novel to this study. Ten families had a founder CRYBB1 deletion (all with bilateral central pulverulent cataract), two had the same missense mutation in CRYAB (both with bilateral juvenile cataract with marked variable expressivity), and two had different mutations in FYCO1 (both with bilateral posterior capsular abnormality). The remaining 12 families each had mutations in 12 different genes (CRYAA, CRYBA1, AKR1E2, AGK, BFSP2, CYP27A1, CYP51A1, EPHA2, GCNT2, LONP1, RNLS, WDR87) with unique phenotypes noted for CYP27A1 (bilateral juvenile fleck with anterior and/or posterior capsular cataract and later cerebrotendinous xanthomatosis), EPHA2 (bilateral anterior persistent fetal vasculature), and BFSP2 (bilateral flecklike with cloudy cortex). Potential carrier signs were documented for several families. Conclusions: In this recessive pediatric cataract case series most identified genes are noncrystallin. Recessive pediatric cataract phenotypes are generally nonspecific, but some notable phenotypes are distinct and associated with specific gene mutations. Marked variable expressivity can occur from a recessive missense CRYAB mutation. Genetic analysis of apparently isolated pediatric cataract can sometimes uncover mutations in a syndromic gene. Some gene mutations seem to be associated with apparent heterozygous carrier signs. PMID:26622071

Spectrum of mutations in leiomyosarcomas identified by clinical targeted next-generation sequencing.

PubMed

Lee, Paul J; Yoo, Naomi S; Hagemann, Ian S; Pfeifer, John D; Cottrell, Catherine E; Abel, Haley J; Duncavage, Eric J

2017-02-01

Recurrent genomic mutations in uterine and non-uterine leiomyosarcomas have not been well established. Using a next generation sequencing (NGS) panel of common cancer-associated genes, 25 leiomyosarcomas arising from multiple sites were examined to explore genetic alterations, including single nucleotide variants (SNV), small insertions/deletions (indels), and copy number alterations (CNA). Sequencing showed 86 non-synonymous, coding region somatic variants within 151 gene targets in 21 cases, with a mean of 4.1 variants per case; 4 cases had no putative mutations in the panel of genes assayed. The most frequently altered genes were TP53 (36%), ATM and ATRX (16%), and EGFR and RB1 (12%). CNA were identified in 85% of cases, with the most frequent copy number losses observed in chromosomes 10 and 13 including PTEN and RB1; the most frequent gains were seen in chromosomes 7 and 17. Our data show that deletions in canonical cancer-related genes are common in leiomyosarcomas. Further, the spectrum of gene mutations observed shows that defects in DNA repair and chromosomal maintenance are central to the biology of leiomyosarcomas, and that activating mutations observed in other common cancer types are rare in leiomyosarcomas. Copyright © 2017 Elsevier Inc. All rights reserved.

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

PubMed

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

2014-04-01

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

Activating PIK3CA mutations coexist with BRAF or NRAS mutations in a limited fraction of melanomas.

PubMed

Manca, Antonella; Lissia, Amelia; Capone, Mariaelena; Ascierto, Paolo A; Botti, Gerardo; Caracò, Corrado; Stanganelli, Ignazio; Colombino, Maria; Sini, MariaCristina; Cossu, Antonio; Palmieri, Giuseppe

2015-01-28

Activated PI3K-AKT pathway may contribute to decrease sensitivity to inhibitors of key pathogenetic effectors (mutated BRAF, active NRAS or MEK) in melanoma. Functional alterations are deeply involved in PI3K-AKT activation, with a minimal role reported for mutations in PIK3CA, the catalytic subunit of the PI3K gene. We here assessed the prevalence of the coexistence of BRAF/NRAS and PIK3CA mutations in a series of melanoma samples. A total of 245 tumor specimens (212 primary melanomas and 33 melanoma cell lines) was screened for mutations in BRAF, NRAS, and PIK3CA genes by automated direct sequencing. Overall, 110 (44.9%) samples carried mutations in BRAF, 26 (10.6%) in NRAS, and 24 (9.8%) in PIK3CA. All identified PIK3CA mutations have been reported to induce PI3K activation; those detected in cultured melanomas were investigated for their interference with the antiproliferative activity of the BRAF-mutant inhibitor vemurafenib. A reduced suppression in cell growth was observed in treated cells carrying both BRAF and PIK3CA mutations as compared with those presenting a mutated BRAF only. Among the analysed melanomas, 12/245 (4.9%) samples presented the coexistence of PIK3CA and BRAF/NRAS mutations. Our study further suggests that PIK3CA mutations account for a small fraction of PI3K pathway activation and have a limited impact in interfering with the BRAF/NRAS-driven growth in melanoma.

Mutation screening of HOXA7 and HOXA9 genes in Chinese women with Müllerian duct abnormalities.

PubMed

Chen, Xinxia; Mu, Yulan; Li, Chunyan; Li, Guangyu; Zhao, Hui; Qin, Yingying; Chen, Zi-Jiang

2014-11-01

HOXA genes in groups 7-13 have been proven to play a role in determining positional identity along the genitalia axis. The aim of the present study was to explore the relationship between HOXA7 and HOXA9 mutations and Müllerian duct abnormalities (MDA). One hundred and ninety-two Chinese patients with MDA abnormalities and 192 healthy controls were recruited. All coding regions of HOXA7 and HOXA9 were amplified and sequenced directly. Rs2301721 and rs2301720 in HOXA7, rs35355140 and rs7810502 in HOXA9 were identified in patients with MDA and controls. One rare single nucleotide polymorphism rs189587233 in 3' UTR of HOXA9 gene was detected in one patient with didelphic uterus and absent in the 192 controls. This polymorphism, however, is known to exist in the normal Chinese population. Our results indicated that variants in the HOXA7 and HOXA9 genes were not common in Chinese women with Müllerian duct abnormalities. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Combining Single Strand Oligodeoxynucleotides and CRISPR/Cas9 to Correct Gene Mutations in β-Thalassemia-induced Pluripotent Stem Cells.

PubMed

Niu, Xiaohua; He, Wenyin; Song, Bing; Ou, Zhanhui; Fan, Di; Chen, Yuchang; Fan, Yong; Sun, Xiaofang

2016-08-05

β-Thalassemia (β-Thal) is one of the most common genetic diseases in the world. The generation of patient-specific β-Thal-induced pluripotent stem cells (iPSCs), correction of the disease-causing mutations in those cells, and then differentiation into hematopoietic stem cells offers a new therapeutic strategy for this disease. Here, we designed a CRISPR/Cas9 to specifically target the Homo sapiens hemoglobin β (HBB) gene CD41/42(-CTTT) mutation. We demonstrated that the combination of single strand oligodeoxynucleotides with CRISPR/Cas9 was capable of correcting the HBB gene CD41/42 mutation in β-Thal iPSCs. After applying a correction-specific PCR assay to purify the corrected clones followed by sequencing to confirm mutation correction, we verified that the purified clones retained full pluripotency and exhibited normal karyotyping. Additionally, whole-exome sequencing showed that the mutation load to the exomes was minimal after CRISPR/Cas9 targeting. Furthermore, the corrected iPSCs were selected for erythroblast differentiation and restored the expression of HBB protein compared with the parental iPSCs. This method provides an efficient and safe strategy to correct the HBB gene mutation in β-Thal iPSCs. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Whole exome sequencing identifies mutations in Usher syndrome genes in profoundly deaf Tunisian patients.

PubMed

Riahi, Zied; Bonnet, Crystel; Zainine, Rim; Lahbib, Saida; Bouyacoub, Yosra; Bechraoui, Rym; Marrakchi, Jihène; Hardelin, Jean-Pierre; Louha, Malek; Largueche, Leila; Ben Yahia, Salim; Kheirallah, Moncef; Elmatri, Leila; Besbes, Ghazi; Abdelhak, Sonia; Petit, Christine

2015-01-01

Usher syndrome (USH) is an autosomal recessive disorder characterized by combined deafness-blindness. It accounts for about 50% of all hereditary deafness blindness cases. Three clinical subtypes (USH1, USH2, and USH3) are described, of which USH1 is the most severe form, characterized by congenital profound deafness, constant vestibular dysfunction, and a prepubertal onset of retinitis pigmentosa. We performed whole exome sequencing in four unrelated Tunisian patients affected by apparently isolated, congenital profound deafness, with reportedly normal ocular fundus examination. Four biallelic mutations were identified in two USH1 genes: a splice acceptor site mutation, c.2283-1G>T, and a novel missense mutation, c.5434G>A (p.Glu1812Lys), in MYO7A, and two previously unreported mutations in USH1G, i.e. a frameshift mutation, c.1195_1196delAG (p.Leu399Alafs*24), and a nonsense mutation, c.52A>T (p.Lys18*). Another ophthalmological examination including optical coherence tomography actually showed the presence of retinitis pigmentosa in all the patients. Our findings provide evidence that USH is under-diagnosed in Tunisian deaf patients. Yet, early diagnosis of USH is of utmost importance because these patients should undergo cochlear implant surgery in early childhood, in anticipation of the visual loss.

The C9ORF72 expansion mutation is a common cause of ALS+/-FTD in Europe and has a single founder.

PubMed

Smith, Bradley N; Newhouse, Stephen; Shatunov, Aleksey; Vance, Caroline; Topp, Simon; Johnson, Lauren; Miller, Jack; Lee, Younbok; Troakes, Claire; Scott, Kirsten M; Jones, Ashley; Gray, Ian; Wright, Jamie; Hortobágyi, Tibor; Al-Sarraj, Safa; Rogelj, Boris; Powell, John; Lupton, Michelle; Lovestone, Simon; Sapp, Peter C; Weber, Markus; Nestor, Peter J; Schelhaas, Helenius J; Asbroek, Anneloor Alm Ten; Silani, Vincenzo; Gellera, Cinzia; Taroni, Franco; Ticozzi, Nicola; Van den Berg, Leonard; Veldink, Jan; Van Damme, Phillip; Robberecht, Wim; Shaw, Pamela J; Kirby, Janine; Pall, Hardev; Morrison, Karen E; Morris, Alex; de Belleroche, Jacqueline; Vianney de Jong, J M B; Baas, Frank; Andersen, Peter M; Landers, John; Brown, Robert H; Weale, Michael E; Al-Chalabi, Ammar; Shaw, Christopher E

2013-01-01

A massive hexanucleotide repeat expansion mutation (HREM) in C9ORF72 has recently been linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we describe the frequency, origin and stability of this mutation in ALS+/-FTD from five European cohorts (total n=1347). Single-nucleotide polymorphisms defining the risk haplotype in linked kindreds were genotyped in cases (n=434) and controls (n=856). Haplotypes were analysed using PLINK and aged using DMLE+. In a London clinic cohort, the HREM was the most common mutation in familial ALS+/-FTD: C9ORF72 29/112 (26%), SOD1 27/112 (24%), TARDBP 1/112 (1%) and FUS 4/112 (4%) and detected in 13/216 (6%) of unselected sporadic ALS cases but was rare in controls (3/856, 0.3%). HREM prevalence was high for familial ALS+/-FTD throughout Europe: Belgium 19/22 (86%), Sweden 30/41 (73%), the Netherlands 10/27 (37%) and Italy 4/20 (20%). The HREM did not affect the age at onset or survival of ALS patients. Haplotype analysis identified a common founder in all 137 HREM carriers that arose around 6300 years ago. The haplotype from which the HREM arose is intrinsically unstable with an increased number of repeats (average 8, compared with 2 for controls, P<10(-8)). We conclude that the HREM has a single founder and is the most common mutation in familial and sporadic ALS in Europe.

Two Novel HOGA1 Splicing Mutations Identified in a Chinese Patient with Primary Hyperoxaluria Type 3.

PubMed

Wang, Xinsheng; Zhao, Xiangzhong; Wang, Xiaoling; Yao, Jian; Zhang, Feifei; Lang, Yanhua; Tuffery-Giraud, Sylvie; Bottillo, Irene; Shao, Leping

2015-01-01

Twenty-six HOGA1 mutations have been reported in primary hyperoxaluria (PH) type 3 (PH3) patients with c.700 + 5G>T accounting for about 50% of the total alleles. However, PH3 has never been described in Asians. A Chinese child with early-onset nephrolithiasis was suspected of having PH. We searched for AGXT, GRHPR and HOGA1 gene mutations in this patient and his parents. All coding regions, including intron-exon boundaries, were analyzed using PCR followed by direct sequence analysis. Two heterozygous mutations not previously described in the literature about HOGA1 were identified (compound heterozygous). One mutation was a successive 2 bp substitution at the last nucleotide of exon 6 and at the first nucleotide of intron 6, respectively (c.834_834 + 1GG>TT), while the other one was a guanine to adenine substitution of the last nucleotide of exon 6 (c.834G>A). Direct sequencing analysis failed to find these mutations in 100 unrelated healthy subjects and the functional role on splicing of both variants found in this study was confirmed by a minigene assay based on the pSPL3 exon trapping vector. In addition, we found a SNP in this family (c.715G>A, p.V239I). There were no mutations detected in AGXT and GRHPR. Two novel HOGA1 mutations were identified in association with PH3. This is the first description and investigation on mutant gene analysis of PH3 in an Asian. © 2015 S. Karger AG, Basel

An in-silico method for identifying aggregation rate enhancer and mitigator mutations in proteins.

PubMed

Rawat, Puneet; Kumar, Sandeep; Michael Gromiha, M

2018-06-24

Newly synthesized polypeptides must pass stringent quality controls in cells to ensure appropriate folding and function. However, mutations, environmental stresses and aging can reduce efficiencies of these controls, leading to accumulation of protein aggregates, amyloid fibrils and plaques. In-vitro experiments have shown that even single amino acid substitutions can drastically enhance or mitigate protein aggregation kinetics. In this work, we have collected a dataset of 220 unique mutations in 25 proteins and classified them as enhancers or mitigators on the basis of their effect on protein aggregation rate. The data were analyzed via machine learning to identify features capable of distinguishing between aggregation rate enhancers and mitigators. Our initial Support Vector Machine (SVM) model separated such mutations with an overall accuracy of 69%. When local secondary structures at the mutation sites were considered, the accuracies further improved by 13-15%. The machine-learnt features are distinct for each secondary structure class at mutation sites. Protein stability and flexibility changes are important features for mutations in α-helices. β-strand propensity, polarity and charge become important when mutations occur in β-strands and ability to form secondary structure, helical tendency and aggregation propensity are important for mutations lying in coils. These results have been incorporated into a sequence-based algorithm (available at http://www.iitm.ac.in/bioinfo/aggrerate-disc/) capable of predicting whether a mutation will enhance or mitigate a protein's aggregation rate. This algorithm will find several applications towards understanding protein aggregation in human diseases, enable in-silico optimization of biopharmaceuticals and enzymes for improved biophysical attributes and de novo design of bio-nanomaterials. Copyright © 2018. Published by Elsevier B.V.

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.

Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung.

PubMed

Martini, Sabrina V; Silva, Adriana L; Ferreira, Debora; Rabelo, Rafael; Ornellas, Felipe M; Gomes, Karina; Rocco, Patricia R M; Petrs-Silva, Hilda; Morales, Marcelo M

2016-01-01

Adeno-associated virus (AAV) vectors are being increasingly used as the vector of choice for in vivo gene delivery and gene therapy for many pulmonary diseases. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. In this study, we evaluated the pulmonary transgene expression efficacy of AAV9 vectors containing point mutations in surface-exposed capsid tyrosine residues. Eighteen C57BL/6 mice were randomly assigned into three groups: (1) a control group (CTRL) animals underwent intratracheal (i.t.) instillation of saline, (2) the wild-type AAV9 group (WT-AAV9, 1010 vg), and (3) the tyrosine-mutant Y731F AAV9 group (M-AAV9, 1010 vg), which received (i.t.) self-complementary AAV9 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP). Four weeks after instillation, lung mechanics, morphometry, tissue cellularity, gene expression, inflammatory cytokines, and growth factor expression were analyzed. No significant differences were observed in lung mechanics and morphometry among the experimental groups. However, the number of polymorphonuclear cells was higher in the WT-AAV9 group than in the CTRL and M-AAV9 groups, suggesting that the administration of tyrosine-mutant AAV9 vectors was better tolerated. Tyrosine-mutant AAV9 vectors significantly improved transgene delivery to the lung (30%) compared with their wild-type counterparts, without eliciting an inflammatory response. Our results provide the impetus for further studies to exploit the use of AAV9 vectors as a tool for pulmonary gene therapy. © 2016 The Author(s) Published by S. Karger AG, Basel.

VCP gene analyses in Japanese patients with sporadic amyotrophic lateral sclerosis identify a new mutation.

PubMed

Hirano, Makito; Nakamura, Yusaku; Saigoh, Kazumasa; Sakamoto, Hikaru; Ueno, Shuichi; Isono, Chiharu; Mitsui, Yoshiyuki; Kusunoki, Susumu

2015-03-01

Accumulating evidence has proven that mutations in the VCP gene encoding valosin-containing protein (VCP) cause inclusion body myopathy with Paget disease of the bone and frontotemporal dementia. This gene was later found to be causative for amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease, occurring typically in elderly persons. We thus sequenced the VCP gene in 75 Japanese patients with sporadic ALS negative for mutations in other genes causative for ALS and found a novel mutation, p.Arg487His, in 1 patient. The newly identified mutant as well as known mutants rendered neuronal cells susceptible to oxidative stress. The presence of the mutation in the Japanese population extends the geographic region for involvement of the VCP gene in sporadic ALS to East Asia. Copyright © 2015 Elsevier Inc. All rights reserved.

Exome Sequencing Identified a Recessive RDH12 Mutation in a Family with Severe Early-Onset Retinitis Pigmentosa

PubMed Central

Gong, Bo; Wei, Bo; Huang, Lulin; Hao, Jilong; Li, Xiulan; Yang, Yin; Zhou, Yu; Hao, Fang; Cui, Zhihua; Zhang, Dingding; Wang, Le

2015-01-01

Retinitis pigmentosa (RP) is the most important hereditary retinal disease caused by progressive degeneration of the photoreceptor cells. This study is to identify gene mutations responsible for autosomal recessive retinitis pigmentosa (arRP) in a Chinese family using next-generation sequencing technology. A Chinese family with 7 members including two individuals affected with severe early-onset RP was studied. All patients underwent a complete ophthalmic examination. Exome sequencing was performed on a single RP patient (the proband of this family) and direct Sanger sequencing on other family members and normal controls was followed to confirm the causal mutations. A homozygous mutation c.437Tidentified as being related to the phenotype of this arRP family. This homozygous mutation was detected in the two affected patients, but not present in other family members and 600 normal controls. Another three normal members in the family were found to carry this heterozygous missense mutation. Our results emphasize the importance of c.437Tmutation in the pathogenesis and clinical diagnosis of RP. PMID:26124963

Phenotypes in siblings with homozygous mutations of TRAPPC9 and/or MCPH1 support a bifunctional model of MCPH1.

PubMed

Duerinckx, Sarah; Meuwissen, Marije; Perazzolo, Camille; Desmyter, Laurence; Pirson, Isabelle; Abramowicz, Marc

2018-04-24

Autosomal recessive intellectual disability (ARID) is vastly heterogeneous. Truncating mutations of TRAPPC9 were reported in 8 ARID families. Autosomal recessive primary microcephaly (MCPH) represents another subgroup of ARID, itself very heterogeneous, where the size of the brain is very small since birth. MCPH1 plays a role at the centrosome via a BRCT1 domain, and in DNA Damage Repair (DDR) via BRCT2 and BRCT3, and it is not clear which of these two mechanisms causes MCPH in man. We studied the phenotype and sequenced the exome in two siblings with MCPH and their unaffected sister. Homozygous mutations of TRAPPC9 (p.Leu178Pro) and of MCPH1 (p.Arg741X) were found in both affected siblings. Brain MRI showed anomalies previously associated with TRAPPC9 defects, supporting the implication of TRAPPC9 in the phenotype. Importantly, the asymptomatic sister with normal head size was homozygous for the MCPH1 truncating mutation and heterozygous for the TRAPPC9 mutation. The affected siblings represent the first ARID cases with a TRAPPC9 missense mutation and with microcephaly of prenatal onset of. Furthermore, their unaffected sister represents strong evidence that the lack of MCPH1 BRCT3 domain does not cause MCPH in man, supporting a bifunctional model of MCPH1 where the centrosomal function is involved in brain volumic development and not the DDR function. © 2018 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.

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

PubMed

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

2013-03-01

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

A Novel Founder Mutation in MYBPC3: Phenotypic Comparison With the Most Prevalent MYBPC3 Mutation in Spain.

PubMed

Sabater-Molina, María; Saura, Daniel; García-Molina Sáez, Esperanza; González-Carrillo, Josefa; Polo, Luis; Pérez-Sánchez, Inmaculada; Olmo, María Del Carmen; Oliva-Sandoval, María José; Barriales-Villa, Roberto; Carbonell, Pablo; Pascual-Figal, Domigo; Gimeno, Juan R

2017-02-01

Mutations in MYBPC3 are the cause of hypertrophic cardiomyopathy (HCM). Although most lead to a truncating protein, the severity of the phenotype differs. We describe the clinical phenotype of a novel MYBPC3 mutation, p.Pro108Alafs*9, present in 13 families from southern Spain and compare it with the most prevalent MYBPC3 mutation in this region (c.2308+1 G>A). We studied 107 relatives of 13 index cases diagnosed as HCM carriers of the p.Pro108Alafs*9 mutation. Pedigree analysis, clinical evaluation, and genotyping were performed. A total of 54 carriers of p.Pro108Alafs*9 were identified, of whom 39 had HCM. There were 5 cases of sudden death in the 13 families. Disease penetrance was greater as age increased and HCM patients were more frequently male and developed disease earlier than female patients. The phenotype was similar in p.Pro108Alafs*9 and in c.2308+1 G>A, but differences were found in several risk factors and in survival. There was a trend toward a higher left ventricular mass in p.Pro108Alafs*9 vs c.2308+1G>A. Cardiac magnetic resonance revealed a similar extent and pattern of fibrosis. The p.Pro108Alafs*9 mutation is associated with HCM, high penetrance, and disease onset in middle age. Copyright © 2016 Sociedad Española de Cardiología. Published by Elsevier España, S.L.U. All rights reserved.

Genome-Wide Linkage Analysis to Identify Genetic Modifiers of ALK Mutation Penetrance in Familial Neuroblastoma

PubMed Central

Devoto, Marcella; Specchia, Claudia; Laudenslager, Marci; Longo, Luca; Hakonarson, Hakon; Maris, John; Mossé, Yael

2011-01-01

Background Neuroblastoma (NB) is an important childhood cancer with a strong genetic component related to disease susceptibility. Approximately 1% of NB cases have a positive family history. Following a genome-wide linkage analysis and sequencing of candidate genes in the critical region, we identified ALK as the major familial NB gene. Dominant mutations in ALK are found in more than 50% of familial NB cases. However, in the families used for the linkage study, only about 50% of carriers of ALK mutations are affected by NB. Methods To test whether genetic variation may explain the reduced penetrance of the disease phenotype, we analyzed genome-wide genotype data in ALK mutation-positive families using a model-based linkage approach with different liability classes for carriers and non-carriers of ALK mutations. Results The region with the highest LOD score was located at chromosome 2p23–p24 and included the ALK locus under models of dominant and recessive inheritance. Conclusions This finding suggests that variants in the non-mutated ALK gene or another gene linked to it may affect penetrance of the ALK mutations and risk of developing NB in familial cases. PMID:21734404

Efficient Knock-in of a Point Mutation in Porcine Fibroblasts Using the CRISPR/Cas9-GMNN Fusion Gene.

PubMed

Gerlach, Max; Kraft, Theresia; Brenner, Bernhard; Petersen, Björn; Niemann, Heiner; Montag, Judith

2018-06-13

During CRISPR/Cas9 mediated genome editing, site-specific double strand breaks are introduced and repaired either unspecific by non-homologous end joining (NHEJ) or sequence dependent by homology directed repair (HDR). Whereas NHEJ-based generation of gene knock-out is widely performed, the HDR-based knock-in of specific mutations remains a bottleneck. Especially in primary cell lines that are essential for the generation of cell culture and animal models of inherited human diseases, knock-in efficacy is insufficient and needs significant improvement. Here, we tested two different approaches to increase the knock-in frequency of a specific point mutation into the MYH7 -gene in porcine fetal fibroblasts. We added a small molecule inhibitor of NHEJ, SCR7 (5,6-bis((E)-benzylideneamino)-2-mercaptopyrimidin-4-ol), during genome editing and screened cell cultures for the point mutation. However, this approach did not yield increased knock-in rates. In an alternative approach, we fused humanized Cas9 (hCas9) to the N-terminal peptide of the Geminin gene ( GMNN ). The fusion protein is degraded in NHEJ-dominated cell cycle phases, which should increase HDR-rates. Using hCas9- GMNN and point mutation-specific real time PCR screening, we found a two-fold increase in genome edited cell cultures. This increase of HDR by hCas9- GMNN provides a promising way to enrich specific knock-in in porcine fibroblast cultures for somatic cloning approaches.

Loss of function mutations in RPL27 and RPS27 identified by whole-exome sequencing in Diamond-Blackfan anaemia.

PubMed

Wang, RuNan; Yoshida, Kenichi; Toki, Tsutomu; Sawada, Takafumi; Uechi, Tamayo; Okuno, Yusuke; Sato-Otsubo, Aiko; Kudo, Kazuko; Kamimaki, Isamu; Kanezaki, Rika; Shiraishi, Yuichi; Chiba, Kenichi; Tanaka, Hiroko; Terui, Kiminori; Sato, Tomohiko; Iribe, Yuji; Ohga, Shouichi; Kuramitsu, Madoka; Hamaguchi, Isao; Ohara, Akira; Hara, Junichi; Goi, Kumiko; Matsubara, Kousaku; Koike, Kenichi; Ishiguro, Akira; Okamoto, Yasuhiro; Watanabe, Kenichiro; Kanno, Hitoshi; Kojima, Seiji; Miyano, Satoru; Kenmochi, Naoya; Ogawa, Seishi; Ito, Etsuro

2015-03-01

Diamond-Blackfan anaemia is a congenital bone marrow failure syndrome that is characterized by red blood cell aplasia. The disease has been associated with mutations or large deletions in 11 ribosomal protein genes including RPS7, RPS10, RPS17, RPS19, RPS24, RPS26, RPS29, RPL5, RPL11, RPL26 and RPL35A as well as GATA1 in more than 50% of patients. However, the molecular aetiology of many Diamond-Blackfan anaemia cases remains to be uncovered. To identify new mutations responsible for Diamond-Blackfan anaemia, we performed whole-exome sequencing analysis of 48 patients with no documented mutations/deletions involving known Diamond-Blackfan anaemia genes except for RPS7, RPL26, RPS29 and GATA1. Here, we identified a de novo splicing error mutation in RPL27 and frameshift deletion in RPS27 in sporadic patients with Diamond-Blackfan anaemia. In vitro knockdown of gene expression disturbed pre-ribosomal RNA processing. Zebrafish models of rpl27 and rps27 mutations showed impairments of erythrocyte production and tail and/or brain development. Additional novel mutations were found in eight patients, including RPL3L, RPL6, RPL7L1T, RPL8, RPL13, RPL14, RPL18A and RPL31. In conclusion, we identified novel germline mutations of two ribosomal protein genes responsible for Diamond-Blackfan anaemia, further confirming the concept that mutations in ribosomal protein genes lead to Diamond-Blackfan anaemia. © 2014 John Wiley & Sons Ltd.

Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip

NASA Astrophysics Data System (ADS)

Li, Ren; Zhou, Mingxing; Li, Jine; Wang, Zihua; Zhang, Weikai; Yue, Chunyan; Ma, Yan; Peng, Hailin; Wei, Zewen; Hu, Zhiyuan

2018-03-01

EGFR mutations companion diagnostics have been proved to be crucial for the efficacy of tyrosine kinase inhibitor targeted cancer therapies. To uncover multiple mutations occurred in minority of EGFR-mutated cells, which may be covered by the noises from majority of un-mutated cells, is currently becoming an urgent clinical requirement. Here we present the validation of a microfluidic-chip-based method for detecting EGFR multi-mutations at single-cell level. By trapping and immunofluorescently imaging single cells in specifically designed silicon microwells, the EGFR-expressed cells were easily identified. By in situ lysing single cells, the cell lysates of EGFR-expressed cells were retrieved without cross-contamination. Benefited from excluding the noise from cells without EGFR expression, the simple and cost-effective Sanger's sequencing, but not the expensive deep sequencing of the whole cell population, was used to discover multi-mutations. We verified the new method with precisely discovering three most important EGFR drug-related mutations from a sample in which EGFR-mutated cells only account for a small percentage of whole cell population. The microfluidic chip is capable of discovering not only the existence of specific EGFR multi-mutations, but also other valuable single-cell-level information: on which specific cells the mutations occurred, or whether different mutations coexist on the same cells. This microfluidic chip constitutes a promising method to promote simple and cost-effective Sanger's sequencing to be a routine test before performing targeted cancer therapy.[Figure not available: see fulltext.

An innovative strategy for the molecular diagnosis of Usher syndrome identifies causal biallelic mutations in 93% of European patients.

PubMed

Bonnet, Crystel; Riahi, Zied; Chantot-Bastaraud, Sandra; Smagghe, Luce; Letexier, Mélanie; Marcaillou, Charles; Lefèvre, Gaëlle M; Hardelin, Jean-Pierre; El-Amraoui, Aziz; Singh-Estivalet, Amrit; Mohand-Saïd, Saddek; Kohl, Susanne; Kurtenbach, Anne; Sliesoraityte, Ieva; Zobor, Ditta; Gherbi, Souad; Testa, Francesco; Simonelli, Francesca; Banfi, Sandro; Fakin, Ana; Glavač, Damjan; Jarc-Vidmar, Martina; Zupan, Andrej; Battelino, Saba; Martorell Sampol, Loreto; Claveria, Maria Antonia; Catala Mora, Jaume; Dad, Shzeena; Møller, Lisbeth B; Rodriguez Jorge, Jesus; Hawlina, Marko; Auricchio, Alberto; Sahel, José-Alain; Marlin, Sandrine; Zrenner, Eberhart; Audo, Isabelle; Petit, Christine

2016-12-01

Usher syndrome (USH), the most prevalent cause of hereditary deafness-blindness, is an autosomal recessive and genetically heterogeneous disorder. Three clinical subtypes (USH1-3) are distinguishable based on the severity of the sensorineural hearing impairment, the presence or absence of vestibular dysfunction, and the age of onset of the retinitis pigmentosa. A total of 10 causal genes, 6 for USH1, 3 for USH2, and 1 for USH3, and an USH2 modifier gene, have been identified. A robust molecular diagnosis is required not only to improve genetic counseling, but also to advance gene therapy in USH patients. Here, we present an improved diagnostic strategy that is both cost- and time-effective. It relies on the sequential use of three different techniques to analyze selected genomic regions: targeted exome sequencing, comparative genome hybridization, and quantitative exon amplification. We screened a large cohort of 427 patients (139 USH1, 282 USH2, and six of undefined clinical subtype) from various European medical centers for mutations in all USH genes and the modifier gene. We identified a total of 421 different sequence variants predicted to be pathogenic, about half of which had not been previously reported. Remarkably, we detected large genomic rearrangements, most of which were novel and unique, in 9% of the patients. Thus, our strategy led to the identification of biallelic and monoallelic mutations in 92.7% and 5.8% of the USH patients, respectively. With an overall 98.5% mutation characterization rate, the diagnosis efficiency was substantially improved compared with previously reported methods.

An innovative strategy for the molecular diagnosis of Usher syndrome identifies causal biallelic mutations in 93% of European patients

PubMed Central

Bonnet, Crystel; Riahi, Zied; Chantot-Bastaraud, Sandra; Smagghe, Luce; Letexier, Mélanie; Marcaillou, Charles; Lefèvre, Gaëlle M; Hardelin, Jean-Pierre; El-Amraoui, Aziz; Singh-Estivalet, Amrit; Mohand-Saïd, Saddek; Kohl, Susanne; Kurtenbach, Anne; Sliesoraityte, Ieva; Zobor, Ditta; Gherbi, Souad; Testa, Francesco; Simonelli, Francesca; Banfi, Sandro; Fakin, Ana; Glavač, Damjan; Jarc-Vidmar, Martina; Zupan, Andrej; Battelino, Saba; Martorell Sampol, Loreto; Claveria, Maria Antonia; Catala Mora, Jaume; Dad, Shzeena; Møller, Lisbeth B; Rodriguez Jorge, Jesus; Hawlina, Marko; Auricchio, Alberto; Sahel, José-Alain; Marlin, Sandrine; Zrenner, Eberhart; Audo, Isabelle; Petit, Christine

2016-01-01

Usher syndrome (USH), the most prevalent cause of hereditary deafness–blindness, is an autosomal recessive and genetically heterogeneous disorder. Three clinical subtypes (USH1–3) are distinguishable based on the severity of the sensorineural hearing impairment, the presence or absence of vestibular dysfunction, and the age of onset of the retinitis pigmentosa. A total of 10 causal genes, 6 for USH1, 3 for USH2, and 1 for USH3, and an USH2 modifier gene, have been identified. A robust molecular diagnosis is required not only to improve genetic counseling, but also to advance gene therapy in USH patients. Here, we present an improved diagnostic strategy that is both cost- and time-effective. It relies on the sequential use of three different techniques to analyze selected genomic regions: targeted exome sequencing, comparative genome hybridization, and quantitative exon amplification. We screened a large cohort of 427 patients (139 USH1, 282 USH2, and six of undefined clinical subtype) from various European medical centers for mutations in all USH genes and the modifier gene. We identified a total of 421 different sequence variants predicted to be pathogenic, about half of which had not been previously reported. Remarkably, we detected large genomic rearrangements, most of which were novel and unique, in 9% of the patients. Thus, our strategy led to the identification of biallelic and monoallelic mutations in 92.7% and 5.8% of the USH patients, respectively. With an overall 98.5% mutation characterization rate, the diagnosis efficiency was substantially improved compared with previously reported methods. PMID:27460420

Spectrum of mismatch repair gene mutations and clinical presentation of Hispanic individuals with Lynch syndrome.

PubMed

Sunga, Annette Y; Ricker, Charité; Espenschied, Carin R; Castillo, Danielle; Melas, Marilena; Herzog, Josef; Bannon, Sarah; Cruz-Correa, Marcia; Lynch, Patrick; Solomon, Ilana; Gruber, Stephen B; Weitzel, Jeffrey N

2017-04-01

Lynch syndrome (LS), the most common hereditary colorectal cancer syndrome, is caused by mismatch repair (MMR) gene mutations. However, data about MMR mutations in Hispanics are limited. This study aims to describe the spectrum of MMR mutations in Hispanics with LS and explore ancestral origins. This case series involved an IRB-approved retrospective chart review of self-identified Hispanic patients (n = 397) seen for genetic cancer risk assessment at four collaborating academic institutions in California, Texas, and Puerto Rico who were evaluated by MMR genotyping and/or tumor analysis. A literature review was conducted for all mutations identified. Of those who underwent clinical genetic testing (n = 176), 71 had MMR gene mutations. Nine mutations were observed more than once. One third (3/9) of recurrent mutations and two additional mutations (seen only once) were previously reported in Spain, confirming the influence of Spanish ancestry on MMR mutations in Hispanic populations. The recurrent mutations identified (n = 9) included both previously reported mutations as well as unique mutations not in the literature. This is the largest report of Hispanic MMR mutations in North America; however, a larger sample and haplotype analyses are needed to better understand recurrent MMR mutations in Hispanic populations. Copyright © 2017. Published by Elsevier Inc.

EIF2AK4 Mutations in Patients Diagnosed With Pulmonary Arterial Hypertension.

PubMed

Best, D Hunter; Sumner, Kelli L; Smith, Benjamin P; Damjanovich-Colmenares, Kristy; Nakayama, Ikue; Brown, Lynette M; Ha, Youna; Paul, Eleri; Morris, Ashley; Jama, Mohamed A; Dodson, Mark W; Bayrak-Toydemir, Pinar; Elliott, C Gregory

2017-04-01

Differentiating pulmonary venoocclusive disease (PVOD) and pulmonary capillary hemangiomatosis (PCH) from idiopathic pulmonary arterial hypertension (IPAH) or heritable pulmonary arterial hypertension (HPAH) is important clinically. Mutations in eukaryotic translation initiation factor 2 alpha kinase 4 (EIF2AK4) cause heritable PVOD and PCH, whereas mutations in other genes cause HPAH. The aim of this study was to describe the frequency of pathogenic EIF2AK4 mutations in patients diagnosed clinically with IPAH or HPAH. Sanger sequencing and deletion/duplication analysis were performed to detect mutations in the bone morphogenetic protein receptor type II (BMPR2) gene in 81 patients diagnosed at 30 North American medical centers with IPAH (n = 72) or HPAH (n = 9). BMPR2 mutation-negative patients (n = 67) were sequenced for mutations in four other genes (ACVRL1, ENG, CAV1, and KCNK3) known to cause HPAH. Patients negative for mutations in all known PAH genes (n = 66) were then sequenced for mutations in EIF2AK4. We assessed the pathogenicity of EIF2AK4 mutations and reviewed clinical characteristics of patients with pathogenic EIF2AK4 mutations. Pathogenic BMPR2 mutations were identified in 8 of 72 (11.1%) patients with IPAH and 6 of 9 (66.7%) patients with HPAH. A novel homozygous EIF2AK4 mutation (c.257+4A>C) was identified in 1 of 9 (11.1%) patients diagnosed with HPAH. The novel EIF2AK4 mutation (c.257+4A>C) was homozygous in two sisters with severe pulmonary hypertension. None of the 72 patients with IPAH had biallelic EIF2AK4 mutations. Pathogenic biallelic EIF2AK4 mutations are rarely identified in patients diagnosed with HPAH. Identification of pathogenic biallelic EIF2AK4 mutations can aid clinicians in differentiating HPAH from heritable PVOD or PCH. Copyright © 2016 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.

Whole exome analysis identifies dominant COL4A1 mutations in patients with complex ocular phenotypes involving microphthalmia.

PubMed

Deml, B; Reis, L M; Maheshwari, M; Griffis, C; Bick, D; Semina, E V

2014-11-01

Anophthalmia/microphthalmia (A/M) is a developmental ocular malformation defined as complete absence or reduction in size of the eye. A/M is a heterogenous disorder with numerous causative genes identified; however, about half the cases lack a molecular diagnosis. We undertook whole exome sequencing in an A/M family with two affected siblings, two unaffected siblings, and unaffected parents; the ocular phenotype was isolated with only mild developmental delay/learning difficulties reported and a normal brain magnetic resonance imaging (MRI) in the proband at 16 months. No pathogenic mutations were identified in 71 known A/M genes. Further analysis identified a shared heterozygous mutation in COL4A1, c.2317G>A, p.(Gly773Arg) that was not seen in the unaffected parents and siblings. Analysis of 24 unrelated A/M exomes identified a novel c.2122G>A, p.(Gly708Arg) mutation in an additional patient with unilateral microphthalmia, bilateral microcornea and Peters anomaly; the mutation was absent in the unaffected mother and the unaffected father was not available. Mutations in COL4A1 have been linked to a spectrum of human disorders; the most consistent feature is cerebrovascular disease with variable ocular anomalies, kidney and muscle defects. This study expands the spectrum of COL4A1 phenotypes and indicates screening in patients with A/M regardless of MRI findings or presumed inheritance pattern. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Eight Mutations of Three Genes (EDA, EDAR, and WNT10A) Identified in Seven Hypohidrotic Ectodermal Dysplasia Patients.

PubMed

Zeng, Binghui; Xiao, Xue; Li, Sijie; Lu, Hui; Lu, Jiaxuan; Zhu, Ling; Yu, Dongsheng; Zhao, Wei

2016-09-19

Hypohidrotic ectodermal dysplasia (HED) is characterized by abnormal development of the teeth, hair, and sweat glands. Ectodysplasin A (EDA), Ectodysplasin A receptor (EDAR), and EDAR-associated death domain (EDARADD) are candidate genes for HED, but the relationship between WNT10A and HED has not yet been validated. In this study, we included patients who presented at least two of the three ectodermal dysplasia features. The four genes were analyzed in seven HED patients by PCR and Sanger sequencing. Five EDA and one EDAR heterozygous mutations were identified in families 1-6. Two WNT10A heterozygous mutations were identified in family 7 as a compound heterozygote. c.662G>A (p.Gly221Asp) in EDA and c.354T>G (p.Tyr118*) in WNT10A are novel mutations. Bioinformatics analyses results confirmed the pathogenicity of the two novel mutations. In family 7, we also identified two single-nucleotide polymorphisms (SNPs) that were predicted to affect the splicing of EDAR. Analysis of the patient's total RNA revealed normal splicing of EDAR. This ascertained that the compound heterozygous WNT10A mutations are the genetic defects that led to the onset of HED. Our data revealed the genetic basis of seven HED patients and expended the mutational spectrum. Interestingly, we confirmed WNT10A as a candidate gene of HED and we propose WNT10A to be tested in EDA-negative HED patients.

GNAQ and GNA11 mutations occur in 9.5% of mucosal melanoma and are associated with poor prognosis.

PubMed

Sheng, Xinan; Kong, Yan; Li, Yiqian; Zhang, Qiannan; Si, Lu; Cui, Chuanliang; Chi, Zhihong; Tang, Bixia; Mao, Lili; Lian, Bin; Wang, Xuan; Yan, Xieqiao; Li, Siming; Dai, Jie; Guo, Jun

2016-09-01

Mucosal melanoma (MM) is a rare subtype of melanoma in Caucasians with extremely poor prognosis, and therapy strategy has not been clearly established for MM. We aimed to investigate the genetic aberrations possibly applicable in targeted therapy of MM. We examined the somatic mutations of GNAQ and GNA11 (GNAQ/11, encoding the guanine nucleotide-binding alpha subunits) in MM and evaluated their correlation to clinicopathologic features of MM. This study collected samples from primary lesions of 284 MM patients. Tissue samples were analysed for mutations in exons 4 and 5 of GNAQ/11 in genomic DNA by polymerase chain reaction amplification and Sanger sequencing. Correlations of GNAQ/11 mutations to clinicopathologic features and prognosis of MM were evaluated. The overall mutation frequency of GNAQ/11 in MM was 9.5% (27 in 284), with a frequency of 4.6% and 4.9% for GNAQ and GNA11 mutations, respectively. The mutations in exon 5 of GNAQ/11 occurred exclusively in codon 209. GNAQ(Q209L) was the most prevalent variation (92.3% of missense GNAQ mutations). GNAQ/11 mutations were not significantly associated with age, gender, ulceration, and primary anatomic site. The median overall survival for MM patients with GNAQ mutations (16.0 versus 26.0 months, P = 0.031) or GNA11 mutations (15.0 versus 26.0 months, P = 0.039) were significantly shorter than those for patients with wild-type GNAQ and GNA11, respectively. Our study suggests that GNAQ and GNA11 mutations occur frequently in MM and may be a prognostic factor for MM. Our data implicate that GNAQ/11 may be potential targets for targeted therapy of MM. Copyright © 2016 Elsevier Ltd. All rights reserved.

Comprehensive Characterization of Cancer Driver Genes and Mutations.

PubMed

Bailey, Matthew H; Tokheim, Collin; Porta-Pardo, Eduard; Sengupta, Sohini; Bertrand, Denis; Weerasinghe, Amila; Colaprico, Antonio; Wendl, Michael C; Kim, Jaegil; Reardon, Brendan; Ng, Patrick Kwok-Shing; Jeong, Kang Jin; Cao, Song; Wang, Zixing; Gao, Jianjiong; Gao, Qingsong; Wang, Fang; Liu, Eric Minwei; Mularoni, Loris; Rubio-Perez, Carlota; Nagarajan, Niranjan; Cortés-Ciriano, Isidro; Zhou, Daniel Cui; Liang, Wen-Wei; Hess, Julian M; Yellapantula, Venkata D; Tamborero, David; Gonzalez-Perez, Abel; Suphavilai, Chayaporn; Ko, Jia Yu; Khurana, Ekta; Park, Peter J; Van Allen, Eliezer M; Liang, Han; Lawrence, Michael S; Godzik, Adam; Lopez-Bigas, Nuria; Stuart, Josh; Wheeler, David; Getz, Gad; Chen, Ken; Lazar, Alexander J; Mills, Gordon B; Karchin, Rachel; Ding, Li

2018-04-05

Identifying molecular cancer drivers is critical for precision oncology. Multiple advanced algorithms to identify drivers now exist, but systematic attempts to combine and optimize them on large datasets are few. We report a PanCancer and PanSoftware analysis spanning 9,423 tumor exomes (comprising all 33 of The Cancer Genome Atlas projects) and using 26 computational tools to catalog driver genes and mutations. We identify 299 driver genes with implications regarding their anatomical sites and cancer/cell types. Sequence- and structure-based analyses identified >3,400 putative missense driver mutations supported by multiple lines of evidence. Experimental validation confirmed 60%-85% of predicted mutations as likely drivers. We found that >300 MSI tumors are associated with high PD-1/PD-L1, and 57% of tumors analyzed harbor putative clinically actionable events. Our study represents the most comprehensive discovery of cancer genes and mutations to date and will serve as a blueprint for future biological and clinical endeavors. Published by Elsevier Inc.

The C9ORF72 expansion mutation is a common cause of ALS+/−FTD in Europe and has a single founder

PubMed Central

Smith, Bradley N; Newhouse, Stephen; Shatunov, Aleksey; Vance, Caroline; Topp, Simon; Johnson, Lauren; Miller, Jack; Lee, Younbok; Troakes, Claire; Scott, Kirsten M; Jones, Ashley; Gray, Ian; Wright, Jamie; Hortobágyi, Tibor; Al-Sarraj, Safa; Rogelj, Boris; Powell, John; Lupton, Michelle; Lovestone, Simon; Sapp, Peter C; Weber, Markus; Nestor, Peter J; Schelhaas, Helenius J; Asbroek, Anneloor ALM ten; Silani, Vincenzo; Gellera, Cinzia; Taroni, Franco; Ticozzi, Nicola; Van den Berg, Leonard; Veldink, Jan; Van Damme, Phillip; Robberecht, Wim; Shaw, Pamela J; Kirby, Janine; Pall, Hardev; Morrison, Karen E; Morris, Alex; de Belleroche, Jacqueline; Vianney de Jong, J M B; Baas, Frank; Andersen, Peter M; Landers, John; Brown, Robert H; Weale, Michael E; Al-Chalabi, Ammar; Shaw, Christopher E

2013-01-01

A massive hexanucleotide repeat expansion mutation (HREM) in C9ORF72 has recently been linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we describe the frequency, origin and stability of this mutation in ALS+/−FTD from five European cohorts (total n=1347). Single-nucleotide polymorphisms defining the risk haplotype in linked kindreds were genotyped in cases (n=434) and controls (n=856). Haplotypes were analysed using PLINK and aged using DMLE+. In a London clinic cohort, the HREM was the most common mutation in familial ALS+/−FTD: C9ORF72 29/112 (26%), SOD1 27/112 (24%), TARDBP 1/112 (1%) and FUS 4/112 (4%) and detected in 13/216 (6%) of unselected sporadic ALS cases but was rare in controls (3/856, 0.3%). HREM prevalence was high for familial ALS+/−FTD throughout Europe: Belgium 19/22 (86%), Sweden 30/41 (73%), the Netherlands 10/27 (37%) and Italy 4/20 (20%). The HREM did not affect the age at onset or survival of ALS patients. Haplotype analysis identified a common founder in all 137 HREM carriers that arose around 6300 years ago. The haplotype from which the HREM arose is intrinsically unstable with an increased number of repeats (average 8, compared with 2 for controls, P<10−8). We conclude that the HREM has a single founder and is the most common mutation in familial and sporadic ALS in Europe. PMID:22692064

Clinical and molecular characterization of a novel INS mutation identified in patients with MODY phenotype.

PubMed

Piccini, Barbara; Artuso, Rosangela; Lenzi, Lorenzo; Guasti, Monica; Braccesi, Giulia; Barni, Federica; Casalini, Emilio; Giglio, Sabrina; Toni, Sonia

2016-11-01

Correct diagnosis of Maturity-Onset Diabetes of the Young (MODY) is based on genetic tests requiring an appropriate subject selection by clinicians. Mutations in the insulin (INS) gene rarely occur in patients with MODY. This study is aimed at determining the genetic background and clinical phenotype in patients with suspected MODY. 34 patients with suspected MODY, negative for mutations in the GCK, HNF1α, HNF4α, HNF1β and PDX1 genes, were screened by next generation sequencing (NGS). A heterozygous INS mutation was identified in 4 members of the same family. First genetic tests performed identified two heterozygous silent nucleotide substitutions in MODY3/HNF1α gene. An ineffective attempt to suspend insulin therapy, administering repaglinide and sulphonylureas, was made. DNA was re-sequenced by NGS investigating a set of 102 genes. Genes implicated in the pathway of pancreatic β-cells, candidate genes for type 2 diabetes mellitus and genes causative of diabetes in mice were selected. A novel heterozygous variant in human preproinsulin INS gene (c.125T > C) was found in the affected family members. The new INS mutation broadens the spectrum of possible INS phenotypes. Screening for INS mutations is warranted not only in neonatal diabetes but also in MODYx patients and in selected patients with type 1 diabetes mellitus negative for autoantibodies. Subjects with complex diseases without a specific phenotype should be studied by NGS because Sanger sequencing is ineffective and time consuming in detecting rare variants. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Immunocytochemistry and fluorescence imaging efficiently identify individual neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures.

PubMed

Tsunematsu, Hiroto; Uyeda, Akiko; Yamamoto, Nobuhiko; Sugo, Noriyuki

2017-08-01

CRISPR/Cas9 system is a powerful method to investigate the role of genes by introducing a mutation selectively and efficiently to specific genome positions in cell and animal lines. However, in primary neuron cultures, this method is affected by the issue that the effectiveness of CRISPR/Cas9 is different in each neuron. Here, we report an easy, quick and reliable method to identify mutants induced by the CRISPR/Cas9 system at a single neuron level, using immunocytochemistry (ICC) and fluorescence imaging. Dissociated cortical cells were transfected with CRISPR/Cas9 plasmids targeting the transcription factor cAMP-response element binding protein (CREB). Fluorescence ICC with CREB antibody and quantitative analysis of fluorescence intensity demonstrated that CREB expression disappeared in a fraction of the transfected neurons. The downstream FOS expression was also decreased in accordance with suppressed CREB expression. Moreover, dendritic arborization was decreased in the transfected neurons which lacked CREB immunoreactivity. Detection of protein expression is efficient to identify individual postmitotic neurons with CRISPR/Cas9-mediated gene disruption in primary cortical cultures. The present method composed of CRISPR/Cas9 system, ICC and fluorescence imaging is applicable to study the function of various genes at a single-neuron level.

Exome sequencing identifies a novel SMCHD1 mutation in facioscapulohumeral muscular dystrophy 2.

PubMed

Mitsuhashi, Satomi; Boyden, Steven E; Estrella, Elicia A; Jones, Takako I; Rahimov, Fedik; Yu, Timothy W; Darras, Basil T; Amato, Anthony A; Folkerth, Rebecca D; Jones, Peter L; Kunkel, Louis M; Kang, Peter B

2013-12-01

FSHD2 is a rare form of facioscapulohumeral muscular dystrophy (FSHD) characterized by the absence of a contraction in the D4Z4 macrosatellite repeat region on chromosome 4q35 that is the hallmark of FSHD1. However, hypomethylation of this region is common to both subtypes. Recently, mutations in SMCHD1 combined with a permissive 4q35 allele were reported to cause FSHD2. We identified a novel p.Lys275del SMCHD1 mutation in a family affected with FSHD2 using whole-exome sequencing and linkage analysis. This mutation alters a highly conserved amino acid in the ATPase domain of SMCHD1. Subject III-11 is a male who developed asymmetrical muscle weakness characteristic of FSHD at 13 years. Physical examination revealed marked bilateral atrophy at biceps brachii, bilateral scapular winging, some asymmetrical weakness at tibialis anterior and peroneal muscles, and mild lower facial weakness. Biopsy of biceps brachii in subject II-5, the father of III-11, demonstrated lobulated fibers and dystrophic changes. Endomysial and perivascular inflammation was found, which has been reported in FSHD1 but not FSHD2. Given the previous report of SMCHD1 mutations in FSHD2 and the clinical presentations consistent with the FSHD phenotype, we conclude that the SMCHD1 mutation is the likely cause of the disease in this family. Copyright © 2013 Elsevier B.V. All rights reserved.

Newly identified CHO ERCC3/XPB mutations and phenotype characterization

PubMed Central

Rybanská, Ivana; Gurský, Ján; Fašková, Miriam; Salazar, Edmund P.; Kimlíčková-Polakovičová, Erika; Kleibl, Karol; Thompson, Larry H.; Piršel, Miroslav

2010-01-01

Nucleotide excision repair (NER) is a complex multistage process involving many interacting gene products to repair a wide range of DNA lesions. Genetic defects in NER cause human hereditary diseases including xeroderma pigmentosum (XP), Cockayne syndrome (CS), trichothiodystrophy and a combined XP/CS overlapping symptom. One key gene product associated with all these disorders is the excision repair cross-complementing 3/xeroderma pigmentosum B (ERCC3/XPB) DNA helicase, a subunit of the transcription factor IIH complex. ERCC3 is involved in initiation of basal transcription and global genome repair as well as in transcription-coupled repair (TCR). The hamster ERCC3 gene shows high degree of homology with the human ERCC3/XPB gene. We identified new mutations in the Chinese hamster ovary cell ERCC3 gene and characterized the role of hamster ERCC3 protein in DNA repair of ultraviolet (UV)-induced and oxidative DNA damage. All but one newly described mutations are located in the protein C-terminal region around the last intron–exon boundary. Due to protein truncations or frameshifts, they lack amino acid Ser751, phosphorylation of which prevents the 5′ incision of the UV-induced lesion during NER. Thus, despite the various locations of the mutations, their phenotypes are similar. All ercc3 mutants are extremely sensitive to UV-C light and lack recovery of RNA synthesis (RRS), confirming a defect in TCR of UV-induced damage. Their limited global genome NER capacity averages ∼8%. We detected modest sensitivity of ercc3 mutants to the photosensitizer Ro19-8022, which primarily introduces 8-oxoguanine lesions into DNA. Ro19-8022-induced damage interfered with RRS, and some of the ercc3 mutants had delayed kinetics. All ercc3 mutants showed efficient base excision repair (BER). Thus, the positions of the mutations have no effect on the sensitivity to, and repair of, Ro19-8022-induced DNA damage, suggesting that the ERCC3 protein is not involved in BER. PMID:19942596

Exome sequencing identifies a novel homozygous mutation in the phosphate transporter SLC34A1 in hypophosphatemia and nephrocalcinosis.

PubMed

Rajagopal, Abbhirami; Braslavsky, Débora; Lu, James T; Kleppe, Soledad; Clément, Florencia; Cassinelli, Hamilton; Liu, David S; Liern, Jose Miguel; Vallejo, Graciela; Bergadá, Ignacio; Gibbs, Richard A; Campeau, Phillipe M; Lee, Brendan H

2014-11-01

Two Argentinean siblings (a boy and a girl) from a nonconsanguineous family presented with hypercalcemia, hypercalciuria, hypophosphatemia, low parathyroid hormone (PTH), and nephrocalcinosis. The goal of this study was to identify genetic causes of the clinical findings in the two siblings. Whole exome sequencing was performed to identify disease-causing mutations in the youngest sibling, and a candidate variant was screened in other family members by Sanger sequencing. In vitro experiments were conducted to determine the effects of the mutation that was identified. Affected siblings (2 y.o. female and 10 y.o male) and their parents were included in the study. Informed consent was obtained for genetic studies. A novel homozygous mutation in the gene encoding the renal sodium-dependent phosphate transporter SLC34A1 was identified in both siblings (c.1484G>A, p.Arg495His). In vitro studies showed that the p.Arg495His mutation resulted in decreased phosphate uptake when compared to wild-type SLC34A1. The homozygous G>A transition that results in the substitution of histidine for arginine at position 495 of the renal sodium-dependent phosphate transporter, SLC34A1, is involved in disease pathogenesis in these patients. Our report of the second family with two mutated SLC34A1 alleles expands the known phenotype of this rare condition.

Novel compound heterozygous mutations in the OTOF Gene identified by whole-exome sequencing in auditory neuropathy spectrum disorder.

PubMed

Tang, Fengzhu; Ma, Dengke; Wang, Yulan; Qiu, Yuecai; Liu, Fei; Wang, Qingqing; Lu, Qiutian; Shi, Min; Xu, Liang; Liu, Min; Liang, Jianping

2017-03-23

Many hearing-loss diseases are demonstrated to have Mendelian inheritance caused by mutations in single gene. However, many deaf individuals have diseases that remain genetically unexplained. Auditory neuropathy is a sensorineural deafness in which sounds are able to be transferred into the inner ear normally but the transmission of the signals from inner ear to auditory nerve and brain is injured, also known as auditory neuropathy spectrum disorder (ANSD). The pathogenic mutations of the genes responsible for the Chinese ANSD population remain poorly understood. A total of 127 patients with non-syndromic hearing loss (NSHL) were enrolled in Guangxi Zhuang Autonomous Region. A hereditary deafness gene mutation screening was performed to identify the mutation sites in four deafness-related genes (GJB2, GJB3, 12S rRNA, and SLC26A4). In addition, whole-exome sequencing (WES) was applied to explore unappreciated mutation sites in the cases with the singularity of its phenotype. Well-characterized mutations were found in only 8.7% (11/127) of the patients. Interestingly, two mutations in the OTOF gene were identified in two affected siblings with ANSD from a Chinese family, including one nonsense mutation c.1273C > T (p.R425X) and one missense mutation c.4994 T > C (p.L1665P). Furthermore, we employed Sanger sequencing to confirm the mutations in each subject. Two compound heterozygous mutations in the OTOF gene were observed in the two affected siblings, whereas the two parents and unaffected sister were heterozygous carriers of c.1273C > T (father and sister) and c.4994 T > C (mother). The nonsense mutation p.R425X, contributes to a premature stop codon, may result in a truncated polypeptide, which strongly suggests its pathogenicity for ANSD. The missense mutation p.L1665P results in a single amino acid substitution in a highly conserved region. Two mutations in the OTOF gene in the Chinese deaf population were recognized for the first time. These

A mutational analysis of the cytosolic domain of the tomato Cf-9 disease-resistance protein shows that membrane-proximal residues are important for Avr9-dependent necrosis.

PubMed

Chakrabarti, Apratim; Velusamy, Thilaga; Tee, Choon Yang; Jones, David A

2016-05-01

The tomato Cf-9 gene encodes a membrane-anchored glycoprotein that imparts race-specific resistance against the tomato leaf mould fungus Cladosporium fulvum in response to the avirulence protein Avr9. Although the N-terminal half of the extracellular leucine-rich repeat (eLRR) domain of the Cf-9 protein determines its specificity for Avr9, the C-terminal half, including its small cytosolic domain, is postulated to be involved in signalling. The cytosolic domain of Cf-9 carries several residues that are potential sites for ubiquitinylation or phosphorylation, or signals for endocytic uptake. A targeted mutagenesis approach was employed to investigate the roles of these residues and cellular processes in Avr9-dependent necrosis triggered by Cf-9. Our results indicate that the membrane-proximal region of the cytosolic domain of Cf-9 plays an important role in Cf-9-mediated necrosis, and two amino acids within this region, a threonine (T835) and a proline (P838), are particularly important for Cf-9 function. An alanine mutation of T835 had no effect on Cf-9 function, but an aspartic acid mutation, which mimics phosphorylation, reduced Cf-9 function. We therefore postulate that phosphorylation/de-phosphorylation of T835 could act as a molecular switch to determine whether Cf-9 is in a primed or inactive state. Yeast two-hybrid analysis was used to show that the cytosolic domain of Cf-9 interacts with the cytosolic domain of tomato VAP27. This interaction could be disrupted by an alanine mutation of P838, whereas interaction with CITRX remained unaffected. We therefore postulate that a proline-induced kink in the membrane-proximal region of the cytosolic domain of Cf-9 may be important for interaction with VAP27, which may, in turn, be important for Cf-9 function. © 2015 BSPP AND JOHN WILEY & SONS LTD.

A histone H3K9M mutation traps histone methyltransferase Clr4 to prevent heterochromatin spreading

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

Shan, Chun-Min; Wang, Jiyong; Xu, Ke

2016-09-20

Histone lysine-to-methionine (K-to-M) mutations are associated with multiple cancers, and they function in a dominant fashion to block the methylation of corresponding lysines on wild type histones. However, their mechanisms of function are controversial. Here we show that in fission yeast, introducing the K9M mutation into one of the three histone H3 genes dominantly blocks H3K9 methylation on wild type H3 across the genome. In addition, H3K9M enhances the interaction of histone H3 tail with the H3K9 methyltransferase Clr4 in a SAM (S-adenosyl-methionine)-dependent manner, and Clr4 is trapped at nucleation sites to prevent its spreading and the formation of largemore » heterochromatin domains. We further determined the crystal structure of an H3K9M peptide in complex with human H3K9 methyltransferase G9a and SAM, which reveales that the methionine side chain had enhanced van der Waals interactions with G9a. Therefore, our results provide a detailed mechanism by which H3K9M regulates H3K9 methylation.« less

Exome sequencing identifies CTSK mutations in patients originally diagnosed as intermediate osteopetrosis☆

PubMed Central

Pangrazio, Alessandra; Puddu, Alessandro; Oppo, Manuela; Valentini, Maria; Zammataro, Luca; Vellodi, Ashok; Gener, Blanca; Llano-Rivas, Isabel; Raza, Jamal; Atta, Irum; Vezzoni, Paolo; Superti-Furga, Andrea; Villa, Anna; Sobacchi, Cristina

2014-01-01

Autosomal Recessive Osteopetrosis is a genetic disorder characterized by increased bone density due to lack of resorption by the osteoclasts. Genetic studies have widely unraveled the molecular basis of the most severe forms, while cases of intermediate severity are more difficult to characterize, probably because of a large heterogeneity. Here, we describe the use of exome sequencing in the molecular diagnosis of 2 siblings initially thought to be affected by “intermediate osteopetrosis”, which identified a homozygous mutation in the CTSK gene. Prompted by this finding, we tested by Sanger sequencing 25 additional patients addressed to us for recessive osteopetrosis and found CTSK mutations in 4 of them. In retrospect, their clinical and radiographic features were found to be compatible with, but not typical for, Pycnodysostosis. We sought to identify modifier genes that might have played a role in the clinical manifestation of the disease in these patients, but our results were not informative. In conclusion, we underline the difficulties of differential diagnosis in some patients whose clinical appearance does not fit the classical malignant or benign picture and recommend that CTSK gene be included in the molecular diagnosis of high bone density conditions. PMID:24269275

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.

Whole exome sequencing identifies driver mutations in asymptomatic computed tomography-detected lung cancers with normal karyotype.

PubMed

Belloni, Elena; Veronesi, Giulia; Rotta, Luca; Volorio, Sara; Sardella, Domenico; Bernard, Loris; Pece, Salvatore; Di Fiore, Pier Paolo; Fumagalli, Caterina; Barberis, Massimo; Spaggiari, Lorenzo; Pelicci, Pier Giuseppe; Riva, Laura

2015-04-01

The efficacy of curative surgery for lung cancer could be largely improved by non-invasive screening programs, which can detect the disease at early stages. We previously showed that 18% of screening-identified lung cancers demonstrate a normal karyotype and, following high-density genome scanning, can be subdivided into samples with 1) numerous; 2) none; and 3) few copy number alterations. Whole exome sequencing was applied to the two normal karyotype, screening-detected lung cancers, constituting group 2, as well as normal controls. We identified mutations in both tumors, including KEAP1 (commonly mutated in lung cancers) in one, and TP53, PMS1, and MSH3 (well-characterized DNA-repair genes) in the other. The two normal karyotype screening-detected lung tumors displayed a typical lung cancer mutational profile that only next generation sequencing could reveal, which offered an additional contribution to the over-diagnosis bias concept hypothesized within lung cancer screening programs. Copyright © 2015 Elsevier Inc. All rights reserved.

Genetic screening of non-classic CAH females with hyperandrogenemia identifies a novel CYP11B1 gene mutation.

PubMed

Shammas, Christos; Byrou, Stefania; Phelan, Marie M; Toumba, Meropi; Stylianou, Charilaos; Skordis, Nicos; Neocleous, Vassos; Phylactou, Leonidas A

2016-04-01

Congenital adrenal hyperplasia (CAH) is an endocrine autosomal recessive disorder with various symptoms of diverse severity. Mild hyperandrogenemia is the most commonclinical feature in non-classic CAH patients and 95% of the cases are identified by mutations in the CYP21A2 gene. In the present study, the second most common cause for non-classic CAH (NC-CAH), 11β-hydroxylase deficiency due to mutations in the CYP11B1 gene, is investigated. Screening of the CYP21A2 and CYP11B1 genes by direct sequencing was carried out for the detection of possible genetic defects in patients with suspected CAH. It wasobserved that CYP11B1 variants co-exist only in rare cases along with mutations in CYP21A2 in patients clinically diagnosed with CAH. A total of 23 NC-CAH female patients out of 75 were identified with only one mutation in the CYP21A2 gene. The novel CYP11B1 gene mutation, p.Val484Asp, was identified in a patient with CAH in the heterozygous state. The structural characterization of the novel p.Val484Asp was found to likely cause distortion of the surrounding beta sheet and indirect destabilization of the cavity that occurs on the opposite face of the structural elements, leading to partial impairment of the enzymatic activity. CYP21A2 gene mutations are the most frequent genetic defects in cases of NC-CAH even when these patients are in the heterozygous state. These mutations have a diverse phenotype giving rise to a variable extent of cortisol synthesis impairment; it is also clear that CYP11B1 mutants are a rare type of defects causing CAH.

A pilot study identifying a potential plasma biomarker for determining EGFR mutations in exons 19 or 21 in lung cancer patients

PubMed Central

Pamungkas, Aryo D.; Medriano, Carl A.; Sim, Eunjung; Lee, Sungyong; Park, Youngja H.

2017-01-01

The most common type of lung cancer is non-small cell lung cancer (NSCLC), which is frequently characterized by a mutation in the epidermal growth factor receptor (EGFR). Determining the presence of an EGFR mutation in lung cancer is important, as it determines the type of treatment that a patients will receive. Therefore, the aim of the present study was to apply high-resolution metabolomics (HRM) using liquid chromatography-mass spectrometry to identify significant compounds in human plasma samples obtained from South Korean NSCLC patients, as potential biomarkers for providing early detection and diagnosis of minimally-invasive NSCLC. The metabolic differences between lung cancer patients without EGFR mutations were compared with patients harboring EGFR mutations. Univariate analysis was performed, with a false discovery rate of q=0.05, in order to identify significant metabolites between the two groups. In addition, hierarchical clustering analysis was performed to discriminate between the metabolic profiles of the two groups. Furthermore, the significant metabolites were identified and mapped using Mummichog software, in order to generate a potential metabolic network model. Using metabolome-wide association studies, metabolic alterations were identified. Linoleic acid [303.23 m/z, (M+Na)+], 5-methyl tetrahydrofolate [231.10 m/z, (M+2H)+] and N-succinyl-L-glutamate-5 semialdehyde [254.06 m/z, (M+Na)+], were observed to be elevated in patients harboring EGFR mutations, whereas tetradecanoyl carnitine [394.29 m/z, (M+Na)+] was observed to be reduced. This suggests that these compounds may be affected by the EGFR mutation. In conclusion, the present study identified four potential biomarkers in patients with EGFR mutations, using HRM combined with pathway analysis. These results may facilitate the development of novel diagnostic tools for EGFR mutation detection in patients with lung cancer. PMID:28487968

Characterization of BRCA2 Mutation in a Series of Functional Assays

DTIC Science & Technology

2005-05-01

9 Appendices .................................................................................... 10 Abstract Mutations in the BRCA2 gene account for...approximately 20% of all hereditary breast cancer. Many individuals undergo expensive clinical testing for mutations in the BRCA2 gene in order to...BRCA2 breast and ovarian cancer predisposition gene was identified in 1995. Mutations in the gene account for approximately 20% of all hereditary breast

Targeted sequencing identifies associations between IL7R-JAK mutations and epigenetic modulators in T-cell acute lymphoblastic leukemia

PubMed Central

Vicente, Carmen; Schwab, Claire; Broux, Michaël; Geerdens, Ellen; Degryse, Sandrine; Demeyer, Sofie; Lahortiga, Idoya; Elliott, Alannah; Chilton, Lucy; La Starza, Roberta; Mecucci, Cristina; Vandenberghe, Peter; Goulden, Nicholas; Vora, Ajay; Moorman, Anthony V.; Soulier, Jean; Harrison, Christine J.; Clappier, Emmanuelle; Cools, Jan

2015-01-01

T-cell acute lymphoblastic leukemia is caused by the accumulation of multiple oncogenic lesions, including chromosomal rearrangements and mutations. To determine the frequency and co-occurrence of mutations in T-cell acute lymphoblastic leukemia, we performed targeted re-sequencing of 115 genes across 155 diagnostic samples (44 adult and 111 childhood cases). NOTCH1 and CDKN2A/B were mutated/deleted in more than half of the cases, while an additional 37 genes were mutated/deleted in 4% to 20% of cases. We found that IL7R-JAK pathway genes were mutated in 27.7% of cases, with JAK3 mutations being the most frequent event in this group. Copy number variations were also detected, including deletions of CREBBP or CTCF and duplication of MYB. FLT3 mutations were rare, but a novel extracellular mutation in FLT3 was detected and confirmed to be transforming. Furthermore, we identified complex patterns of pairwise associations, including a significant association between mutations in IL7R-JAK genes and epigenetic regulators (WT1, PRC2, PHF6). Our analyses showed that IL7R-JAK genetic lesions did not confer adverse prognosis in T-cell acute lymphoblastic leukemia cases enrolled in the UK ALL2003 trial. Overall, these results identify interconnections between the T-cell acute lymphoblastic leukemia genome and disease biology, and suggest a potential clinical application for JAK inhibitors in a significant proportion of patients with T-cell acute lymphoblastic leukemia. PMID:26206799

Mutations in a delta9-Stearoyl-ACP-Desaturase Gene Are Associated with Enhanced Stearic Acid Levels in Soybean Seeds

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

Zhang, P.; Shanklin, J.; Burton, J. W.

2008-11-01

Stearic acid (18:0) is typically a minor component of soybean [Glycine max (L.) Merr.] oil, accounting for only 2 to 4% of the total fatty acid content. Increasing stearic acid levels of soybean oil would lead to enhanced oxidative stability, potentially reducing the need for hydrogenation, a process leading to the formation of undesirable trans fatty acids. Although mutagenesis strategies have been successful in developing soybean germplasm with elevated 18:0 levels in the seed oil, the specific gene mutations responsible for this phenotype were not known. We report a newly identified soybean gene, designated SACPD-C, that encodes a unique isoformmore » of {Delta}{sup 9}-stearoyl-ACP-desaturase, the enzyme responsible for converting stearic acid to oleic acid (18:1). High levels of SACPD-C transcript were only detected in developing seed tissue, suggesting that the encoded desaturase functions to enhance oleic acid biosynthetic capacity as the immature seed is actively engaged in triacylglycerol production and storage. The participation of SACPD-C in storage triacylglycerol synthesis is further supported by the observation of mutations in this gene in two independent sources of elevated 18:0 soybean germplasm, A6 (30% 18:0) and FAM94-41 (9% 18:0). A molecular marker diagnostic for the FAM94-41 SACPD-C gene mutation strictly associates with the elevated 18:0 phenotype in a segregating population, and could thus serve as a useful tool in the development of cultivars with oils possessing enhanced oxidative stability.« less

Whole exome sequencing identifies a recurrent RQCD1 P131L mutation in cutaneous melanoma

PubMed Central

Wong, Stephen Q.; Behren, Andreas; Mar, Victoria J.; Woods, Katherine; Li, Jason; Martin, Claire; Sheppard, Karen E.; Wolfe, Rory; Kelly, John; Cebon, Jonathan; Dobrovic, Alexander; McArthur, Grant A.

2015-01-01

Melanoma is often caused by mutations due to exposure to ultraviolet radiation. This study reports a recurrent somatic C > T change causing a P131L mutation in the RQCD1 (Required for Cell Differentiation1 Homolog) gene identified through whole exome sequencing of 20 metastatic melanomas. Screening in 715 additional primary melanomas revealed a prevalence of ~4%. This represents the first reported recurrent mutation in a member of the CCR4-NOT complex in cancer. Compared to tumors without the mutation, the P131L mutant positive tumors were associated with increased thickness (p = 0.02), head and neck (p = 0.009) and upper limb (p = 0.03) location, lentigo maligna melanoma subtype (p = 0.02) and BRAF V600K (p = 0.04) but not V600E or NRAS codon 61 mutations. There was no association with nodal disease (p = 0.3). Mutually exclusive mutations of other members of the CCR4-NOT complex were found in ~20% of the TCGA melanoma dataset suggesting the complex may play an important role in melanoma biology. Mutant RQCD1 was predicted to bind strongly to HLA-A0201 and HLA-Cw3 MHC1 complexes. From thirteen patients with mutant RQCD1, an anti-tumor CD8+ T cell response was observed from a single patient's peripheral blood mononuclear cell population stimulated with mutated peptide compared to wildtype indicating a neoantigen may be formed. PMID:25544760

Mutations in the Norrie disease gene.

PubMed

Schuback, D E; Chen, Z Y; Craig, I W; Breakefield, X O; Sims, K B

1995-01-01

We report our experience to date in mutation identification in the Norrie disease (ND) gene. We carried out mutational analysis in 26 kindreds in an attempt to identify regions presumed critical to protein function and potentially correlated with generation of the disease phenotype. All coding exons, as well as noncoding regions of exons 1 and 2, 636 nucleotides in the noncoding region of exon 3, and 197 nucleotides of 5' flanking sequence, were analyzed for single-strand conformation polymorphisms (SSCP) by polymerase chain reaction (PCR) amplification of genomic DNA. DNA fragments that showed altered SSCP band mobilities were sequenced to locate the specific mutations. In addition to three previously described submicroscopic deletions encompassing the entire ND gene, we have now identified 6 intragenic deletions, 8 missense (seven point mutations, one 9-bp deletion), 6 nonsense (three point mutations, three single bp deletions/frameshift) and one 10-bp insertion, creating an expanded repeat in the 5' noncoding region of exon 1. Thus, mutations have been identified in a total of 24 of 26 (92%) of the kindreds we have studied to date. With the exception of two different mutations, each found in two apparently unrelated kindreds, these mutations are unique and expand the genotype database. Localization of the majority of point mutations at or near cysteine residues, potentially critical in protein tertiary structure, supports a previous protein model for norrin as member of a cystine knot growth factor family (Meitinger et al., 1993). Genotype-phenotype correlations were not evident with the limited clinical data available, except in the cases of larger submicroscopic deletions associated with a more severe neurologic syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)

Arabidopsis cop8 and fus4 mutations define the same gene that encodes subunit 4 of the COP9 signalosome.

PubMed Central

Serino, G; Tsuge, T; Kwok, S; Matsui, M; Wei, N; Deng, X W

1999-01-01

The pleiotropic constitutive photomorphogenic/deetiolated/fusca (cop/det/fus) mutants of Arabidopsis exhibit features of light-grown seedlings when grown in the dark. Cloning and biochemical analysis of COP9 have revealed that it is a component of a multiprotein complex, the COP9 signalosome (previously known as the COP9 complex). Here, we compare the immunoaffinity and the biochemical purification of the COP9 signalosome from cauliflower and confirm its eight-subunit composition. Molecular cloning of subunit 4 of the complex revealed that it is a proteasome-COP9 complex-eIF3 domain protein encoded by a gene that maps to chromosome 5, near the chromosomal location of the cop8 and fus4 mutations. Genetic complementation tests showed that the cop8 and fus4 mutations define the same locus, now designated as COP8. Molecular analysis of the subunit 4-encoding gene in both cop8 and fus4 mutants identified specific molecular lesions, and overexpression of the subunit 4 cDNA in a cop8 mutant background resulted in complete rescue of the mutant phenotype. Thus, we conclude that COP8 encodes subunit 4 of the COP9 signalosome. Examination of possible molecular interactions by using the yeast two-hybrid assay indicated that COP8 is capable of strong self-association as well as interaction with COP9, FUS6/COP11, FUS5, and Arabidopsis JAB1 homolog 1, the latter four proteins being previously defined subunits of the Arabidopsis COP9 signalosome. A comparative sequence analysis indicated that COP8 is highly conserved among multicellular eukaryotes and is also similar to a subunit of the 19S regulatory particle of the 26S proteasome. PMID:10521526

Exome-wide Association Study Identifies GREB1L Mutations in Congenital Kidney Malformations.

PubMed

Sanna-Cherchi, Simone; Khan, Kamal; Westland, Rik; Krithivasan, Priya; Fievet, Lorraine; Rasouly, Hila Milo; Ionita-Laza, Iuliana; Capone, Valentina P; Fasel, David A; Kiryluk, Krzysztof; Kamalakaran, Sitharthan; Bodria, Monica; Otto, Edgar A; Sampson, Matthew G; Gillies, Christopher E; Vega-Warner, Virginia; Vukojevic, Katarina; Pediaditakis, Igor; Makar, Gabriel S; Mitrotti, Adele; Verbitsky, Miguel; Martino, Jeremiah; Liu, Qingxue; Na, Young-Ji; Goj, Vinicio; Ardissino, Gianluigi; Gigante, Maddalena; Gesualdo, Loreto; Janezcko, Magdalena; Zaniew, Marcin; Mendelsohn, Cathy Lee; Shril, Shirlee; Hildebrandt, Friedhelm; van Wijk, Joanna A E; Arapovic, Adela; Saraga, Marijan; Allegri, Landino; Izzi, Claudia; Scolari, Francesco; Tasic, Velibor; Ghiggeri, Gian Marco; Latos-Bielenska, Anna; Materna-Kiryluk, Anna; Mane, Shrikant; Goldstein, David B; Lifton, Richard P; Katsanis, Nicholas; Davis, Erica E; Gharavi, Ali G

2017-11-02

Renal agenesis and hypodysplasia (RHD) are major causes of pediatric chronic kidney disease and are highly genetically heterogeneous. We conducted whole-exome sequencing in 202 case subjects with RHD and identified diagnostic mutations in genes known to be associated with RHD in 7/202 case subjects. In an additional affected individual with RHD and a congenital heart defect, we found a homozygous loss-of-function (LOF) variant in SLIT3, recapitulating phenotypes reported with Slit3 inactivation in the mouse. To identify genes associated with RHD, we performed an exome-wide association study with 195 unresolved case subjects and 6,905 control subjects. The top signal resided in GREB1L, a gene implicated previously in Hoxb1 and Shha signaling in zebrafish. The significance of the association, which was p = 2.0 × 10 -5 for novel LOF, increased to p = 4.1 × 10 -6 for LOF and deleterious missense variants combined, and augmented further after accounting for segregation and de novo inheritance of rare variants (joint p = 2.3 × 10 -7 ). Finally, CRISPR/Cas9 disruption or knockdown of greb1l in zebrafish caused specific pronephric defects, which were rescued by wild-type human GREB1L mRNA, but not mRNA containing alleles identified in case subjects. Together, our study provides insight into the genetic landscape of kidney malformations in humans, presents multiple candidates, and identifies SLIT3 and GREB1L as genes implicated in the pathogenesis of RHD. Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Exome Sequencing Identifies a Novel Homozygous Mutation in the Phosphate Transporter SLC34A1 in Hypophosphatemia and Nephrocalcinosis

PubMed Central

Rajagopal, Abbhirami; Braslavsky, Débora; Lu, James T.; Kleppe, Soledad; Clément, Florencia; Cassinelli, Hamilton; Liu, David S.; Liern, Jose Miguel; Vallejo, Graciela; Bergadá, Ignacio; Gibbs, Richard A.; Campeau, Phillipe M.

2014-01-01

Context: Two Argentinean siblings (a boy and a girl) from a nonconsanguineous family presented with hypercalcemia, hypercalciuria, hypophosphatemia, low parathyroid hormone (PTH), and nephrocalcinosis. Objective: The goal of this study was to identify genetic causes of the clinical findings in the two siblings. Design: Whole exome sequencing was performed to identify disease-causing mutations in the youngest sibling, and a candidate variant was screened in other family members by Sanger sequencing. In vitro experiments were conducted to determine the effects of the mutation that was identified. Patients and Other Participants: Affected siblings (2 y.o. female and 10 y.o male) and their parents were included in the study. Informed consent was obtained for genetic studies. Results: A novel homozygous mutation in the gene encoding the renal sodium-dependent phosphate transporter SLC34A1 was identified in both siblings (c.1484G>A, p.Arg495His). In vitro studies showed that the p.Arg495His mutation resulted in decreased phosphate uptake when compared to wild-type SLC34A1. Conclusions: The homozygous G>A transition that results in the substitution of histidine for arginine at position 495 of the renal sodium-dependent phosphate transporter, SLC34A1, is involved in disease pathogenesis in these patients. Our report of the second family with two mutated SLC34A1 alleles expands the known phenotype of this rare condition. PMID:25050900

An Evolutionary Approach for Identifying Driver Mutations in Colorectal Cancer

PubMed Central

Leder, Kevin; Riester, Markus; Iwasa, Yoh; Lengauer, Christoph; Michor, Franziska

2015-01-01

The traditional view of cancer as a genetic disease that can successfully be treated with drugs targeting mutant onco-proteins has motivated whole-genome sequencing efforts in many human cancer types. However, only a subset of mutations found within the genomic landscape of cancer is likely to provide a fitness advantage to the cell. Distinguishing such “driver” mutations from innocuous “passenger” events is critical for prioritizing the validation of candidate mutations in disease-relevant models. We design a novel statistical index, called the Hitchhiking Index, which reflects the probability that any observed candidate gene is a passenger alteration, given the frequency of alterations in a cross-sectional cancer sample set, and apply it to a mutational data set in colorectal cancer. Our methodology is based upon a population dynamics model of mutation accumulation and selection in colorectal tissue prior to cancer initiation as well as during tumorigenesis. This methodology can be used to aid in the prioritization of candidate mutations for functional validation and contributes to the process of drug discovery. PMID:26379039

Oncogenic PIK3CA mutations occur in epidermal nevi and seborrheic keratoses with a characteristic mutation pattern

PubMed Central

Hafner, Christian; López-Knowles, Elena; Luis, Nuno M.; Toll, Agustí; Baselga, Eulàlia; Fernández-Casado, Alex; Hernández, Silvia; Ribé, Adriana; Mentzel, Thomas; Stoehr, Robert; Hofstaedter, Ferdinand; Landthaler, Michael; Vogt, Thomas; Pujol, Ramòn M.; Hartmann, Arndt; Real, Francisco X.

2007-01-01

Activating mutations of the p110 α subunit of PI3K (PIK3CA) oncogene have been identified in a broad spectrum of malignant tumors. However, their role in benign or preneoplastic conditions is unknown. Activating FGF receptor 3 (FGFR3) mutations are common in benign skin lesions, either as embryonic mutations in epidermal nevi (EN) or as somatic mutations in seborrheic keratoses (SK). FGFR3 mutations are also common in low-grade malignant bladder tumors, where they often occur in association with PIK3CA mutations. Therefore, we examined exons 9 and 20 of PIK3CA and FGFR3 hotspot mutations in EN (n = 33) and SK (n = 62), two proliferative skin lesions lacking malignant potential. Nine of 33 (27%) EN harbored PIK3CA mutations; all cases showed the E545G substitution, which is uncommon in cancers. In EN, R248C was the only FGFR3 mutation identified. By contrast, 10 of 62 (16%) SK revealed the typical cancer-associated PIK3CA mutations E542K, E545K, and H1047R. The same lesions displayed a wide range of FGFR3 mutations. Corresponding unaffected tissue was available for four EN and two mutant SK: all control samples displayed a WT sequence, confirming the somatic nature of the mutations found in lesional tissue. Forty of 95 (42%) lesions showed at least one mutation in either gene. PIK3CA and FGFR3 mutations displayed an independent distribution; 5/95 lesions harbored mutations in both genes. Our findings suggest that, in addition to their role in cancer, oncogenic PIK3CA mutations contribute to the pathogenesis of skin tumors lacking malignant potential. The remarkable genotype–phenotype correlation as observed in this study points to a distinct etiopathogenesis of the mutations in keratinocytes occuring either during fetal development or in adult life. PMID:17673550

DFNA8/12 caused by TECTA mutations is the most identified subtype of nonsyndromic autosomal dominant hearing loss.

PubMed

Hildebrand, Michael S; Morín, Matías; Meyer, Nicole C; Mayo, Fernando; Modamio-Hoybjor, Silvia; Mencía, Angeles; Olavarrieta, Leticia; Morales-Angulo, Carmelo; Nishimura, Carla J; Workman, Heather; DeLuca, Adam P; del Castillo, Ignacio; Taylor, Kyle R; Tompkins, Bruce; Goodman, Corey W; Schrauwen, Isabelle; Wesemael, Maarten Van; Lachlan, K; Shearer, A Eliot; Braun, Terry A; Huygen, Patrick L M; Kremer, Hannie; Van Camp, Guy; Moreno, Felipe; Casavant, Thomas L; Smith, Richard J H; Moreno-Pelayo, Miguel A

2011-07-01

The prevalence of DFNA8/DFNA12 (DFNA8/12), a type of autosomal dominant nonsyndromic hearing loss (ADNSHL), is unknown as comprehensive population-based genetic screening has not been conducted. We therefore completed unbiased screening for TECTA mutations in a Spanish cohort of 372 probands from ADNSHL families. Three additional families (Spanish, Belgian, and English) known to be linked to DFNA8/12 were also included in the screening. In an additional cohort of 835 American ADNSHL families, we preselected 73 probands for TECTA screening based on audiometric data. In aggregate, we identified 23 TECTA mutations in this process. Remarkably, 20 of these mutations are novel, more than doubling the number of reported TECTA ADNSHL mutations from 13 to 33. Mutations lie in all domains of the α-tectorin protein, including those for the first time identified in the entactin domain, as well as the vWFD1, vWFD2, and vWFD3 repeats, and the D1-D2 and TIL2 connectors. Although the majority are private mutations, four of them-p.Cys1036Tyr, p.Cys1837Gly, p.Thr1866Met, and p.Arg1890Cys-were observed in more than one unrelated family. For two of these mutations founder effects were also confirmed. Our data validate previously observed genotype-phenotype correlations in DFNA8/12 and introduce new correlations. Specifically, mutations in the N-terminal region of α-tectorin (entactin domain, vWFD1, and vWFD2) lead to mid-frequency NSHL, a phenotype previously associated only with mutations in the ZP domain. Collectively, our results indicate that DFNA8/12 hearing loss is a frequent type of ADNSHL. © 2011 Wiley-Liss, Inc.

Epidermal growth factor receptor mutation in gastric cancer.

PubMed

Liu, Zhimin; Liu, Lina; Li, Mei; Wang, Zhaohui; Feng, Lu; Zhang, Qiuping; Cheng, Shihua; Lu, Shen

2011-04-01

Epidermal growth factor receptor (EGFR) and Kirsten-RAS (KRAS) mutations have been identified as predictors of response to EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer. We aimed to screen the mutations of both genes in gastric carcinoma to detect the suitability of EGFR TKIs for patients with gastric carcinoma. We screened EGFR mutation in exons 19-21 and KRAS mutation in exon 2 in 58 gastric adenocarcinomas from China using high resolution melting analysis (HRMA). Positive samples were confirmed by DNA sequencing. Three EGFR missense mutations (5.2%) and 22 single nucleotide polymorphisms (SNP, Q787Q, 37.9%) were identified. To our knowledge, we report for the first time three mutation patterns of EGFR, Y801C, L858R and G863D, in gastric carcinoma. Two samples with EGFR mutation were mucinous adenocarcinoma. These three samples were collected from male patients aged over 75 years old. The frequency of KRAS mutation was 10.3% (6/58). The exclusiveness of EGFR and KRAS mutations was proven for the first time in gastric cancer. Gastric carcinoma of the mucinous adenocarcinoma type collected from older male patients may harbour EGFR mutations. The small subset of gastric adenocarcinoma patients may respond to EGFR TKIs.

Cancer Susceptibility Gene Mutations in Individuals With Colorectal Cancer

PubMed Central

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

2017-01-01

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

Non-coding cancer driver candidates identified with a sample- and position-specific model of the somatic mutation rate

PubMed Central

Juul, Malene; Bertl, Johanna; Guo, Qianyun; Nielsen, Morten Muhlig; Świtnicki, Michał; Hornshøj, Henrik; Madsen, Tobias; Hobolth, Asger; Pedersen, Jakob Skou

2017-01-01

Non-coding mutations may drive cancer development. Statistical detection of non-coding driver regions is challenged by a varying mutation rate and uncertainty of functional impact. Here, we develop a statistically founded non-coding driver-detection method, ncdDetect, which includes sample-specific mutational signatures, long-range mutation rate variation, and position-specific impact measures. Using ncdDetect, we screened non-coding regulatory regions of protein-coding genes across a pan-cancer set of whole-genomes (n = 505), which top-ranked known drivers and identified new candidates. For individual candidates, presence of non-coding mutations associates with altered expression or decreased patient survival across an independent pan-cancer sample set (n = 5454). This includes an antigen-presenting gene (CD1A), where 5’UTR mutations correlate significantly with decreased survival in melanoma. Additionally, mutations in a base-excision-repair gene (SMUG1) correlate with a C-to-T mutational-signature. Overall, we find that a rich model of mutational heterogeneity facilitates non-coding driver identification and integrative analysis points to candidates of potential clinical relevance. DOI: http://dx.doi.org/10.7554/eLife.21778.001 PMID:28362259

Exome sequencing identifies CTSK mutations in patients originally diagnosed as intermediate osteopetrosis.

PubMed

Pangrazio, Alessandra; Puddu, Alessandro; Oppo, Manuela; Valentini, Maria; Zammataro, Luca; Vellodi, Ashok; Gener, Blanca; Llano-Rivas, Isabel; Raza, Jamal; Atta, Irum; Vezzoni, Paolo; Superti-Furga, Andrea; Villa, Anna; Sobacchi, Cristina

2014-02-01

Autosomal Recessive Osteopetrosis is a genetic disorder characterized by increased bone density due to lack of resorption by the osteoclasts. Genetic studies have widely unraveled the molecular basis of the most severe forms, while cases of intermediate severity are more difficult to characterize, probably because of a large heterogeneity. Here, we describe the use of exome sequencing in the molecular diagnosis of 2 siblings initially thought to be affected by "intermediate osteopetrosis", which identified a homozygous mutation in the CTSK gene. Prompted by this finding, we tested by Sanger sequencing 25 additional patients addressed to us for recessive osteopetrosis and found CTSK mutations in 4 of them. In retrospect, their clinical and radiographic features were found to be compatible with, but not typical for, Pycnodysostosis. We sought to identify modifier genes that might have played a role in the clinical manifestation of the disease in these patients, but our results were not informative. In conclusion, we underline the difficulties of differential diagnosis in some patients whose clinical appearance does not fit the classical malignant or benign picture and recommend that CTSK gene be included in the molecular diagnosis of high bone density conditions. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Analysis of SOX10 mutations identified in Waardenburg-Hirschsprung patients: Differential effects on target gene regulation.

PubMed

Chan, Kwok Keung; Wong, Corinne Kung Yen; Lui, Vincent Chi Hang; Tam, Paul Kwong Hang; Sham, Mai Har

2003-10-15

SOX10 is a member of the SOX gene family related by homology to the high-mobility group (HMG) box region of the testis-determining gene SRY. Mutations of the transcription factor gene SOX10 lead to Waardenburg-Hirschsprung syndrome (Waardenburg-Shah syndrome, WS4) in humans. A number of SOX10 mutations have been identified in WS4 patients who suffer from different extents of intestinal aganglionosis, pigmentation, and hearing abnormalities. Some patients also exhibit signs of myelination deficiency in the central and peripheral nervous systems. Although the molecular bases for the wide range of symptoms displayed by the patients are still not clearly understood, a few target genes for SOX10 have been identified. We have analyzed the impact of six different SOX10 mutations on the activation of SOX10 target genes by yeast one-hybrid and mammalian cell transfection assays. To investigate the transactivation activities of the mutant proteins, three different SOX target binding sites were introduced into luciferase reporter gene constructs and examined in our series of transfection assays: consensus HMG domain protein binding sites; SOX10 binding sites identified in the RET promoter; and Sox10 binding sites identified in the P0 promoter. We found that the same mutation could have different transactivation activities when tested with different target binding sites and in different cell lines. The differential transactivation activities of the SOX10 mutants appeared to correlate with the intestinal and/or neurological symptoms presented in the patients. Among the six mutant SOX10 proteins tested, much reduced transactivation activities were observed when tested on the SOX10 binding sites from the RET promoter. Of the two similar mutations X467K and 1400del12, only the 1400del12 mutant protein exhibited an increase of transactivation through the P0 promoter. While the lack of normal SOX10 mediated activation of RET transcription may lead to intestinal aganglionosis

Common pathogenic effects of missense mutations in the P-type ATPase ATP13A2 (PARK9) associated with early-onset parkinsonism.

PubMed

Podhajska, Agata; Musso, Alessandra; Trancikova, Alzbeta; Stafa, Klodjan; Moser, Roger; Sonnay, Sarah; Glauser, Liliane; Moore, Darren J

2012-01-01

Mutations in the ATP13A2 gene (PARK9) cause autosomal recessive, juvenile-onset Kufor-Rakeb syndrome (KRS), a neurodegenerative disease characterized by parkinsonism. KRS mutations produce truncated forms of ATP13A2 with impaired protein stability resulting in a loss-of-function. Recently, homozygous and heterozygous missense mutations in ATP13A2 have been identified in subjects with early-onset parkinsonism. The mechanism(s) by which missense mutations potentially cause parkinsonism are not understood at present. Here, we demonstrate that homozygous F182L, G504R and G877R missense mutations commonly impair the protein stability of ATP13A2 leading to its enhanced degradation by the proteasome. ATP13A2 normally localizes to endosomal and lysosomal membranes in neurons and the F182L and G504R mutations disrupt this vesicular localization and promote the mislocalization of ATP13A2 to the endoplasmic reticulum. Heterozygous T12M, G533R and A746T mutations do not obviously alter protein stability or subcellular localization but instead impair the ATPase activity of microsomal ATP13A2 whereas homozygous missense mutations disrupt the microsomal localization of ATP13A2. The overexpression of ATP13A2 missense mutants in SH-SY5Y neural cells does not compromise cellular viability suggesting that these mutant proteins lack intrinsic toxicity. However, the overexpression of wild-type ATP13A2 may impair neuronal integrity as it causes a trend of reduced neurite outgrowth of primary cortical neurons, whereas the majority of disease-associated missense mutations lack this ability. Finally, ATP13A2 overexpression sensitizes cortical neurons to neurite shortening induced by exposure to cadmium or nickel ions, supporting a functional interaction between ATP13A2 and heavy metals in post-mitotic neurons, whereas missense mutations influence this sensitizing effect. Collectively, our study provides support for common loss-of-function effects of homozygous and heterozygous missense

Common Pathogenic Effects of Missense Mutations in the P-Type ATPase ATP13A2 (PARK9) Associated with Early-Onset Parkinsonism

PubMed Central

Podhajska, Agata; Musso, Alessandra; Trancikova, Alzbeta; Stafa, Klodjan; Moser, Roger; Sonnay, Sarah; Glauser, Liliane; Moore, Darren J.

2012-01-01

Mutations in the ATP13A2 gene (PARK9) cause autosomal recessive, juvenile-onset Kufor-Rakeb syndrome (KRS), a neurodegenerative disease characterized by parkinsonism. KRS mutations produce truncated forms of ATP13A2 with impaired protein stability resulting in a loss-of-function. Recently, homozygous and heterozygous missense mutations in ATP13A2 have been identified in subjects with early-onset parkinsonism. The mechanism(s) by which missense mutations potentially cause parkinsonism are not understood at present. Here, we demonstrate that homozygous F182L, G504R and G877R missense mutations commonly impair the protein stability of ATP13A2 leading to its enhanced degradation by the proteasome. ATP13A2 normally localizes to endosomal and lysosomal membranes in neurons and the F182L and G504R mutations disrupt this vesicular localization and promote the mislocalization of ATP13A2 to the endoplasmic reticulum. Heterozygous T12M, G533R and A746T mutations do not obviously alter protein stability or subcellular localization but instead impair the ATPase activity of microsomal ATP13A2 whereas homozygous missense mutations disrupt the microsomal localization of ATP13A2. The overexpression of ATP13A2 missense mutants in SH-SY5Y neural cells does not compromise cellular viability suggesting that these mutant proteins lack intrinsic toxicity. However, the overexpression of wild-type ATP13A2 may impair neuronal integrity as it causes a trend of reduced neurite outgrowth of primary cortical neurons, whereas the majority of disease-associated missense mutations lack this ability. Finally, ATP13A2 overexpression sensitizes cortical neurons to neurite shortening induced by exposure to cadmium or nickel ions, supporting a functional interaction between ATP13A2 and heavy metals in post-mitotic neurons, whereas missense mutations influence this sensitizing effect. Collectively, our study provides support for common loss-of-function effects of homozygous and heterozygous missense

Computational Assay of H7N9 Influenza Neuraminidase Reveals R292K Mutation Reduces Drug Binding Affinity

NASA Astrophysics Data System (ADS)

Woods, Christopher J.; Malaisree, Maturos; Long, Ben; McIntosh-Smith, Simon; Mulholland, Adrian J.

2013-12-01

The emergence of a novel H7N9 avian influenza that infects humans is a serious cause for concern. Of the genome sequences of H7N9 neuraminidase available, one contains a substitution of arginine to lysine at position 292, suggesting a potential for reduced drug binding efficacy. We have performed molecular dynamics simulations of oseltamivir, zanamivir and peramivir bound to H7N9, H7N9-R292K, and a structurally related H11N9 neuraminidase. They show that H7N9 neuraminidase is structurally homologous to H11N9, binding the drugs in identical modes. The simulations reveal that the R292K mutation disrupts drug binding in H7N9 in a comparable manner to that observed experimentally for H11N9-R292K. Absolute binding free energy calculations with the WaterSwap method confirm a reduction in binding affinity. This indicates that the efficacy of antiviral drugs against H7N9-R292K will be reduced. Simulations can assist in predicting disruption of binding caused by mutations in neuraminidase, thereby providing a computational `assay.'

MSH6 and PMS2 mutation positive Australian Lynch syndrome families: novel mutations, cancer risk and age of diagnosis of colorectal cancer.

PubMed

Talseth-Palmer, Bente A; McPhillips, Mary; Groombridge, Claire; Spigelman, Allan; Scott, Rodney J

2010-05-21

Approximately 10% of Lynch syndrome families have a mutation in MSH6 and fewer families have a mutation in PMS2. It is assumed that the cancer incidence is the same in families with mutations in MSH6 as in families with mutations in MLH1/MSH2 but that the disease tends to occur later in life, little is known about families with PMS2 mutations. This study reports on our findings on mutation type, cancer risk and age of diagnosis in MSH6 and PMS2 families. A total of 78 participants (from 29 families) with a mutation in MSH6 and 7 participants (from 6 families) with a mutation in PMS2 were included in the current study. A database of de-identified patient information was analysed to extract all relevant information such as mutation type, cancer incidence, age of diagnosis and cancer type in this Lynch syndrome cohort. Cumulative lifetime risk was calculated utilising Kaplan-Meier survival analysis. MSH6 and PMS2 mutations represent 10.3% and 1.9%, respectively, of the pathogenic mutations in our Australian Lynch syndrome families. We identified 26 different MSH6 and 4 different PMS2 mutations in the 35 families studied. We report 15 novel MSH6 and 1 novel PMS2 mutations. The estimated cumulative risk of CRC at age 70 years was 61% (similar in males and females) and 65% for endometrial cancer in MSH6 mutation carriers. The risk of developing CRC is different between males and females at age 50 years, which is 34% for males and 21% for females. Novel MSH6 and PMS2 mutations are being reported and submitted to the current databases for identified Lynch syndrome mutations. Our data provides additional information to add to the genotype-phenotype spectrum for both MSH6 and PMS2 mutations.

Amplicon Resequencing Identified Parental Mosaicism for Approximately 10% of “de novo” SCN1A Mutations in Children with Dravet Syndrome

PubMed Central

Xu, Xiaojing; Yang, Xiaoxu; Wu, Qixi; Liu, Aijie; Yang, Xiaoling; Ye, Adam Yongxin; Huang, August Yue; Li, Jiarui; Wang, Meng; Yu, Zhe; Wang, Sheng; Zhang, Zhichao; Wu, Xiru

2015-01-01

ABSTRACT The majority of children with Dravet syndrome (DS) are caused by de novo SCN1A mutations. To investigate the origin of the mutations, we developed and applied a new method that combined deep amplicon resequencing with a Bayesian model to detect and quantify allelic fractions with improved sensitivity. Of 174 SCN1A mutations in DS probands which were considered “de novo” by Sanger sequencing, we identified 15 cases (8.6%) of parental mosaicism. We identified another five cases of parental mosaicism that were also detectable by Sanger sequencing. Fraction of mutant alleles in the 20 cases of parental mosaicism ranged from 1.1% to 32.6%. Thirteen (65% of 20) mutations originated paternally and seven (35% of 20) maternally. Twelve (60% of 20) mosaic parents did not have any epileptic symptoms. Their mutant allelic fractions were significantly lower than those in mosaic parents with epileptic symptoms (P = 0.016). We identified mosaicism with varied allelic fractions in blood, saliva, urine, hair follicle, oral epithelium, and semen, demonstrating that postzygotic mutations could affect multiple somatic cells as well as germ cells. Our results suggest that more sensitive tools for detecting low‐level mosaicism in parents of families with seemingly “de novo” mutations will allow for better informed genetic counseling. PMID:26096185

Identifying uniformly mutated segments within repeats.

PubMed

Sahinalp, S Cenk; Eichler, Evan; Goldberg, Paul; Berenbrink, Petra; Friedetzky, Tom; Ergun, Funda

2004-12-01

Given a long string of characters from a constant size alphabet we present an algorithm to determine whether its characters have been generated by a single i.i.d. random source. More specifically, consider all possible n-coin models for generating a binary string S, where each bit of S is generated via an independent toss of one of the n coins in the model. The choice of which coin to toss is decided by a random walk on the set of coins where the probability of a coin change is much lower than the probability of using the same coin repeatedly. We present a procedure to evaluate the likelihood of a n-coin model for given S, subject a uniform prior distribution over the parameters of the model (that represent mutation rates and probabilities of copying events). In the absence of detailed prior knowledge of these parameters, the algorithm can be used to determine whether the a posteriori probability for n=1 is higher than for any other n>1. Our algorithm runs in time O(l4logl), where l is the length of S, through a dynamic programming approach which exploits the assumed convexity of the a posteriori probability for n. Our test can be used in the analysis of long alignments between pairs of genomic sequences in a number of ways. For example, functional regions in genome sequences exhibit much lower mutation rates than non-functional regions. Because our test provides means for determining variations in the mutation rate, it may be used to distinguish functional regions from non-functional ones. Another application is in determining whether two highly similar, thus evolutionarily related, genome segments are the result of a single copy event or of a complex series of copy events. This is particularly an issue in evolutionary studies of genome regions rich with repeat segments (especially tandemly repeated segments).

Exome sequencing identifies SUCO mutations in mesial temporal lobe epilepsy.

PubMed

Sha, Zhiqiang; Sha, Longze; Li, Wenting; Dou, Wanchen; Shen, Yan; Wu, Liwen; Xu, Qi

2015-03-30

Mesial temporal lobe epilepsy (mTLE) is the main type and most common medically intractable form of epilepsy. Severity of disease-based stratified samples may help identify new disease-associated mutant genes. We analyzed mRNA expression profiles from patient hippocampal tissue. Three of the seven patients had severe mTLE with generalized-onset convulsions and consciousness loss that occurred over many years. We found that compared with other groups, patients with severe mTLE were classified into a distinct group. Whole-exome sequencing and Sanger sequencing validation in all seven patients identified three novel SUN domain-containing ossification factor (SUCO) mutations in severely affected patients. Furthermore, SUCO knock down significantly reduced dendritic length in vitro. Our results indicate that mTLE defects may affect neuronal development, and suggest that neurons have abnormal development due to lack of SUCO, which may be a generalized-onset epilepsy-related gene. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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.

Next-generation sequencing identifies a novel compound heterozygous mutation in MYO7A in a Chinese patient with Usher Syndrome 1B.

PubMed

Wei, Xiaoming; Sun, Yan; Xie, Jiansheng; Shi, Quan; Qu, Ning; Yang, Guanghui; Cai, Jun; Yang, Yi; Liang, Yu; Wang, Wei; Yi, Xin

2012-11-20

Targeted enrichment and next-generation sequencing (NGS) have been employed for detection of genetic diseases. The purpose of this study was to validate the accuracy and sensitivity of our method for comprehensive mutation detection of hereditary hearing loss, and identify inherited mutations involved in human deafness accurately and economically. To make genetic diagnosis of hereditary hearing loss simple and timesaving, we designed a 0.60 MB array-based chip containing 69 nuclear genes and mitochondrial genome responsible for human deafness and conducted NGS toward ten patients with five known mutations and a Chinese family with hearing loss (never genetically investigated). Ten patients with five known mutations were sequenced using next-generation sequencing to validate the sensitivity of the method. We identified four known mutations in two nuclear deafness causing genes (GJB2 and SLC26A4), one in mitochondrial DNA. We then performed this method to analyze the variants in a Chinese family with hearing loss and identified compound heterozygosity for two novel mutations in gene MYO7A. The compound heterozygosity identified in gene MYO7A causes Usher Syndrome 1B with severe phenotypes. The results support that the combination of enrichment of targeted genes and next-generation sequencing is a valuable molecular diagnostic tool for hereditary deafness and suitable for clinical application. Copyright © 2012 Elsevier B.V. All rights reserved.

Recurrent PTPRB and PLCG1 mutations in angiosarcoma.

PubMed

Behjati, Sam; Tarpey, Patrick S; Sheldon, Helen; Martincorena, Inigo; Van Loo, Peter; Gundem, Gunes; Wedge, David C; Ramakrishna, Manasa; Cooke, Susanna L; Pillay, Nischalan; Vollan, Hans Kristian M; Papaemmanuil, Elli; Koss, Hans; Bunney, Tom D; Hardy, Claire; Joseph, Olivia R; Martin, Sancha; Mudie, Laura; Butler, Adam; Teague, Jon W; Patil, Meena; Steers, Graham; Cao, Yu; Gumbs, Curtis; Ingram, Davis; Lazar, Alexander J; Little, Latasha; Mahadeshwar, Harshad; Protopopov, Alexei; Al Sannaa, Ghadah A; Seth, Sahil; Song, Xingzhi; Tang, Jiabin; Zhang, Jianhua; Ravi, Vinod; Torres, Keila E; Khatri, Bhavisha; Halai, Dina; Roxanis, Ioannis; Baumhoer, Daniel; Tirabosco, Roberto; Amary, M Fernanda; Boshoff, Chris; McDermott, Ultan; Katan, Matilda; Stratton, Michael R; Futreal, P Andrew; Flanagan, Adrienne M; Harris, Adrian; Campbell, Peter J

2014-04-01

Angiosarcoma is an aggressive malignancy that arises spontaneously or secondarily to ionizing radiation or chronic lymphoedema. Previous work has identified aberrant angiogenesis, including occasional somatic mutations in angiogenesis signaling genes, as a key driver of angiosarcoma. Here we employed whole-genome, whole-exome and targeted sequencing to study the somatic changes underpinning primary and secondary angiosarcoma. We identified recurrent mutations in two genes, PTPRB and PLCG1, which are intimately linked to angiogenesis. The endothelial phosphatase PTPRB, a negative regulator of vascular growth factor tyrosine kinases, harbored predominantly truncating mutations in 10 of 39 tumors (26%). PLCG1, a signal transducer of tyrosine kinases, encoded a recurrent, likely activating p.Arg707Gln missense variant in 3 of 34 cases (9%). Overall, 15 of 39 tumors (38%) harbored at least one driver mutation in angiogenesis signaling genes. Our findings inform and reinforce current therapeutic efforts to target angiogenesis signaling in angiosarcoma.

Abnormal RNA splicing and genomic instability after induction of DNMT3A mutations by CRISPR/Cas9 gene editing.

PubMed

Banaszak, Lauren G; Giudice, Valentina; Zhao, Xin; Wu, Zhijie; Gao, Shouguo; Hosokawa, Kohei; Keyvanfar, Keyvan; Townsley, Danielle M; Gutierrez-Rodrigues, Fernanda; Fernandez Ibanez, Maria Del Pilar; Kajigaya, Sachiko; Young, Neal S

2018-03-01

DNA methyltransferase 3A (DNMT3A) mediates de novo DNA methylation. Mutations in DNMT3A are associated with hematological malignancies, most frequently acute myeloid leukemia. DNMT3A mutations are hypothesized to establish a pre-leukemic state, rendering cells vulnerable to secondary oncogenic mutations and malignant transformation. However, the mechanisms by which DNMT3A mutations contribute to leukemogenesis are not well-defined. Here, we successfully created four DNMT3A-mutated K562 cell lines with frameshift mutations resulting in truncated DNMT3A proteins. DNMT3A-mutated cell lines exhibited significantly impaired growth and increased apoptotic activity compared to wild-type (WT) cells. Consistent with previous studies, DNMT3A-mutated cells displayed impaired differentiation capacity. RNA-seq was used to compare transcriptomes of DNMT3A-mutated and WT cells; DNMT3A ablation resulted in downregulation of genes involved in spliceosome function, causing dysfunction of RNA splicing. Unexpectedly, we observed DNMT3A-mutated cells to exhibit marked genomic instability and an impaired DNA damage response compared to WT. CRISPR/Cas9-mediated DNMT3A-mutated K562 cells may be used to model effects of DNMT3A mutations in human cells. Our findings implicate aberrant splicing and induction of genomic instability as potential mechanisms by which DNMT3A mutations might predispose to malignancy. Published by Elsevier Inc.

Both TALENs and CRISPR/Cas9 directly target the HBB IVS2-654 (C > T) mutation in β-thalassemia-derived iPSCs.

PubMed

Xu, Peng; Tong, Ying; Liu, Xiu-zhen; Wang, Ting-ting; Cheng, Li; Wang, Bo-yu; Lv, Xiang; Huang, Yue; Liu, De-pei

2015-07-09

β-Thalassemia is one of the most common genetic blood diseases and is caused by either point mutations or deletions in the β-globin (HBB) gene. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations may be a potential therapeutic strategy for this disease. Due to the low efficiency of typical homologous recombination, endonucleases, including TALENs and CRISPR/Cas9, have been widely used to enhance the gene correction efficiency in patient-derived iPSCs. Here, we designed TALENs and CRISPR/Cas9 to directly target the intron2 mutation site IVS2-654 in the globin gene. We observed different frequencies of double-strand breaks (DSBs) at IVS2-654 loci using TALENs and CRISPR/Cas9, and TALENs mediated a higher homologous gene targeting efficiency compared to CRISPR/Cas9 when combined with the piggyBac transposon donor. In addition, more obvious off-target events were observed for CRISPR/Cas9 compared to TALENs. Finally, TALENs-corrected iPSC clones were selected for erythroblast differentiation using the OP9 co-culture system and detected relatively higher transcription of HBB than the uncorrected cells. This comparison of using TALENs or CRISPR/Cas9 to correct specific HBB mutations in patient-derived iPSCs will guide future applications of TALENs- or CRISPR/Cas9-based gene therapies in monogenic diseases.

Somatic mosaicism of a CDKL5 mutation identified by next-generation sequencing.

PubMed

Kato, Takeshi; Morisada, Naoya; Nagase, Hiroaki; Nishiyama, Masahiro; Toyoshima, Daisaku; Nakagawa, Taku; Maruyama, Azusa; Fu, Xue Jun; Nozu, Kandai; Wada, Hiroko; Takada, Satoshi; Iijima, Kazumoto

2015-10-01

CDKL5-related encephalopathy is an X-linked dominantly inherited disorder that is characterized by early infantile epileptic encephalopathy or atypical Rett syndrome. We describe a 5-year-old Japanese boy with intractable epilepsy, severe developmental delay, and Rett syndrome-like features. Onset was at 2 months, when his electroencephalogram showed sporadic single poly spikes and diffuse irregular poly spikes. We conducted a genetic analysis using an Illumina® TruSight™ One sequencing panel on a next-generation sequencer. We identified two epilepsy-associated single nucleotide variants in our case: CDKL5 p.Ala40Val and KCNQ2 p.Glu515Asp. CDKL5 p.Ala40Val has been previously reported to be responsible for early infantile epileptic encephalopathy. In our case, the CDKL5 heterozygous mutation showed somatic mosaicism because the boy's karyotype was 46,XY. The KCNQ2 variant p.Glu515Asp is known to cause benign familial neonatal seizures-1, and this variant showed paternal inheritance. Although we believe that the somatic mosaic CDKL5 mutation is mainly responsible for the neurological phenotype in the patient, the KCNQ2 variant might have some neurological effect. Genetic analysis by next-generation sequencing is capable of identifying multiple variants in a patient. Copyright © 2015 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

A9 region in EPHB2 mutation is frequent in tumors with microsatellite instability. Analysis of prognosis.

PubMed

Rafael, Sara; Vidaurreta, Marta; Veganzones, Silvia; De La Orden, Virgnia; Mediero, Beatriz; Gutierrez, Maria Luisa; Maestro, Maria Luisa

2013-11-01

The aim of the present study was to determine the relation of EPH tyrosine kinase receptor B2 (EPHB2) A9 region mutation and microsatellite instability (MSI); and to analyze their influence in prognosis of patients with sporadic colorectal cancer (CRC). A total of 481 patients with CRC were examined. MSI (NCI criteria) and EPHB2 were analyzed using PCR and fragment analysis software. EPHB2 mutation was detected in 3.1% of patients. Mutation of EPHB2 was associated with location and with MSI status. We considered low instability (L-MSI) when only one marker showed instability, high instability (H-MSI) when two or more markers were positive and microsatelllite stable (MSS) when no instability was detected. The stratified analysis of overall survival (OS) and disease-free survival (DFS) in MSI according to EPHB2 status revealed no statistically significant differences. However, the risk of recurrence of H-MSI tumors with EPHB2 mutation carriers was 3.6-times higher than in non-mutation carriers. The frequency of EPHB2 mutation is higher in patients with H-MSI than MSS tumors. Promising results were found regarding the prognostic influence of EPHB2 in H-MSI.

[Pain and analgesia : Mutations of voltage-gated sodium channels].

PubMed

Eberhardt, M J; Leffler, A

2017-02-01

Voltage-gated sodium channels (Navs) are crucial for the generation and propagation of action potentials in all excitable cells, and therefore for the function of sensory neurons as well. Preclinical research over the past 20 years identified three Nav-isoforms in sensory neurons, namely Nav1.7, Nav1.8 and Nav1.9. A specific role for the function of nociceptive neurons was postulated for each. Whereas no selective sodium channel inhibitors have been established in the clinic so far, the relevance of all three isoforms regarding the pain sensitivity in humans is currently undergoing a remarkable verification through the translation of preclinical data into clinically manifest pictures. For the last ten years, Nav1.7 has been the main focus of clinical interest, as a large number of hereditary mutants were identified. The so-called "gain-of-function" mutations of Nav1.7 cause the pain syndromes hereditary erythromelalgia and paroxysmal extreme pain disorder. In addition, several Nav1.7 mutants were shown to be associated with small-fiber neuropathies. On the contrary, "loss-of-function" Nav1.7 mutants lead to a congenital insensitivity to pain. Recently, several gain-of-function mutations in Nav1.8 and Nav1.9 have been identified in patients suffering from painful peripheral neuropathies. However, another gain-of-function Nav1.9 mutation is associated with congenital insensitivity to pain. This review offers an overview of published work on painful Nav mutations with clinical relevance, and proposes possible consequences for the therapy of different pain symptoms resulting from these findings.

Splice-site mutations identified in PDE6A responsible for retinitis pigmentosa in consanguineous Pakistani families

PubMed Central

Khan, Shahid Y.; Ali, Shahbaz; Naeem, Muhammad Asif; Khan, Shaheen N.; Husnain, Tayyab; Butt, Nadeem H.; Qazi, Zaheeruddin A.; Akram, Javed; Riazuddin, Sheikh; Ayyagari, Radha; Hejtmancik, J. Fielding

2015-01-01

Purpose This study was conducted to localize and identify causal mutations associated with autosomal recessive retinitis pigmentosa (RP) in consanguineous familial cases of Pakistani origin. Methods Ophthalmic examinations that included funduscopy and electroretinography (ERG) were performed to confirm the affectation status. Blood samples were collected from all participating individuals, and genomic DNA was extracted. A genome-wide scan was performed, and two-point logarithm of odds (LOD) scores were calculated. Sanger sequencing was performed to identify the causative variants. Subsequently, we performed whole exome sequencing to rule out the possibility of a second causal variant within the linkage interval. Sequence conservation was performed with alignment analyses of PDE6A orthologs, and in silico splicing analysis was completed with Human Splicing Finder version 2.4.1. Results A large multigenerational consanguineous family diagnosed with early-onset RP was ascertained. An ophthalmic clinical examination consisting of fundus photography and electroretinography confirmed the diagnosis of RP. A genome-wide scan was performed, and suggestive two-point LOD scores were observed with markers on chromosome 5q. Haplotype analyses identified the region; however, the region did not segregate with the disease phenotype in the family. Subsequently, we performed a second genome-wide scan that excluded the entire genome except the chromosome 5q region harboring PDE6A. Next-generation whole exome sequencing identified a splice acceptor site mutation in intron 16: c.2028–1G>A, which was completely conserved in PDE6A orthologs and was absent in ethnically matched 350 control chromosomes, the 1000 Genomes database, and the NHLBI Exome Sequencing Project. Subsequently, we investigated our entire cohort of RP familial cases and identified a second family who harbored a splice acceptor site mutation in intron 10: c.1408–2A>G. In silico analysis suggested that these

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

PubMed Central

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

2009-01-01

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

Molecular spectrum of c-KIT and PDGFRA gene mutations in gastro intestinal stromal tumor: determination of frequency, distribution pattern and identification of novel mutations in Indian patients.

PubMed

Ahmad, Firoz; Lad, Purnima; Bhatia, Simi; Das, Bibhu Ranjan

2015-01-01

KIT and PDGFRA gene mutations are the major genetic alterations seen in gastrointestinal stromal tumors (GISTs) and are being used clinically for predicting response to imatinib therapy. In the current study, we set out to explore the frequency and distribution pattern of c-KIT (exons 9, 11 and 13) and PDGFRA (exons 12 and 18) by direct sequencing in a series of 70 Indian GIST cases. Overall, 27 (38.5 %) and 4 (5.7 %) of the cases had c-KIT and PDGFRA mutations, respectively. Majority of KIT mutations involved exon 11 (85.7 %), followed by exon 9 (14.3 %), while none showed exon 13 mutation. Most exon 9 mutations showed Ala503-Tyr504 duplication, while one had novel point mutation at codon 476 (S476G). In contrast to exon 9 mutations, most exon 11 mutations were in-frame deletions (79 %, 19/24), predominantly at codons 550-560, while remaining exon 11 mutant cases were point mutations at codons 559, 560, 568, 573 and 575. Interestingly, P573T, Q556_V560delinsH, Q575H and Q575_P577 were novel variations observed in exon 11. The PDGFRA mutations were seen mostly in exon 18, which showed point mutation at codon 842 (D842V), while exon 12 showed a novel indel variation (V561_H570delinsT). No significant correlation between c-KIT/PDGFRA mutations and clinicopathological data was observed. In conclusion, this study highlights the frequency and distribution pattern of c-KIT/PDGFRA mutation in Indian cohort. The current study identified novel variations that added new insights into the genetic heterogeneity of GIST patients. Furthermore, this is the first study to report the presence of PDGFRA mutation from Indian subcontinent.

Identifying Attenuating Mutations: Tools for a New Vaccine Design against Flaviviruses.

PubMed

Khou, Cécile; Pardigon, Nathalie

2017-01-01

Emerging Flaviviruses pose an increasing threat to global human health. To date, human vaccines against yellow fever virus (YFV), Japanese encephalitis virus (JEV), dengue virus (DV), and tick-borne encephalitis virus (TBEV) exist. However, there is no human vaccine against other Flaviviruses such as Zika virus (ZIKV) and West Nile virus (WNV). In order to restrict their spread and to protect populations against the diseases they induce, vaccines against these emerging viruses must be designed. Obtaining new live attenuated Flavivirus vaccines using molecular biology methods is now possible. Molecular infectious clones of the parental viruses are relatively easy to generate. Key mutations present in live attenuated vaccines or mutations known to have a key role in the Flavivirus life cycle and/or interactions with their hosts can be identified by sequencing, and are then inserted in infectious clones by site-directed mutagenesis. More recently, the use of chimeric viruses and large-scale reencoding and introduction of microRNA target sequences have also been tested. Indeed, a combination of these methods will help in designing new generations of vaccines against emerging and reemerging Flaviviruses. © 2017 S. Karger AG, Basel.

Novel mutations and phenotypic associations identified through APC, MUTYH, NTHL1, POLD1, POLE gene analysis in Indian Familial Adenomatous Polyposis cohort.

PubMed

Khan, Nikhat; Lipsa, Anuja; Arunachal, Gautham; Ramadwar, Mukta; Sarin, Rajiv

2017-05-22

Colo-Rectal Cancer is a common cancer worldwide with 5-10% cases being hereditary. Familial Adenomatous Polyposis (FAP) syndrome is due to germline mutations in the APC or rarely MUTYH gene. NTHL1, POLD1, POLE have been recently reported in previously unexplained FAP cases. Unlike the Caucasian population, FAP phenotype and its genotypic associations have not been widely studied in several geoethnic groups. We report the first FAP cohort from South Asia and the only non-Caucasian cohort with comprehensive analysis of APC, MUTYH, NTHL1, POLD1, POLE genes. In this cohort of 112 individuals from 53 FAP families, we detected germline APC mutations in 60 individuals (45 families) and biallelic MUTYH mutations in 4 individuals (2 families). No NTHL1, POLD1, POLE mutations were identified. Fifteen novel APC mutations and a new Indian APC mutational hotspot at codon 935 were identified. Eight very rare FAP phenotype or phenotypes rarely associated with mutations outside specific APC regions were observed. APC genotype-phenotype association studies in different geo-ethnic groups can enrich the existing knowledge about phenotypic consequences of distinct APC mutations and guide counseling and risk management in different populations. A stepwise cost-effective mutation screening approach is proposed for genetic testing of south Asian FAP patients.

Analysis of recombinant H7N9 wild-type and mutant viruses in pigs shows that the Q226L mutation in HA is important for transmission.

PubMed

Liu, Qinfang; Zhou, Bin; Ma, Wenjun; Bawa, Bhupinder; Ma, Jingjiao; Wang, Wei; Lang, Yuekun; Lyoo, Young; Halpin, Rebecca A; Lin, Xudong; Stockwell, Timothy B; Webby, Richard; Wentworth, David E; Richt, Juergen A

2014-07-01

The fact that there have been more than 300 human infections with a novel avian H7N9 virus in China indicates that this emerging strain has pandemic potential. Furthermore, many of the H7N9 viruses circulating in animal reservoirs contain putative mammalian signatures in the HA and PB2 genes that are believed to be important in the adaptation of other avian strains to humans. To date, the definitive roles of these mammalian-signature substitutions in transmission and pathogenesis of H7N9 viruses remain unclear. To address this we analyzed the biological characteristics, pathogenicity, and transmissibility of A/Anhui/1/2013 (H7N9) virus and variants in vitro and in vivo using a synthetically created wild-type virus (rAnhui-WT) and two mutants (rAnhui-HA-226Q and rAnhui-PB2-627E). All three viruses replicated in lungs of intratracheally inoculated pigs, yet nasal shedding was limited. The rAnhui-WT and rAnhui-PB2-627E viruses were transmitted to contact animals. In contrast, the rAnhui-HA-226Q virus was not transmitted to sentinel pigs. Deep sequencing of viruses from the lungs of infected pigs identified substitutions arising in the viral population (e.g., PB2-T271A, PB2-D701N, HA-V195I, and PB2-E627K reversion) that may enhance viral replication in pigs. Collectively, the results demonstrate that critical mutations (i.e., HA-Q226L) enable the H7N9 viruses to be transmitted in a mammalian host and suggest that the myriad H7N9 genotypes circulating in avian species in China and closely related strains (e.g., H7N7) have the potential for further adaptation to human or other mammalian hosts (e.g., pigs), leading to strains capable of sustained human-to-human transmission. Importance: The genomes of the zoonotic avian H7N9 viruses emerging in China have mutations in critical genes (PB2-E627K and HA-Q226L) that may be important in their pandemic potential. This study shows that (i) HA-226L of zoonotic H7N9 strains is critical for binding the α-2,6-linked receptor and

A novel NOTCH3 mutation identified in patients with oral cancer by whole exome sequencing.

PubMed

Yi, Yanjun; Tian, Zhuowei; Ju, Houyu; Ren, Guoxin; Hu, Jingzhou

2017-06-01

Oral cancer is a serious disease caused by environmental factors and/or susceptible genes. In the present study, in order to identify useful genetic biomarkers for cancer prediction and prevention, and for personalized treatment, we detected somatic mutations in 5 pairs of oral cancer tissues and blood samples using whole exome sequencing (WES). Finally, we confirmed a novel nonsense single-nucleotide polymorphism (SNP; chr19:15288426A>C) in the NOTCH3 gene with sanger sequencing, which resulted in a N1438T mutation in the protein sequence. Using multiple in silico analyses, this variant was found to mildly damaging effects on the NOTCH3 gene, which was supported by the results from analyses using PANTHER, SNAP and SNPs&GO. However, further analysis using Mutation Taster revealed that this SNP had a probability of 0.9997 to be 'disease causing'. In addition, we performed 3D structure simulation analysis and the results suggested that this variant had little effect on the solubility and hydrophobicity of the protein and thus on its function; however, it decreased the stability of the protein by increasing the total energy following minimization (-1,051.39 kcal/mol for the mutant and -1,229.84 kcal/mol for the native) and decreasing one stabilizing residue of the protein. Less stability of the N1438T mutant was also supported by analysis using I-Mutant with a DDG value of -1.67. Overall, the present study identified and confirmed a novel mutation in the NOTCH3 gene, which may decrease the stability of NOTCH3, and may thus prove to be helpful in cancer prognosis.

Next-Generation Sequencing-based genomic profiling of brain metastases of primary ovarian cancer identifies high number of BRCA-mutations.

PubMed

Balendran, S; Liebmann-Reindl, S; Berghoff, A S; Reischer, T; Popitsch, N; Geier, C B; Kenner, L; Birner, P; Streubel, B; Preusser, M

2017-07-01

Ovarian cancer represents the most common gynaecological malignancy and has the highest mortality of all female reproductive cancers. It has a rare predilection to develop brain metastases (BM). In this study, we evaluated the mutational profile of ovarian cancer metastases through Next-Generation Sequencing (NGS) with the aim of identifying potential clinically actionable genetic alterations with options for small molecule targeted therapy. Library preparation was conducted using Illumina TruSight Rapid Capture Kit in combination with a cancer specific enrichment kit covering 94 genes. BRCA-mutations were confirmed by using TruSeq Custom Amplicon Low Input Kit in combination with a custom-designed BRCA gene panel. In our cohort all eight sequenced BM samples exhibited a multitude of variant alterations, each with unique molecular profiles. The 37 identified variants were distributed over 22 cancer-related genes (23.4%). The number of mutated genes per sample ranged from 3 to 7 with a median of 4.5. The most commonly altered genes were BRCA1/2, TP53, and ATM. In total, 7 out of 8 samples revealed either a BRCA1 or a BRCA2 pathogenic mutation. Furthermore, all eight BM samples showed mutations in at least one DNA repair gene. Our NGS study of BM of ovarian carcinoma revealed a significant number of BRCA-mutations beside TP53, ATM and CHEK2 mutations. These findings strongly suggest the implication of BRCA and DNA repair malfunction in ovarian cancer metastasizing to the brain. Based on these findings, pharmacological PARP inhibition could be one potential targeted therapeutic for brain metastatic ovarian cancer patients.

Interaction among variants in the SLC gene family (SLC6A14, SLC26A9, SLC11A1, and SLC9A3) and CFTR mutations with clinical markers of cystic fibrosis.

PubMed

Pereira, Stephanie V N; Ribeiro, Jose D; Bertuzzo, Carmen S; Marson, Fernando A L

2018-04-10

Cystic fibrosis (CF) is due to dysfunction of the CFTR channel and function of this channel is, in turn, affected by modifier genes that can impact the clinical phenotype. In this context, we analyzed the interaction among rs3788766*SLC6A14, rs7512462*SLC26A9, rs17235416*SLC11A1, and rs17563161*SLC9A3 variants, CFTR mutations and 40 CF severity markers by the Multifactor Dimensionality Reduction (MDR) model. A total of 164 patients with CF were included in the study. The variants in the modifier genes were identified by real-time PCR and the genotype of the CFTR gene in the diagnostic routine. Analysis of interaction between variants, CFTR mutations groupings and demographic, clinical and laboratory data were performed by the MDR. There were interaction between the rs3788766, rs7512462, rs17235416, and rs17563161 variants, and CFTR mutations with pancreatic insufficiency (PI), onset of digestive symptoms, and presence of mucoid Pseudomonas aeruginosa. Regarding PI, the interaction was observed for CFTR*rs17563161 (P-value = 0.015). Also, for onset of digestive symptoms the interaction was observed for CFTR*rs3788766*rs7512462*rs17235416*rs17563161 (P-value = 0.036). Considering the presence of mucoid P. aeruginosa, the interaction occurred for CFTR*rs3788766*rs7512462*rs17563161 (P-value = 0.035). Interaction between variants in the SLC family genes and the grouping for CFTR mutations were associated with PI, onset of digestive symptoms and mucoid P. aeruginosa, being important to determine one of the factors that may cause the diversity among the patients with CF. © 2018 Wiley Periodicals, Inc.

An atypical case of SCN9A mutation presenting with global motor delay and a severe pain disorder.

PubMed

Meijer, Inge Anita; Vanasse, Michel; Nizard, Sonia; Robitaille, Yves; Rossignol, Elsa

2014-01-01

Erythromelalgia due to heterozygous gain-of-function SCN9A mutations usually presents as a pure sensory-autonomic disorder characterized by recurrent episodes of burning pain and redness of the extremities. We describe a patient with an unusual phenotypic presentation of gross motor delay, childhood-onset erythromelalgia, extreme visceral pain episodes, hypesthesia, and self-mutilation. The investigation of the patient's motor delay included various biochemical analyses, a comparative genomic hybridization array (CGH), electromyogram (EMG), and muscle biopsy. Once erythromelalgia was suspected clinically, the SCN9A gene was sequenced. The EMG, CGH, and biochemical tests were negative. The biopsy showed an axonal neuropathy and neurogenic atrophy. Sequencing of SCN9A revealed a heterozygous missense mutation in exon 7; p.I234T. This is a case of global motor delay and erythromelalgia associated with SCN9A. The motor delay may be attributed to the extreme pain episodes or to a developmental perturbation of proprioceptive inputs. Copyright © 2013 Wiley Periodicals, Inc.

Splice Site Mutations in the ATP7A Gene

PubMed Central

Møller, Lisbeth Birk

2011-01-01

Menkes disease (MD) is caused by mutations in the ATP7A gene. We describe 33 novel splice site mutations detected in patients with MD or the milder phenotypic form, Occipital Horn Syndrome. We review these 33 mutations together with 28 previously published splice site mutations. We investigate 12 mutations for their effect on the mRNA transcript in vivo. Transcriptional data from another 16 mutations were collected from the literature. The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation to in vivo results. Ninety-six percent of the mutations identified in 45 patients with classical MD were predicted to have a significant effect on splicing, which concurs with the absence of any detectable wild-type transcript in all 19 patients investigated in vivo. Sixty-seven percent of the mutations identified in 12 patients with milder phenotypes were predicted to have no significant effect on splicing, which concurs with the presence of wild-type transcript in 7 out of 9 patients investigated in vivo. Both the in silico predictions and the in vivo results support the hypothesis previously suggested by us and others, that the presence of some wild-type transcript is correlated to a milder phenotype. PMID:21494555

CRISPR/Cas9 and TALENs generate heritable mutations for genes involved in small RNA processing of Glycine max and Medicago truncatula.

PubMed

Curtin, Shaun J; Xiong, Yer; Michno, Jean-Michel; Campbell, Benjamin W; Stec, Adrian O; Čermák, Tomas; Starker, Colby; Voytas, Daniel F; Eamens, Andrew L; Stupar, Robert M

2018-06-01

Processing of double-stranded RNA precursors into small RNAs is an essential regulator of gene expression in plant development and stress response. Small RNA processing requires the combined activity of a functionally diverse group of molecular components. However, in most of the plant species, there are insufficient mutant resources to functionally characterize each encoding gene. Here, mutations in loci encoding protein machinery involved in small RNA processing in soya bean and Medicago truncatula were generated using the CRISPR/Cas9 and TAL-effector nuclease (TALEN) mutagenesis platforms. An efficient CRISPR/Cas9 reagent was used to create a bi-allelic double mutant for the two soya bean paralogous Double-stranded RNA-binding2 (GmDrb2a and GmDrb2b) genes. These mutations, along with a CRISPR/Cas9-generated mutation of the M. truncatula Hua enhancer1 (MtHen1) gene, were determined to be germ-line transmissible. Furthermore, TALENs were used to generate a mutation within the soya bean Dicer-like2 gene. CRISPR/Cas9 mutagenesis of the soya bean Dicer-like3 gene and the GmHen1a gene was observed in the T 0 generation, but these mutations failed to transmit to the T 1 generation. The irregular transmission of induced mutations and the corresponding transgenes was investigated by whole-genome sequencing to reveal a spectrum of non-germ-line-targeted mutations and multiple transgene insertion events. Finally, a suite of combinatorial mutant plants were generated by combining the previously reported Gmdcl1a, Gmdcl1b and Gmdcl4b mutants with the Gmdrb2ab double mutant. Altogether, this study demonstrates the synergistic use of different genome engineering platforms to generate a collection of useful mutant plant lines for future study of small RNA processing in legume crops. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

Novel germline mutations in FLCN gene identified in two Chinese patients with Birt-Hogg-Dubé syndrome.

PubMed

Li, Teng; Ning, Xianghui; He, Qun; Gong, Kan

2017-01-09

Birt-Hogg-Dubé (BHD) syndrome, a hereditary renal cancer syndrome caused by mutations in the folliculin (FLCN) gene, is characterized by the presence of fibrofolliculomas, pulmonary cysts, spontaneous pneumothorax, and renal cell carcinoma (RCC). Few BHD syndrome cases have been reported in Asian countries, and cutaneous presentations are relatively rare in Asian patients. Asian BHD patients may be misdiagnosed due to their atypical manifestations. Here, we report two Chinese BHD patients with novel FLCN mutations (c.946-947delAG in exon 9 and c.770-772delCCT in exon 7). Both of them had RCC and spontaneous pneumothorax without fibrofolliculomas. In patients with RCC and pulmonary cysts but without cutaneous lesions, screening for mutations in the FLCN gene should be performed, especially for those with a family history of RCC or pulmonary cysts (pneumothorax).

Epidermal growth factor receptor mutations in adenocarcinoma in situ and minimally invasive adenocarcinoma detected using mutation-specific monoclonal antibodies.

PubMed

Nakamura, Haruhiko; Koizumi, Hirotaka; Kimura, Hiroyuki; Marushima, Hideki; Saji, Hisashi; Takagi, Masayuki

2016-09-01

Epidermal growth factor receptor (EGFR) mutation rates in adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA) were studied using both DNA analysis and mutation-specific immunohistochemistry. The peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp method was used to detect mutations in exons 18, 19, 20, and 21 of the EGFR gene in DNA samples extracted from paraffin-embedded tissue sections. Simultaneously, immunohistochemical analysis with two EGFR mutation-specific monoclonal antibodies was used to identify proteins resulting from an in-frame deletion in exon 19 (E746_A750del) and a point mutation replacing leucine with arginine at codon 858 of exon 21 (L858R). Forty-three tumors (22 AIS and 21 MIA) were examined. The EGFR mutation rate in AIS detected by DNA analysis was 27.3% (L858R, 5/22; exon 19 deletion,1/22), whereas that detected in MIA was 42.9% (L858R,4/21; exon 19 deletion,5/21). Mutations detected by immunohistochemical analysis included 22.7% (L858R, 4/22; exon 19 deletion, 1/22) in AIS and 42.9% (L858R, 4/21; exon 19 deletion, 5/21) in MIA. Although some results were contradictory, concordant results were obtained using both assays in 38 of 43 cases (88.4%). DNA and immunohistochemical analyses revealed similar EGFR mutation rates in both MIA and AIS, suggesting that mutation-specific monoclonal antibodies are useful to confirm DNA assay results. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Establishment of Mouse Model of MYH9 Disorders: Heterozygous R702C Mutation Provokes Macrothrombocytopenia with Leukocyte Inclusion Bodies, Renal Glomerulosclerosis and Hearing Disability

PubMed Central

Suzuki, Nobuaki; Kunishima, Shinji; Ikejiri, Makoto; Maruyama, Shoichi; Sone, Michihiko; Takagi, Akira; Ikawa, Masahito; Okabe, Masaru; Kojima, Tetsuhito; Saito, Hidehiko; Naoe, Tomoki; Matsushita, Tadashi

2013-01-01

Nonmuscle myosin heavy chain IIA (NMMHCIIA) encoded by MYH9 is associated with autosomal dominantly inherited diseases called MYH9 disorders. MYH9 disorders are characterized by macrothrombocytopenia and very characteristic inclusion bodies in granulocytes. MYH9 disorders frequently cause nephritis, sensorineural hearing disability and cataracts. One of the most common and deleterious mutations causing these disorders is the R702C missense mutation. We generated knock-in mice expressing the Myh9 R702C mutation. R702C knock-in hetero mice (R702C+/− mice) showed macrothrombocytopenia. We studied megakaryopoiesis of cultured fetal liver cells of R702C+/− mice and found that proplatelet formation was impaired: the number of proplatelet tips was decreased, proplatelet size was increased, and proplatelet shafts were short and enlarged. Although granulocyte inclusion bodies were not visible by May–Grünwald Giemsa staining, immunofluorescence analysis indicated that NMMHCIIA proteins aggregated and accumulated in the granulocyte cytoplasm. In other organs, R702C+/− mice displayed albuminuria which increased with age. Renal pathology examination revealed glomerulosclerosis. Sensory hearing loss was indicated by lowered auditory brainstem response. These findings indicate that Myh9 R702C knock-in mice mirror features of human MYH9 disorders arising from the R702C mutation. PMID:23976996

Establishment of mouse model of MYH9 disorders: heterozygous R702C mutation provokes macrothrombocytopenia with leukocyte inclusion bodies, renal glomerulosclerosis and hearing disability.

PubMed

Suzuki, Nobuaki; Kunishima, Shinji; Ikejiri, Makoto; Maruyama, Shoichi; Sone, Michihiko; Takagi, Akira; Ikawa, Masahito; Okabe, Masaru; Kojima, Tetsuhito; Saito, Hidehiko; Naoe, Tomoki; Matsushita, Tadashi

2013-01-01

Nonmuscle myosin heavy chain IIA (NMMHCIIA) encoded by MYH9 is associated with autosomal dominantly inherited diseases called MYH9 disorders. MYH9 disorders are characterized by macrothrombocytopenia and very characteristic inclusion bodies in granulocytes. MYH9 disorders frequently cause nephritis, sensorineural hearing disability and cataracts. One of the most common and deleterious mutations causing these disorders is the R702C missense mutation. We generated knock-in mice expressing the Myh9 R702C mutation. R702C knock-in hetero mice (R702C+/- mice) showed macrothrombocytopenia. We studied megakaryopoiesis of cultured fetal liver cells of R702C+/- mice and found that proplatelet formation was impaired: the number of proplatelet tips was decreased, proplatelet size was increased, and proplatelet shafts were short and enlarged. Although granulocyte inclusion bodies were not visible by May-Grünwald Giemsa staining, immunofluorescence analysis indicated that NMMHCIIA proteins aggregated and accumulated in the granulocyte cytoplasm. In other organs, R702C+/- mice displayed albuminuria which increased with age. Renal pathology examination revealed glomerulosclerosis. Sensory hearing loss was indicated by lowered auditory brainstem response. These findings indicate that Myh9 R702C knock-in mice mirror features of human MYH9 disorders arising from the R702C mutation.

WholeGenome Sequencing of High-Risk Families to Identify New Mutational Mechanisms of Breast Cancer Predisposition

DTIC Science & Technology

2014-10-01

4 APPENDICES 4 INTRODUCTION: Despite tremendous advances in mutation detection with gene panels...population frequency and overlap with ENCODE regions. 2a. Align reads to the reference sequence (months 4-10) 2b. Identify SNPs, indels, CNVs and

Exome Sequencing Identifies Truncating Mutations in Human SERPINF1 in Autosomal-Recessive Osteogenesis Imperfecta

PubMed Central

Becker, Jutta; Semler, Oliver; Gilissen, Christian; Li, Yun; Bolz, Hanno Jörn; Giunta, Cecilia; Bergmann, Carsten; Rohrbach, Marianne; Koerber, Friederike; Zimmermann, Katharina; de Vries, Petra; Wirth, Brunhilde; Schoenau, Eckhard; Wollnik, Bernd; Veltman, Joris A.; Hoischen, Alexander; Netzer, Christian

2011-01-01

Osteogenesis imperfecta (OI) is a heterogeneous genetic disorder characterized by bone fragility and susceptibility to fractures after minimal trauma. After mutations in all known OI genes had been excluded by Sanger sequencing, we applied next-generation sequencing to analyze the exome of a single individual who has a severe form of the disease and whose parents are second cousins. A total of 26,922 variations from the human reference genome sequence were subjected to several filtering steps. In addition, we extracted the genotypes of all dbSNP130-annotated SNPs from the exome sequencing data and used these 299,494 genotypes as markers for the genome-wide identification of homozygous regions. A single homozygous truncating mutation, affecting SERPINF1 on chromosome 17p13.3, that was embedded into a homozygous stretch of 2.99 Mb remained. The mutation was also homozygous in the affected brother of the index patient. Subsequently, we identified homozygosity for two different truncating SERPINF1 mutations in two unrelated patients with OI and parental consanguinity. All four individuals with SERPINF1 mutations have severe OI. Fractures of long bones and severe vertebral compression fractures with resulting deformities were observed as early as the first year of life in these individuals. Collagen analyses with cultured dermal fibroblasts displayed no evidence for impaired collagen folding, posttranslational modification, or secretion. SERPINF1 encodes pigment epithelium-derived factor (PEDF), a secreted glycoprotein of the serpin superfamily. PEDF is a multifunctional protein and one of the strongest inhibitors of angiogenesis currently known in humans. Our data provide genetic evidence for PEDF involvement in human bone homeostasis. PMID:21353196

Newly identified mutations at the CSN1S1 gene in Ethiopian goats affect casein content and coagulation properties of their milk.

PubMed

Mestawet, T A; Girma, A; Adnøy, T; Devold, T G; Vegarud, G E

2013-08-01

Very high casein content and good coagulation properties previously observed in some Ethiopian goat breeds led to investigating the αs1-casein (CSN1S1) gene in these breeds. Selected regions of the CSN1S1 gene were sequenced in 115 goats from 5 breeds (2 indigenous: Arsi-Bale and Somali, 1 exotic: Boer, and 2 crossbreeds: Boer × Arsi-Bale and Boer × Somali). The DNA analysis resulted in 35 new mutations: 3 in exons, 3 in the 5' untranslated region (UTR), and 29 in the introns. The mutations in exons that resulted in an amino acid shift were then picked to evaluate their influence on individual casein content (αs1-, αs2-, β-, and κ-CN), micellar size, and coagulation properties in the milk from the 5 goat breeds. A mutation at nucleotide 10657 (exon 10) involved a transversion: CAG→CCG, resulting in an amino acid exchange Gln77→Pro77. This mutation was associated with the indigenous breeds only. Two new mutations, at nucleotide 6072 (exon 4) and 12165 (exon 12), revealed synonymous transitions: GTC→GTT in Val15 and AGA→AGG in Arg100 of the mature protein. Transitions G→A and C→T at nucleotides 1374 and 1866, respectively, occurred in the 5' UTR, whereas the third mutation involved a transversion T→G at nucleotide location 1592. The goats were grouped into homozygote new (CC), homozygote reference (AA), and heterozygote (CA) based on the nucleotide that involved the transversion. The content of αs1-CN (15.32g/kg) in milk samples of goats homozygous (CC) for this newly identified mutation, Gln77→Pro77 was significantly higher than in milks of heterozygous (CA; 9.05g/kg) and reference (AA; 7.61g/kg) genotype animals. The αs2-, β-, and κ-CN contents showed a similar pattern. Milk from goats with a homozygous new mutation had significantly lower micellar size. Milk from both homozygote and heterozygote new-mutation goats had significantly shorter coagulation rate and stronger gel than the reference genotype. Except the transversion, the

Target enrichment and high-throughput sequencing of 80 ribosomal protein genes to identify mutations associated with Diamond-Blackfan anaemia.

PubMed

Gerrard, Gareth; Valgañón, Mikel; Foong, Hui En; Kasperaviciute, Dalia; Iskander, Deena; Game, Laurence; Müller, Michael; Aitman, Timothy J; Roberts, Irene; de la Fuente, Josu; Foroni, Letizia; Karadimitris, Anastasios

2013-08-01

Diamond-Blackfan anaemia (DBA) is caused by inactivating mutations in ribosomal protein (RP) genes, with mutations in 13 of the 80 RP genes accounting for 50-60% of cases. The remaining 40-50% cases may harbour mutations in one of the remaining RP genes, but the very low frequencies render conventional genetic screening as challenging. We, therefore, applied custom enrichment technology combined with high-throughput sequencing to screen all 80 RP genes. Using this approach, we identified and validated inactivating mutations in 15/17 (88%) DBA patients. Target enrichment combined with high-throughput sequencing is a robust and improved methodology for the genetic diagnosis of DBA. © 2013 John Wiley & Sons Ltd.

BCOR and BCORL1 mutations in myelodysplastic syndromes and related disorders.

PubMed

Damm, Frederik; Chesnais, Virginie; Nagata, Yasunobu; Yoshida, Kenichi; Scourzic, Laurianne; Okuno, Yusuke; Itzykson, Raphael; Sanada, Masashi; Shiraishi, Yuichi; Gelsi-Boyer, Véronique; Renneville, Aline; Miyano, Satoru; Mori, Hiraku; Shih, Lee-Yung; Park, Sophie; Dreyfus, François; Guerci-Bresler, Agnes; Solary, Eric; Rose, Christian; Cheze, Stéphane; Prébet, Thomas; Vey, Norbert; Legentil, Marion; Duffourd, Yannis; de Botton, Stéphane; Preudhomme, Claude; Birnbaum, Daniel; Bernard, Olivier A; Ogawa, Seishi; Fontenay, Michaela; Kosmider, Olivier

2013-10-31

Patients with low-risk myelodysplastic syndromes (MDS) that rapidly progress to acute myeloid leukemia (AML) remain a challenge in disease management. Using whole-exome sequencing of an MDS patient, we identified a somatic mutation in the BCOR gene also mutated in AML. Sequencing of BCOR and related BCORL1 genes in a cohort of 354 MDS patients identified 4.2% and 0.8% of mutations respectively. BCOR mutations were associated with RUNX1 (P = .002) and DNMT3A mutations (P = .015). BCOR is also mutated in chronic myelomonocytic leukemia patients (7.4%) and BCORL1 in AML patients with myelodysplasia-related changes (9.1%). Using deep sequencing, we show that BCOR mutations arise after mutations affecting genes involved in splicing machinery or epigenetic regulation. In univariate analysis, BCOR mutations were associated with poor prognosis in MDS (overall survival [OS]: P = .013; cumulative incidence of AML transformation: P = .005). Multivariate analysis including age, International Prognostic Scoring System, transfusion dependency, and mutational status confirmed a significant inferior OS to patients with a BCOR mutation (hazard ratio, 3.3; 95% confidence interval, 1.4-8.1; P = .008). These data suggest that BCOR mutations define the clinical course rather than disease initiation. Despite infrequent mutations, BCOR analyses should be considered in risk stratification.

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

PubMed

Tomar, Swati; Sethi, Raman; Sundar, Gangadhara; Quah, Thuan Chong; Quah, Boon Long; Lai, Poh San

2017-01-01

Retinoblastoma (RB) is a rare childhood malignant disorder caused by the biallelic inactivation of RB1 gene. Early diagnosis and identification of carriers of heritable RB1 mutations can improve disease outcome and management. In this study, mutational analysis was conducted on fifty-nine matched tumor and peripheral blood samples from 18 bilateral and 41 unilateral unrelated RB cases by a combinatorial approach of Multiplex Ligation-dependent Probe Amplification (MLPA) assay, deletion screening, direct sequencing, copy number gene dosage analysis and methylation assays. Screening of both blood and tumor samples yielded a mutation detection rate of 94.9% (56/59) while only 42.4% (25/59) of mutations were detected if blood samples alone were analyzed. Biallelic mutations were observed in 43/59 (72.9%) of tumors screened. There were 3 cases (5.1%) in which no mutations could be detected and germline mutations were detected in 19.5% (8/41) of unilateral cases. A total of 61 point mutations were identified, of which 10 were novel. There was a high incidence of previously reported recurrent mutations, occurring at 38.98% (23/59) of all cases. Of interest were three cases of mosaic RB1 mutations detected in the blood from patients with unilateral retinoblastoma. Additionally, two germline mutations previously reported to be associated with low-penetrance phenotypes: missense-c.1981C>T and splice variant-c.607+1G>T, were observed in a bilateral and a unilateral proband, respectively. These findings have implications for genetic counselling and risk prediction for the affected families. This is the first published report on the spectrum of mutations in RB patients from Singapore and shows that further improved mutation screening strategies are required in order to provide a definitive molecular diagnosis for every case of RB. Our findings also underscore the importance of genetic testing in supporting individualized disease management plans for patients and asymptomatic

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

PubMed Central

Tomar, Swati; Sethi, Raman; Sundar, Gangadhara; Quah, Thuan Chong; Quah, Boon Long; Lai, Poh San

2017-01-01

Retinoblastoma (RB) is a rare childhood malignant disorder caused by the biallelic inactivation of RB1 gene. Early diagnosis and identification of carriers of heritable RB1 mutations can improve disease outcome and management. In this study, mutational analysis was conducted on fifty-nine matched tumor and peripheral blood samples from 18 bilateral and 41 unilateral unrelated RB cases by a combinatorial approach of Multiplex Ligation-dependent Probe Amplification (MLPA) assay, deletion screening, direct sequencing, copy number gene dosage analysis and methylation assays. Screening of both blood and tumor samples yielded a mutation detection rate of 94.9% (56/59) while only 42.4% (25/59) of mutations were detected if blood samples alone were analyzed. Biallelic mutations were observed in 43/59 (72.9%) of tumors screened. There were 3 cases (5.1%) in which no mutations could be detected and germline mutations were detected in 19.5% (8/41) of unilateral cases. A total of 61 point mutations were identified, of which 10 were novel. There was a high incidence of previously reported recurrent mutations, occurring at 38.98% (23/59) of all cases. Of interest were three cases of mosaic RB1 mutations detected in the blood from patients with unilateral retinoblastoma. Additionally, two germline mutations previously reported to be associated with low-penetrance phenotypes: missense-c.1981C>T and splice variant-c.607+1G>T, were observed in a bilateral and a unilateral proband, respectively. These findings have implications for genetic counselling and risk prediction for the affected families. This is the first published report on the spectrum of mutations in RB patients from Singapore and shows that further improved mutation screening strategies are required in order to provide a definitive molecular diagnosis for every case of RB. Our findings also underscore the importance of genetic testing in supporting individualized disease management plans for patients and asymptomatic

PB2-Q591K Mutation Determines the Pathogenicity of Avian H9N2 Influenza Viruses for Mammalian Species

PubMed Central

Wang, Congrong; Lee, Horace Hok Yeung; Yang, Zi Feng; Mok, Chris Ka Pun; Zhang, Zhi

2016-01-01

Background Influenza A subtype H9N2 is widespread and prevalent in poultry. It has repeatedly transmitted zoonotically to cause mild influenza-like illness in humans and is regarded as a potential pandemic candidate. In additon, the six internal genes of H7N9 and H10N8 viruses which caused infection in human in China as well as some of the highly pathogenic H5N1 strains are origined from H9N2. Previous studies have shown that the mammalian adaptation PB2-Q591K contributes to the pathogenicity of H5N1 and H7N9 viruses. However, the role of the PB2-Q591K mutation in H9N2 subtype is still not well understood. Methods To define and compare the individual role of PB2-Q591K substitution in the PB2 gene segment of H9N2 in relation to polymerase activity, replication competence and the pathogenicity using in vitro and in vivo models. Results The PB2-Q591K mutation in H9N2 virus enhanced the polymerase activity and virus replication in human NHBE cells when compared to the wild type strain. Mice infected with the PB2 mutant showed significant weight loss, higher virus replication and immune responses in the lungs. Conclusions Our evidences suggest that the PB2-Q591K, in addition to the -E627K mutation in H9N2 enhanced the pathogenicity in mammalian host. PMID:27684944

Targeted high-throughput sequencing identifies mutations in atlastin-1 as a cause of hereditary sensory neuropathy type I.

PubMed

Guelly, Christian; Zhu, Peng-Peng; Leonardis, Lea; Papić, Lea; Zidar, Janez; Schabhüttl, Maria; Strohmaier, Heimo; Weis, Joachim; Strom, Tim M; Baets, Jonathan; Willems, Jan; De Jonghe, Peter; Reilly, Mary M; Fröhlich, Eleonore; Hatz, Martina; Trajanoski, Slave; Pieber, Thomas R; Janecke, Andreas R; Blackstone, Craig; Auer-Grumbach, Michaela

2011-01-07

Hereditary sensory neuropathy type I (HSN I) is an axonal form of autosomal-dominant hereditary motor and sensory neuropathy distinguished by prominent sensory loss that leads to painless injuries. Unrecognized, these can result in delayed wound healing and osteomyelitis, necessitating distal amputations. To elucidate the genetic basis of an HSN I subtype in a family in which mutations in the few known HSN I genes had been excluded, we employed massive parallel exon sequencing of the 14.3 Mb disease interval on chromosome 14q. We detected a missense mutation (c.1065C>A, p.Asn355Lys) in atlastin-1 (ATL1), a gene that is known to be mutated in early-onset hereditary spastic paraplegia SPG3A and that encodes the large dynamin-related GTPase atlastin-1. The mutant protein exhibited reduced GTPase activity and prominently disrupted ER network morphology when expressed in COS7 cells, strongly supporting pathogenicity. An expanded screen in 115 additional HSN I patients identified two further dominant ATL1 mutations (c.196G>C [p.Glu66Gln] and c.976 delG [p.Val326TrpfsX8]). This study highlights an unexpected major role for atlastin-1 in the function of sensory neurons and identifies HSN I and SPG3A as allelic disorders.

Survival from breast cancer in patients with CHEK2 mutations.

PubMed

Huzarski, T; Cybulski, C; Wokolorczyk, D; Jakubowska, A; Byrski, T; Gronwald, J; Domagała, P; Szwiec, M; Godlewski, D; Kilar, E; Marczyk, E; Siołek, M; Wiśniowski, R; Janiszewska, H; Surdyka, D; Sibilski, R; Sun, P; Lubiński, J; Narod, S A

2014-04-01

The purpose of this study is to estimate 10-year survival rates for patients with early onset breast cancer, with and without a CHEK2 mutation and to identify prognostic factors among CHEK2-positive breast cancer patients. 3,592 women with stage I to stage III breast cancer, diagnosed at or below age 50, were tested for four founder mutations in the CHEK2 gene. Information on tumor characteristics and on treatments received was retrieved from medical records. Dates of death were obtained from the Poland Vital Statistics Registry. Survival curves were generated for the mutation-positive and -negative sub-cohorts. Predictors of survival were determined among CHEK2 carriers using the Cox proportional hazards model. 3,592 patients were eligible for the study, of whom 140 (3.9 %) carried a CHEK2-truncating mutation and 347 (9.7 %) carried a missense mutation. The mean follow-up was 8.9 years. The 10-year survival for all CHEK2 mutation carriers was 78.8 % (95 % CI 74.6-83.2 %) and for non-carriers was 80.1 % (95 % CI 78.5-81.8 %). Among women with a CHEK2-positive breast cancer, the adjusted hazard ratio associated with ER-positive status was 0.88 (95 % CI 0.48-1.62). Among women with an ER-positive breast cancer, the adjusted hazard ratio associated with a CHEK2 mutation was 1.31 (95 % CI 0.97-1.77). The survival of women with breast cancer and a CHEK2 mutation is similar to that of patients without a CHEK2 mutation.

Whole-exome sequencing identifies novel compound heterozygous mutations in USH2A in Spanish patients with autosomal recessive retinitis pigmentosa.

PubMed

Méndez-Vidal, Cristina; González-Del Pozo, María; Vela-Boza, Alicia; Santoyo-López, Javier; López-Domingo, Francisco J; Vázquez-Marouschek, Carmen; Dopazo, Joaquin; Borrego, Salud; Antiñolo, Guillermo

2013-01-01

Retinitis pigmentosa (RP) is an inherited retinal dystrophy characterized by extreme genetic and clinical heterogeneity. Thus, the diagnosis is not always easily performed due to phenotypic and genetic overlap. Current clinical practices have focused on the systematic evaluation of a set of known genes for each phenotype, but this approach may fail in patients with inaccurate diagnosis or infrequent genetic cause. In the present study, we investigated the genetic cause of autosomal recessive RP (arRP) in a Spanish family in which the causal mutation has not yet been identified with primer extension technology and resequencing. We designed a whole-exome sequencing (WES)-based approach using NimbleGen SeqCap EZ Exome V3 sample preparation kit and the SOLiD 5500×l next-generation sequencing platform. We sequenced the exomes of both unaffected parents and two affected siblings. Exome analysis resulted in the identification of 43,204 variants in the index patient. All variants passing filter criteria were validated with Sanger sequencing to confirm familial segregation and absence in the control population. In silico prediction tools were used to determine mutational impact on protein function and the structure of the identified variants. Novel Usher syndrome type 2A (USH2A) compound heterozygous mutations, c.4325T>C (p.F1442S) and c.15188T>G (p.L5063R), located in exons 20 and 70, respectively, were identified as probable causative mutations for RP in this family. Family segregation of the variants showed the presence of both mutations in all affected members and in two siblings who were apparently asymptomatic at the time of family ascertainment. Clinical reassessment confirmed the diagnosis of RP in these patients. Using WES, we identified two heterozygous novel mutations in USH2A as the most likely disease-causing variants in a Spanish family diagnosed with arRP in which the cause of the disease had not yet been identified with commonly used techniques. Our data

Whole-exome sequencing identifies novel compound heterozygous mutations in USH2A in Spanish patients with autosomal recessive retinitis pigmentosa

PubMed Central

Méndez-Vidal, Cristina; González-del Pozo, María; Vela-Boza, Alicia; Santoyo-López, Javier; López-Domingo, Francisco J.; Vázquez-Marouschek, Carmen; Dopazo, Joaquin; Borrego, Salud

2013-01-01

Purpose Retinitis pigmentosa (RP) is an inherited retinal dystrophy characterized by extreme genetic and clinical heterogeneity. Thus, the diagnosis is not always easily performed due to phenotypic and genetic overlap. Current clinical practices have focused on the systematic evaluation of a set of known genes for each phenotype, but this approach may fail in patients with inaccurate diagnosis or infrequent genetic cause. In the present study, we investigated the genetic cause of autosomal recessive RP (arRP) in a Spanish family in which the causal mutation has not yet been identified with primer extension technology and resequencing. Methods We designed a whole-exome sequencing (WES)-based approach using NimbleGen SeqCap EZ Exome V3 sample preparation kit and the SOLiD 5500×l next-generation sequencing platform. We sequenced the exomes of both unaffected parents and two affected siblings. Exome analysis resulted in the identification of 43,204 variants in the index patient. All variants passing filter criteria were validated with Sanger sequencing to confirm familial segregation and absence in the control population. In silico prediction tools were used to determine mutational impact on protein function and the structure of the identified variants. Results Novel Usher syndrome type 2A (USH2A) compound heterozygous mutations, c.4325T>C (p.F1442S) and c.15188T>G (p.L5063R), located in exons 20 and 70, respectively, were identified as probable causative mutations for RP in this family. Family segregation of the variants showed the presence of both mutations in all affected members and in two siblings who were apparently asymptomatic at the time of family ascertainment. Clinical reassessment confirmed the diagnosis of RP in these patients. Conclusions Using WES, we identified two heterozygous novel mutations in USH2A as the most likely disease-causing variants in a Spanish family diagnosed with arRP in which the cause of the disease had not yet been identified with

Identification of two poorly prognosed ovarian carcinoma subtypes associated with CHEK2 germ-line mutation and non-CHEK2 somatic mutation gene signatures.

PubMed

Ow, Ghim Siong; Ivshina, Anna V; Fuentes, Gloria; Kuznetsov, Vladimir A

2014-01-01

High-grade serous ovarian cancer (HG-SOC), a major histologic type of epithelial ovarian cancer (EOC), is a poorly-characterized, heterogeneous and lethal disease where somatic mutations of TP53 are common and inherited loss-of-function mutations in BRCA1/2 predispose to cancer in 9.5-13% of EOC patients. However, the overall burden of disease due to either inherited or sporadic mutations is not known. We performed bioinformatics analyses of mutational and clinical data of 334 HG-SOC tumor samples from The Cancer Genome Atlas to identify novel tumor-driving mutations, survival-significant patient subgroups and tumor subtypes potentially driven by either hereditary or sporadic factors. We identified a sub-cluster of high-frequency mutations in 22 patients and 58 genes associated with DNA damage repair, apoptosis and cell cycle. Mutations of CHEK2, observed with the highest intensity, were associated with poor therapy response and overall survival (OS) of these patients (P = 8.00e-05), possibly due to detrimental effect of mutations at the nuclear localization signal. A 21-gene mutational prognostic signature significantly stratifies patients into relatively low or high-risk subgroups with 5-y OS of 37% or 6%, respectively (P = 7.31e-08). Further analysis of these genes and high-risk subgroup revealed 2 distinct classes of tumors characterized by either germline mutations of genes such as CHEK2, RPS6KA2 and MLL4, or somatic mutations of other genes in the signature. Our results could provide improvement in prediction and clinical management of HG-SOC, facilitate our understanding of this complex disease, guide the design of targeted therapeutics and improve screening efforts to identify women at high-risk of hereditary ovarian cancers distinct from those associated with BRCA1/2 mutations.

Identification of two poorly prognosed ovarian carcinoma subtypes associated with CHEK2 germ-line mutation and non-CHEK2 somatic mutation gene signatures

PubMed Central

Ow, Ghim Siong; Ivshina, Anna V; Fuentes, Gloria; Kuznetsov, Vladimir A

2014-01-01

High-grade serous ovarian cancer (HG-SOC), a major histologic type of epithelial ovarian cancer (EOC), is a poorly-characterized, heterogeneous and lethal disease where somatic mutations of TP53 are common and inherited loss-of-function mutations in BRCA1/2 predispose to cancer in 9.5–13% of EOC patients. However, the overall burden of disease due to either inherited or sporadic mutations is not known.     We performed bioinformatics analyses of mutational and clinical data of 334 HG-SOC tumor samples from The Cancer Genome Atlas to identify novel tumor-driving mutations, survival-significant patient subgroups and tumor subtypes potentially driven by either hereditary or sporadic factors. We identified a sub-cluster of high-frequency mutations in 22 patients and 58 genes associated with DNA damage repair, apoptosis and cell cycle. Mutations of CHEK2, observed with the highest intensity, were associated with poor therapy response and overall survival (OS) of these patients (P = 8.00e-05), possibly due to detrimental effect of mutations at the nuclear localization signal. A 21-gene mutational prognostic signature significantly stratifies patients into relatively low or high-risk subgroups with 5-y OS of 37% or 6%, respectively (P = 7.31e-08). Further analysis of these genes and high-risk subgroup revealed 2 distinct classes of tumors characterized by either germline mutations of genes such as CHEK2, RPS6KA2 and MLL4, or somatic mutations of other genes in the signature. Our results could provide improvement in prediction and clinical management of HG-SOC, facilitate our understanding of this complex disease, guide the design of targeted therapeutics and improve screening efforts to identify women at high-risk of hereditary ovarian cancers distinct from those associated with BRCA1/2 mutations. PMID:24879340

F8 and F9 mutations in US haemophilia patients: correlation with history of inhibitor and race/ethnicity.

PubMed

Miller, C H; Benson, J; Ellingsen, D; Driggers, J; Payne, A; Kelly, F M; Soucie, J M; Craig Hooper, W

2012-05-01

Both genetic and treatment-related risk factors contribute to the development of inhibitors in haemophilia. An inhibitor surveillance system piloted at 12 US sites has the goal of assessing risk factors through prospective data collection. This report examines the relationship of genotype and race/ethnicity to history of inhibitor in a large cohort of US haemophilia patients. Mutation analysis was performed on 676 haemophilia A (HA) and 153 haemophilia B (HB) patients by sequencing, Multiplex Ligation-dependent Probe Amplification, and PCR for inversions in F8 introns 22 (inv22) and 1 (inv1). Two HB patients with deletions had history of inhibitor. In severe HA, frequency of history of inhibitor was: large deletion 57.1%, splice site 35.7%, inv22 26.8%, nonsense 24.5%, frameshift 12.9%, inv1 11.1% and missense 9.5%. In HA, 19.6% of 321 White non-Hispanics (Whites), 37.1% of 35 Black non-Hispanics (Blacks) and 46.9% of 32 Hispanics had history of inhibitor (P = 0.0003). Mutation types and novel mutation rates were similar across ethnicities. When F8 haplotypes were constructed, Whites and Hispanics showed only H1 and H2. Within H1, history of inhibitor was 12.4% in Whites, 40.0% in Blacks (P = 0.009) and 32.4% in Hispanics (P = 0.002). Inhibitor frequency is confirmed to vary by mutation type and race in a large US population. White patients with history of inhibitor did not exhibit rare F8 haplotypes. F8 gene analysis did not reveal a cause for the higher inhibitor frequencies in Black and Hispanic patients. © 2011 Blackwell Publishing Ltd.

Key Clinical Features to Identify Girls with "CDKL5" Mutations

ERIC Educational Resources Information Center

Bahi-Buisson, Nadia; Nectoux, Juliette; Rosas-Vargas, Haydee; Milh, Mathieu; Boddaert, Nathalie; Girard, Benoit; Cances, Claude; Ville, Dorothee; Afenjar, Alexandra; Rio, Marlene; Heron, Delphine; Morel, Marie Ange N'Guyen; Arzimanoglou, Alexis; Philippe, Christophe; Jonveaux, Philippe; Chelly, Jamel; Bienvenu, Thierry

2008-01-01

Mutations in the human X-linked cyclin-dependent kinase-like 5 ("CDKL5") gene have been shown to cause infantile spasms as well as Rett syndrome (RTT)-like phenotype. To date, less than 25 different mutations have been reported. So far, there are still little data on the key clinical diagnosis criteria and on the natural history of…

Whole Exome Sequencing Identifies RAI1 Mutation in a Morbidly Obese Child Diagnosed With ROHHAD Syndrome

PubMed Central

Esteves, Kristyn M.; Towne, Meghan C.; Brownstein, Catherine A.; James, Philip M.; Crowley, Laura; Hirschhorn, Joel N.; Elsea, Sarah H.; Beggs, Alan H.; Picker, Jonathan

2015-01-01

Context: The current obesity epidemic is attributed to complex interactions between genetic and environmental factors. However, a limited number of cases, especially those with early-onset severe obesity, are linked to single gene defects. Rapid-onset obesity with hypothalamic dysfunction, hypoventilation and autonomic dysregulation (ROHHAD) is one of the syndromes that presents with abrupt-onset extreme weight gain with an unknown genetic basis. Objective: To identify the underlying genetic etiology in a child with morbid early-onset obesity, hypoventilation, and autonomic and behavioral disturbances who was clinically diagnosed with ROHHAD syndrome. Design/Setting/Intervention: The index patient was evaluated at an academic medical center. Whole-exome sequencing was performed on the proband and his parents. Genetic variants were validated by Sanger sequencing. Results: We identified a novel de novo nonsense mutation, c.3265 C>T (p.R1089X), in the retinoic acid-induced 1 (RAI1) gene in the proband. Mutations in the RAI1 gene are known to cause Smith-Magenis syndrome (SMS). On further evaluation, his clinical features were not typical of either SMS or ROHHAD syndrome. Conclusions: This study identifies a de novo RAI1 mutation in a child with morbid obesity and a clinical diagnosis of ROHHAD syndrome. Although extreme early-onset obesity, autonomic disturbances, and hypoventilation are present in ROHHAD, several of the clinical findings are consistent with SMS. This case highlights the challenges in the diagnosis of ROHHAD syndrome and its potential overlap with SMS. We also propose RAI1 as a candidate gene for children with morbid obesity. PMID:25781356

Whole exome sequencing identifies RAI1 mutation in a morbidly obese child diagnosed with ROHHAD syndrome.

PubMed

Thaker, Vidhu V; Esteves, Kristyn M; Towne, Meghan C; Brownstein, Catherine A; James, Philip M; Crowley, Laura; Hirschhorn, Joel N; Elsea, Sarah H; Beggs, Alan H; Picker, Jonathan; Agrawal, Pankaj B

2015-05-01

The current obesity epidemic is attributed to complex interactions between genetic and environmental factors. However, a limited number of cases, especially those with early-onset severe obesity, are linked to single gene defects. Rapid-onset obesity with hypothalamic dysfunction, hypoventilation and autonomic dysregulation (ROHHAD) is one of the syndromes that presents with abrupt-onset extreme weight gain with an unknown genetic basis. To identify the underlying genetic etiology in a child with morbid early-onset obesity, hypoventilation, and autonomic and behavioral disturbances who was clinically diagnosed with ROHHAD syndrome. Design/Setting/Intervention: The index patient was evaluated at an academic medical center. Whole-exome sequencing was performed on the proband and his parents. Genetic variants were validated by Sanger sequencing. We identified a novel de novo nonsense mutation, c.3265 C>T (p.R1089X), in the retinoic acid-induced 1 (RAI1) gene in the proband. Mutations in the RAI1 gene are known to cause Smith-Magenis syndrome (SMS). On further evaluation, his clinical features were not typical of either SMS or ROHHAD syndrome. This study identifies a de novo RAI1 mutation in a child with morbid obesity and a clinical diagnosis of ROHHAD syndrome. Although extreme early-onset obesity, autonomic disturbances, and hypoventilation are present in ROHHAD, several of the clinical findings are consistent with SMS. This case highlights the challenges in the diagnosis of ROHHAD syndrome and its potential overlap with SMS. We also propose RAI1 as a candidate gene for children with morbid obesity.

Both TALENs and CRISPR/Cas9 directly target the HBB IVS2–654 (C > T) mutation in β-thalassemia-derived iPSCs

PubMed Central

Xu, Peng; Tong, Ying; Liu, Xiu-zhen; Wang, Ting-ting; Cheng, Li; Wang, Bo-yu; Lv, Xiang; Huang, Yue; Liu, De-pei

2015-01-01

β-Thalassemia is one of the most common genetic blood diseases and is caused by either point mutations or deletions in the β-globin (HBB) gene. The generation of patient-specific induced pluripotent stem cells (iPSCs) and subsequent correction of the disease-causing mutations may be a potential therapeutic strategy for this disease. Due to the low efficiency of typical homologous recombination, endonucleases, including TALENs and CRISPR/Cas9, have been widely used to enhance the gene correction efficiency in patient-derived iPSCs. Here, we designed TALENs and CRISPR/Cas9 to directly target the intron2 mutation site IVS2-654 in the globin gene. We observed different frequencies of double-strand breaks (DSBs) at IVS2-654 loci using TALENs and CRISPR/Cas9, and TALENs mediated a higher homologous gene targeting efficiency compared to CRISPR/Cas9 when combined with the piggyBac transposon donor. In addition, more obvious off-target events were observed for CRISPR/Cas9 compared to TALENs. Finally, TALENs-corrected iPSC clones were selected for erythroblast differentiation using the OP9 co-culture system and detected relatively higher transcription of HBB than the uncorrected cells. This comparison of using TALENs or CRISPR/Cas9 to correct specific HBB mutations in patient-derived iPSCs will guide future applications of TALENs- or CRISPR/Cas9-based gene therapies in monogenic diseases. PMID:26156589

A novel cell line generated using the CRISPR/Cas9 technology as universal quality control material for KRAS G12V mutation testing.

PubMed

Jia, Shiyu; Zhang, Rui; Lin, Guigao; Peng, Rongxue; Gao, Peng; Han, Yanxi; Fu, Yu; Ding, Jiansheng; Wu, Qisheng; Zhang, Kuo; Xie, Jiehong; Li, Jinming

2018-06-01

KRAS mutations are the key indicator for EGFR monoclonal antibody-targeted therapy and acquired drug resistance, and their accurate detection is critical to the clinical decision-making of colorectal cancer. However, no proper quality control material is available for the current detection methods, particularly next-generation sequencing (NGS). The ideal quality control material for NGS needs to provide both the tumor mutation gene and the matched background genomic DNA, which is uncataloged in public databases, to accurately distinguish germline polymorphisms and somatic mutations. We developed a novel KRAS G12V mutant cell line using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technique to make up for the deficiencies in existing quality control material and further validated the feasibility of the cell line as quality control material by amplification refractory mutation system (ARMS), Sanger sequencing, digital PCR (dPCR), and NGS. We verified that the edited cell line specifically had the G12V mutation, and the validation results presented a high consistency among the four methods of detection. The three cell lines screened contained the G12V mutation and the mutation allele fractions of G12V-1, G12V-2, and G12V-3 were 52.01%, 82.06%, and 17.29%, respectively. The novel KRAS G12V cell line generated using the CRISPR/Cas9 gene editing system is suitable as a quality control material for all current detection methods and provides a new direction in the development of quality control material. © 2018 Wiley Periodicals, Inc.

Mutations in Cas9 Enhance the Rate of Acquisition of Viral Spacer Sequences during the CRISPR-Cas Immune Response.

PubMed

Heler, Robert; Wright, Addison V; Vucelja, Marija; Bikard, David; Doudna, Jennifer A; Marraffini, Luciano A

2017-01-05

CRISPR loci and their associated (Cas) proteins encode a prokaryotic immune system that protects against viruses and plasmids. Upon infection, a low fraction of cells acquire short DNA sequences from the invader. These sequences (spacers) are integrated in between the repeats of the CRISPR locus and immunize the host against the matching invader. Spacers specify the targets of the CRISPR immune response through transcription into short RNA guides that direct Cas nucleases to the invading DNA molecules. Here we performed random mutagenesis of the RNA-guided Cas9 nuclease to look for variants that provide enhanced immunity against viral infection. We identified a mutation, I473F, that increases the rate of spacer acquisition by more than two orders of magnitude. Our results highlight the role of Cas9 during CRISPR immunization and provide a useful tool to study this rare process and develop it as a biotechnological application. Copyright © 2017 Elsevier Inc. All rights reserved.

Novel growth hormone receptor mutation in a Chinese patient with Laron syndrome.

PubMed

Hui, Hamilton N T; Metherell, Louise A; Ng, K L; Savage, Martin O; Camacho-Hübner, Cecilia; Clark, Adrian J L

2005-02-01

Laron syndrome, growth hormone (GH) insensitivity syndrome, caused by a mutation of the GH receptor (GHR) gene, is extremely rare in the Chinese population. We report a Chinese girl diagnosed with Laron syndrome at age 1.9 years with height -4.9 SDS, basal GH 344 mIU/ml, IGF-I <12 ng/ml, IGFBP-3 <0.2 mg/ml, and undetectable GHBP. A novel mutation of the GHR, not previously described, was identified at the donor splice site of intron 6.

Novel Mutations in the ZEB1 Gene Identified in Czech and British Patients With Posterior Polymorphous Corneal Dystrophy

PubMed Central

Liskova, Petra; Tuft, Stephen J.; Gwilliam, Rhian; Ebenezer, Neil D.; Jirsova, Katerina; Prescott, Quincy; Martincova, Radka; Pretorius, Marike; Sinclair, Neil; Boase, David L.; Jeffrey, Margaret J.; Deloukas, Panos; Hardcastle, Alison J.; Filipec, Martin; Bhattacharya, Shomi S.

2009-01-01

We describe the search for mutations in six unrelated Czech and four unrelated British families with posterior polymorphous corneal dystrophy (PPCD); a relatively rare eye disorder. Coding exons and intron/exon boundaries of all three genes (VSX1, COL8A2, and ZEB1/TCF8) previously reported to be implicated in the pathogenesis of this disorder were screened by DNA sequencing. Four novel pathogenic mutations were identified in four families; two deletions, one nonsense, and one duplication within exon 7 in the ZEB1 gene located at 10p11.2. We also genotyped the Czech patients to test for a founder haplotype and lack of disease segregation with the 20p11.2 locus we previously described. Although a systematic clinical examination was not performed, our investigation does not support an association between ZEB1 changes and self reported non-ocular anomalies. In the remaining six families no disease causing mutations were identified thereby indicating that as yet unidentified gene(s) are likely to be responsible for PPCD. PMID:17437275

Assessment of canine BEST1 variations identifies new mutations and establishes an independent bestrophinopathy model (cmr3)

PubMed Central

Wickström, Kaisa; Slavik, Julianna; Lindauer, Sarah J.; Ahonen, Saija; Schelling, Claude; Lohi, Hannes; Guziewicz, Karina E.; Aguirre, Gustavo D.

2010-01-01

Purpose Mutations in bestrophin 1 (BEST1) are associated with a group of retinal disorders known as bestrophinopathies in man and canine multifocal retinopathies (cmr) in the dog. To date, the dog is the only large animal model suitable for the complex characterization and in-depth studies of Best-related disorders. In the first report of cmr, the disease was described in a group of mastiff-related breeds (cmr1) and the Coton de Tulear (cmr2). Additional breeds, e.g., the Lapponian herder (LH) and others, subsequently were recognized with similar phenotypes, but linked loci are unknown. Analysis of the BEST1 gene aimed to identify mutations in these additional populations and extend our understanding of genotype–phenotype associations. Methods Animals were subjected to routine eye exams, phenotypically characterized, and samples were collected for molecular studies. Known BEST1 mutations were assessed, and the canine BEST1 coding exons were amplified and sequenced in selected individuals that exhibited a cmr compatible phenotype but that did not carry known mutations. Resulting sequence changes were genotyped in several different breeds and evaluated in the context of the phenotype. Results Seven novel coding variants were identified in exon 10 of cBEST1. Two linked mutations were associated with cmr exclusive to the LH breed (cmr3). Two individuals of Jämthund and Norfolk terrier breeds were heterozygous for two conservative changes, but these were unlikely to have disease-causing potential. Another three substitutions were found in the Bernese mountain dog that were predicted to have a deleterious effect on protein function. Previously reported mutations were excluded from segregation in these populations, but cmr1 was confirmed in another mastiff-related breed, the Italian cane corso. Conclusions A third independent canine model for human bestrophinopathies has been established in the LH breed. While exhibiting a phenotype comparable to cmr1 and cmr2, the

Novel compound heterozygous mutations in the GPR98 (USH2C) gene identified by whole exome sequencing in a Moroccan deaf family.

PubMed

Bousfiha, Amale; Bakhchane, Amina; Charoute, Hicham; Detsouli, Mustapha; Rouba, Hassan; Charif, Majida; Lenaers, Guy; Barakat, Abdelhamid

2017-10-01

In the present work, we identified two novel compound heterozygote mutations in the GPR98 (G protein-coupled receptor 98) gene causing Usher syndrome. Whole-exome sequencing was performed to study the genetic causes of Usher syndrome in a Moroccan family with three affected siblings. We identify two novel compound heterozygote mutations (c.1054C > A, c.16544delT) in the GPR98 gene in the three affected siblings carrying post-linguale bilateral moderate hearing loss with normal vestibular functions and before installing visual disturbances. This is the first time that mutations in the GPR98 gene are described in the Moroccan deaf patients.

The CDC Hemophilia A Mutation Project (CHAMP) Mutation List: a New Online Resource

PubMed Central

Payne, Amanda B.; Miller, Connie H.; Kelly, Fiona M.; Soucie, J. Michael; Hooper, W. Craig

2015-01-01

Genotyping efforts in hemophilia A (HA) populations in many countries have identified large numbers of unique mutations in the Factor VIII gene (F8). To assist HA researchers conducting genotyping analyses, we have developed a listing of F8 mutations including those listed in existing locus-specific databases as well as those identified in patient populations and reported in the literature. Each mutation was reviewed and uniquely identified using Human Genome Variation Society (HGVS) nomenclature standards for coding DNA and predicted protein changes as well as traditional nomenclature based on the mature, processed protein. Listings also include the associated hemophilia severity classified by International Society of Thrombosis and Haemostasis (ISTH) criteria, associations of the mutations with inhibitors, and reference information. The mutation list currently contains 2,537 unique mutations known to cause HA. HA severity caused by the mutation is available for 2,022 mutations (80%) and information on inhibitors is available for 1,816 mutations (72%). The CDC Hemophilia A Mutation Project (CHAMP) Mutation List is available at http://www.cdc.gov/hemophiliamutations for download and search and will be updated quarterly based on periodic literature reviews and submitted reports. PMID:23280990

Whole Genome Sequencing of High-Risk Families to Identify New Mutational Mechanisms of Breast Cancer Predisposition

DTIC Science & Technology

2014-10-01

INTRODUCTION: Despite tremendous advances in mutation detection with gene panels and exome sequencing the majority of high risk breast...2a. Align reads to the reference sequence (months 4-10) 2b. Identify SNPs, indels, CNVs and rearrangements by bioinformatic tools (months 4-10) 2c

Mitochondrial DNA mutations in diabetes mellitus patients in Chinese Han population.

PubMed

Wang, Suijun; Wu, Songhua; Zheng, Taishan; Yang, Zhen; Ma, Xiaojing; Jia, Weiping; Xiang, Kunsan

2013-12-01

Mutations of mitochondrial DNA are associated with diabetes mellitus (DM). The present case-control study aimed to investigate the mutations of mitochondrial DNA in DM patients of Chinese Han ethnicity. A total of 770 DM patients and 309 healthy control individuals were enrolled. The mitochondrial DNA was extracted from blood cells and analyzed by the polymerase chain reaction-restriction fragment length polymorphism assay. In the diabetes group, there were 13 (1.69%) individuals carrying the mt3243 A → G mutation while none of the healthy control had this mutation. Though the 14709, 3316, 3394, and 12026 mutation variants were identified in 9, 17, 18 and 28 in DM patients respectively, there were no significant differences compared with control group. And the 3256, 8296, 8344, 8363, 3426 and 12258 mutations were not detected in either group. In the diabetes group, two double mutations were identified: A3243G+T3394C and A3243G+A12026G. Our data suggested that mitochondrial gene tRNA(Leu(UUR)) 3243 A → G mutation may be one risk of prevalence of DM and associated with worse clinical status in Chinese Han population. © 2013 Elsevier B.V. All rights reserved.

Targeted High-Throughput Sequencing Identifies Mutations in atlastin-1 as a Cause of Hereditary Sensory Neuropathy Type I

PubMed Central

Guelly, Christian; Zhu, Peng-Peng; Leonardis, Lea; Papić, Lea; Zidar, Janez; Schabhüttl, Maria; Strohmaier, Heimo; Weis, Joachim; Strom, Tim M.; Baets, Jonathan; Willems, Jan; De Jonghe, Peter; Reilly, Mary M.; Fröhlich, Eleonore; Hatz, Martina; Trajanoski, Slave; Pieber, Thomas R.; Janecke, Andreas R.; Blackstone, Craig; Auer-Grumbach, Michaela

2011-01-01

Hereditary sensory neuropathy type I (HSN I) is an axonal form of autosomal-dominant hereditary motor and sensory neuropathy distinguished by prominent sensory loss that leads to painless injuries. Unrecognized, these can result in delayed wound healing and osteomyelitis, necessitating distal amputations. To elucidate the genetic basis of an HSN I subtype in a family in which mutations in the few known HSN I genes had been excluded, we employed massive parallel exon sequencing of the 14.3 Mb disease interval on chromosome 14q. We detected a missense mutation (c.1065C>A, p.Asn355Lys) in atlastin-1 (ATL1), a gene that is known to be mutated in early-onset hereditary spastic paraplegia SPG3A and that encodes the large dynamin-related GTPase atlastin-1. The mutant protein exhibited reduced GTPase activity and prominently disrupted ER network morphology when expressed in COS7 cells, strongly supporting pathogenicity. An expanded screen in 115 additional HSN I patients identified two further dominant ATL1 mutations (c.196G>C [p.Glu66Gln] and c.976 delG [p.Val326TrpfsX8]). This study highlights an unexpected major role for atlastin-1 in the function of sensory neurons and identifies HSN I and SPG3A as allelic disorders. PMID:21194679

Functional examination of MLH1, MSH2, and MSH6 intronic mutations identified in Danish colorectal cancer patients.

PubMed

Petersen, Sanne M; Dandanell, Mette; Rasmussen, Lene J; Gerdes, Anne-Marie; Krogh, Lotte N; Bernstein, Inge; Okkels, Henrik; Wikman, Friedrik; Nielsen, Finn C; Hansen, Thomas V O

2013-10-03

Germ-line mutations in the DNA mismatch repair genes MLH1, MSH2, and MSH6 predispose to the development of colorectal cancer (Lynch syndrome or hereditary nonpolyposis colorectal cancer). These mutations include disease-causing frame-shift, nonsense, and splicing mutations as well as large genomic rearrangements. However, a large number of mutations, including missense, silent, and intronic variants, are classified as variants of unknown clinical significance. Intronic MLH1, MSH2, or MSH6 variants were investigated using in silico prediction tools and mini-gene assay to asses the effect on splicing. We describe in silico and in vitro characterization of nine intronic MLH1, MSH2, or MSH6 mutations identified in Danish colorectal cancer patients, of which four mutations are novel. The analysis revealed aberrant splicing of five mutations (MLH1 c.588 + 5G > A, MLH1 c.677 + 3A > T, MLH1 c.1732-2A > T, MSH2 c.1276 + 1G > T, and MSH2 c.1662-2A > C), while four mutations had no effect on splicing compared to wild type (MLH1 c.117-34A > T, MLH1 c.1039-8 T > A, MSH2 c.2459-18delT, and MSH6 c.3439-16C > T). In conclusion, we classify five MLH1/MSH2 mutations as pathogenic, whereas four MLH1/MSH2/MSH6 mutations are classified as neutral. This study supports the notion that in silico prediction tools and mini-gene assays are important for the classification of intronic variants, and thereby crucial for the genetic counseling of patients and their family members.

AB071. Mutations of AR gene in Vietnamese patients: genotype and phenotype

PubMed Central

Dung, Vu Chi; Fukami, Maki; Ngoc, Can Thi Bich; Thao, Bui Phuong; Khanh, Nguyen Ngoc; Nga, Pham Thu; Dat, Nguyen Phu; Ogata, Tsutomu

2015-01-01

Androgen insensitivity syndrome (AIS) is the most common specific cause of 46,XY disorder in sex development. The androgen signaling pathway is complex but so far, the only gene linked with AIS is the androgen receptor (AR). Mutations in the AR are found in most subjects with complete AIS but in partial AIS, the rate has varied 28-73%, depending on the case selection. More than over 800 entries of mutations causing AIS, representing over 500 different AR mutations from more than 850 patients with AIS have been reported. We aim to describe clinical manifestations and to identify mutation of AR in Vietnamese patients with AIS. This case series study included 12 patients from 9 unrelated families with AIS. The gonadal position and external genitalia were evaluated clinically and using ultrasound. The mutation analysis of AR was performed using PCR and direct sequencing. The age of diagnosis was 1 to 83 years old. 8/12 cases were complete androgen insensitivity syndrome (CAIS) (female external genitalia) and 4 cases were predominantly female partial AIS phenotype. Four cases had two labial testes, six cases had inguinal testes and two cases had abdominal testes. Five different mutations of AR were identified from seven cases of three unrelated families including three novel ones. The novel missense mutation p.L701F (c.2103G > T) was identified in a patient of 83 years of age. The novel missense mutation p.L705F (c.2113C > T) was identified in two sibs. The novel mutation p. W752S (c.2256G > T) was identified in a child with CAIS phenotype and had family history. The reported missense mutation p.V747M was identified in two sibs. The reported mutation p.V867M (c.2599G > A) was identified in a child with female phenotype. Our study identified three novel and two reported mutation in the AR gene that may provide us new insights into the molecular mechanisms of AIS. The expanded database of these mutations should benefit patients in the diagnosis and treatment of this

Identifying RNA splicing factors using IFT genes in Chlamydomonas reinhardtii.

PubMed

Lin, Huawen; Zhang, Zhengyan; Iomini, Carlo; Dutcher, Susan K

2018-03-01

Intraflagellar transport moves proteins in and out of flagella/cilia and it is essential for the assembly of these organelles. Using whole-genome sequencing, we identified splice site mutations in two IFT genes, IFT81 ( fla9 ) and IFT121 ( ift121-2 ), which lead to flagellar assembly defects in the unicellular green alga Chlamydomonas reinhardtii The splicing defects in these ift mutants are partially corrected by mutations in two conserved spliceosome proteins, DGR14 and FRA10. We identified a dgr14 deletion mutant, which suppresses the 3' splice site mutation in IFT81 , and a frameshift mutant of FRA10 , which suppresses the 5' splice site mutation in IFT121 Surprisingly, we found dgr14-1 and fra10 mutations suppress both splice site mutations. We suggest these two proteins are involved in facilitating splice site recognition/interaction; in their absence some splice site mutations are tolerated. Nonsense mutations in SMG1 , which is involved in nonsense-mediated decay, lead to accumulation of aberrant transcripts and partial restoration of flagellar assembly in the ift mutants. The high density of introns and the conservation of noncore splicing factors, together with the ease of scoring the ift mutant phenotype, make Chlamydomonas an attractive organism to identify new proteins involved in splicing through suppressor screening. © 2018 The Authors.

Prevalence and Characterization of Somatic Mutations in Chinese Aldosterone-Producing Adenoma Patients

PubMed Central

Wang, Baojun; Li, Xintao; Zhang, Xu; Ma, Xin; Chen, Luyao; Zhang, Yu; Lyu, Xiangjun; Tang, Yuzhe; Huang, Qingbo; Gao, Yu; Fan, Yang; Ouyang, Jinzhi

2015-01-01

Abstract Recently somatic mutations of KCNJ5, ATP1A1, ATP2B3, and CACNA1D have been identified in patients with aldosterone-producing adenoma (APA). The present study sequenced the DNA in the tissues and blood samples from Chinese patients with APA for KCNJ5, ATP1A1, ATP2B3, and CACNA1D gene mutations. Among the 114 patients, 86 (75.4%) were identified with KCNJ5 somatic mutations, including 3 previously reported (G151R, L168R, T158A) and 2 other unreported mutations. One patient presented with both a point mutation (E147) and an insertion mutation, whereas another had a 36-base duplication, G153_G164dup. No mutation of ATP1A1 and ATP2B3 in the known hotspots was identified and only 1 male patient was detected with a novel CACNA1D mutation, V748I. Unlike other studies, male and female patients had similar KCNJ5 mutation rates (76.9% vs 74.2%). Mutation carriers were younger and had lower preoperative potassium level, whereas male (but not female) mutation carriers had higher preoperative plasma aldosterone concentration and preoperative blood pressures. Mutation carriers also had higher LV mass index (LVMI) than nonmutation carriers. After surgery, LVMI improved significantly in the KCNJ5 mutation group but not in the nonmutation group. The mRNA expression of KCNJ5, CYP11B2, and ATP2B3 was higher in the KCNJ5-mutated APA tissues. Functional characterization of the 2 novel KCNJ5 mutations showed that they were associated with decreased proliferation, membrane depolarization, elevated secretion of aldosterone, and increased expression of CYP11B1 and CYP11B2. In conclusion, Chinese APA patients appear to have a high frequency of somatic KCNJ5 mutation. Mutation prevalence rates are similar among men and women and 2 novel mutations are identified. KCNJ5-mutated patients benefit more from surgical resection of APA than nonmutated patients. PMID:25906099

Whole genome re-sequencing identifies a mutation in an ABC transporter (mdr2) in a Plasmodium chabaudi clone with altered susceptibility to antifolate drugs☆

PubMed Central

Martinelli, Axel; Henriques, Gisela; Cravo, Pedro; Hunt, Paul

2011-01-01

In malaria parasites, mutations in two genes of folate biosynthesis encoding dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) modify responses to antifolate therapies which target these enzymes. However, the involvement of other genes which modify the availability of exogenous folate, for example, has been proposed. Here, we used short-read whole-genome re-sequencing to determine the mutations in a clone of the rodent malaria parasite, Plasmodium chabaudi, which has altered susceptibility to both sulphadoxine and pyrimethamine. This clone bears a previously identified S106N mutation in dhfr and no mutation in dhps. Instead, three additional point mutations in genes on chromosomes 2, 13 and 14 were identified. The mutated gene on chromosome 13 (mdr2 K392Q) encodes an ABC transporter. Because Quantitative Trait Locus analysis previously indicated an association of genetic markers on chromosome 13 with responses to individual and combined antifolates, MDR2 is proposed to modulate antifolate responses, possibly mediated by the transport of folate intermediates. PMID:20858498

Mutation Spectrum and Phenotypic Features in Noonan Syndrome with PTPN11 Mutations: Definition of Two Novel Mutations.

PubMed

Atik, Tahir; Aykut, Ayca; Hazan, Filiz; Onay, Huseyin; Goksen, Damla; Darcan, Sukran; Tukun, Ajlan; Ozkinay, Ferda

2016-06-01

To evaluate the spectrum of PTPN11 gene mutations in Noonan syndrome patients and to study the genotype-phenotype associations. In this study, twenty Noonan syndrome patients with PTPN11 mutations were included. The patients underwent a detailed clinical and physical evaluation. To identify inherited cases, parents of all mutation positive patients were analyzed. Thirteen different PTPN11 mutations, two of them being novel, were detected in the study group. These mutations included eleven missense mutations: p.G60A, p.D61N, p.Y62D, p.Y63C, p.E69Q, p.Q79R, p.Y279C,p.N308D, p.N308S, p.M504V, p.Q510R and two novel missense mutations: p.I56V and p.I282M. The frequency of cardiac abnormalities and short stature were found to be 80 % and 80 %, respectively. Mental retardation was not observed in patients having exon 8 mutations. No significant correlations were detected between other phenotypic features and genotypes. By identifying genotype-phenotype correlations, this study provides information on phenotypes observed in NS patients with different PTPN11 mutations.

Functional analysis of a nonstop mutation in MITF gene identified in a patient with Waardenburg syndrome type 2

PubMed Central

Sun, Jie; Hao, Ziqi; Luo, Hunjin; He, Chufeng; Mei, Lingyun; Liu, Yalan; Wang, Xueping; Niu, Zhijie; Chen, Hongsheng; Li, Jia-Da; Feng, Yong

2017-01-01

Waardenburg syndrome (WS) is an autosomal dominant inherited neurogenic disorder with the combination of various degrees of sensorineural deafness and pigmentary abnormalities affecting the skin, hair and eye. The four subtypes of WS were defined on the basis of the presence or absence of additional symptoms. Mutation of human microphthalmia-associated transcription factor (MITF) gene gives rise to WS2. Here, we identified a novel WS-associated mutation at the stop codon of MITF (p.X420Y) in a Chinese WS2 patient. This mutation resulted in an extension of extra 33 amino-acid residues in MITF. The mutant MITF appeared in both the nucleus and the cytoplasm, whereas the wild-type MITF was localized in the nucleus exclusively. The mutation led to a reduction in the transcriptional activities, whereas the DNA-binding activity was not altered. We show that the foremost mechanism was haploinsufficiency for the mild phenotypes of WS2 induced in X420Y MITF. PMID:28356565

Functional analysis of a nonstop mutation in MITF gene identified in a patient with Waardenburg syndrome type 2.

PubMed

Sun, Jie; Hao, Ziqi; Luo, Hunjin; He, Chufeng; Mei, Lingyun; Liu, Yalan; Wang, Xueping; Niu, Zhijie; Chen, Hongsheng; Li, Jia-Da; Feng, Yong

2017-07-01

Waardenburg syndrome (WS) is an autosomal dominant inherited neurogenic disorder with the combination of various degrees of sensorineural deafness and pigmentary abnormalities affecting the skin, hair and eye. The four subtypes of WS were defined on the basis of the presence or absence of additional symptoms. Mutation of human microphthalmia-associated transcription factor (MITF) gene gives rise to WS2. Here, we identified a novel WS-associated mutation at the stop codon of MITF (p.X420Y) in a Chinese WS2 patient. This mutation resulted in an extension of extra 33 amino-acid residues in MITF. The mutant MITF appeared in both the nucleus and the cytoplasm, whereas the wild-type MITF was localized in the nucleus exclusively. The mutation led to a reduction in the transcriptional activities, whereas the DNA-binding activity was not altered. We show that the foremost mechanism was haploinsufficiency for the mild phenotypes of WS2 induced in X420Y MITF.

Germline loss-of-function mutations in LZTR1 predispose to an inherited disorder of multiple schwannomas

PubMed Central

Piotrowski, Arkadiusz; Xie, Jing; Liu, Ying F; Poplawski, Andrzej B; Gomes, Alicia R; Madanecki, Piotr; Fu, Chuanhua; Crowley, Michael R; Crossman, David K; Armstrong, Linlea; Babovic-Vuksanovic, Dusica; Bergner, Amanda; Blakeley, Jaishri O; Blumenthal, Andrea L; Daniels, Molly S; Feit, Howard; Gardner, Kathy; Hurst, Stephanie; Kobelka, Christine; Lee, Chung; Nagy, Rebecca; Rauen, Katherine A; Slopis, John M; Suwannarat, Pim; Westman, Judith A; Zanko, Andrea; Korf, Bruce R; Messiaen, Ludwine M

2015-01-01

Constitutional SMARCB1 mutations at 22q11.23 have been found in ~50% of familial and <10% of sporadic schwannomatosis cases1. We sequenced highly conserved regions along 22q from eight individuals with schwannomatosis whose schwannomas involved somatic loss of one copy of 22q, encompassing SMARCB1 and NF2, with a different somatic mutation of the other NF2 allele in every schwannoma but no mutation of the remaining SMARCB1 allele in blood and tumor samples. LZTR1 germline mutations were identified in seven of the eight cases. LZTR1 sequencing in 12 further cases with the same molecular signature identified 9 additional germline mutations. Loss of heterozygosity with retention of an LZTR1 mutation was present in all 25 schwannomas studied. Mutations segregated with disease in all available affected first-degree relatives, although four asymptomatic parents also carried an LZTR1 mutation. Our findings identify LZTR1 as a gene predisposing to an autosomal dominant inherited disorder of multiple schwannomas in ~80% of 22q-related schwannomatosis cases lacking mutation in SMARCB1. PMID:24362817

Identification of Germline Genetic Mutations in Pancreatic Cancer Patients

PubMed Central

Salo-Mullen, Erin E.; O’Reilly, Eileen; Kelsen, David; Ashraf, Asad M.; Lowery, Maeve; Yu, Kenneth; Reidy, Diane; Epstein, Andrew S.; Lincoln, Anne; Saldia, Amethyst; Jacobs, Lauren M.; Rau-Murthy, Rohini; Zhang, Liying; Kurtz, Robert; Saltz, Leonard; Offit, Kenneth; Robson, Mark; Stadler, Zsofia K.

2016-01-01

Background Pancreatic adenocarcinoma (PAC) is part of several cancer predisposition syndromes; however, indications for genetic counseling/testing are not well-defined. We sought to determine mutation prevalence and characteristics that predict for inherited predisposition to PAC. Methods We identified 175 consecutive PAC patients who underwent clinical genetics assessment at Memorial Sloan Kettering between 2011–2014. Clinical data, family history, and germline results were evaluated. Results Among 159 PAC patients who pursued genetic testing, 24 pathogenic mutations were identified (15.1%; 95%CI, 9.5%–20.7%), including BRCA2(n=13), BRCA1(n=4), p16(n=2), PALB2(n=1), and Lynch syndrome(n=4). BRCA1/BRCA2 prevalence was 13.7% in Ashkenazi Jewish(AJ) (n=95) and 7.1% in non-AJ(n=56) patients. In AJ patients with strong, weak, or absent family history of BRCA-associated cancers, mutation prevalence was 16.7%, 15.8%, and 7.4%, respectively. Mean age at diagnosis in all mutation carriers was 58.5y(range 45–75y) compared to 64y(range 27–87y) in non-mutation carriers(P=0.02). Although BRCA2 was the most common mutation identified, no patients with early-onset PAC(≤50y) harbored a BRCA2 mutation and the mean age at diagnosis in BRCA2 carriers was equivalent to non-mutation carriers(P=0.34). Mutation prevalence in early-onset patients(n=21) was 28.6%, including BRCA1(n=2), p16(n=2), MSH2(n=1) and MLH1(n=1). Conclusion Mutations in BRCA2 account for over 50% of PAC patients with an identified susceptibility syndrome. AJ patients had high BRCA1/BRCA2 prevalence regardless of personal/family history, suggesting that ancestry alone indicates a need for genetic evaluation. With the exception of BRCA2-associated PAC, inherited predisposition to PAC is associated with earlier age at PAC diagnosis suggesting that this subset of patients may also represent a population warranting further evaluation. PMID:26440929

Gene expression profiling and candidate gene resequencing identifies pathways and mutations important for malignant transformation caused by leukemogenic fusion genes.

PubMed

Novak, Rachel L; Harper, David P; Caudell, David; Slape, Christopher; Beachy, Sarah H; Aplan, Peter D

2012-12-01

NUP98-HOXD13 (NHD13) and CALM-AF10 (CA10) are oncogenic fusion proteins produced by recurrent chromosomal translocations in patients with acute myeloid leukemia (AML). Transgenic mice that express these fusions develop AML with a long latency and incomplete penetrance, suggesting that collaborating genetic events are required for leukemic transformation. We employed genetic techniques to identify both preleukemic abnormalities in healthy transgenic mice as well as collaborating events leading to leukemic transformation. Candidate gene resequencing revealed that 6 of 27 (22%) CA10 AMLs spontaneously acquired a Ras pathway mutation and 8 of 27 (30%) acquired an Flt3 mutation. Two CA10 AMLs acquired an Flt3 internal-tandem duplication, demonstrating that these mutations can be acquired in murine as well as human AML. Gene expression profiles revealed a marked upregulation of Hox genes, particularly Hoxa5, Hoxa9, and Hoxa10 in both NHD13 and CA10 mice. Furthermore, mir196b, which is embedded within the Hoxa locus, was overexpressed in both CA10 and NHD13 samples. In contrast, the Hox cofactors Meis1 and Pbx3 were differentially expressed; Meis1 was increased in CA10 AMLs but not NHD13 AMLs, whereas Pbx3 was consistently increased in NHD13 but not CA10 AMLs. Silencing of Pbx3 in NHD13 cells led to decreased proliferation, increased apoptosis, and decreased colony formation in vitro, suggesting a previously unexpected role for Pbx3 in leukemic transformation. Published by Elsevier Inc.

Whole-exome sequencing identifies recurrent SF3B1 R625 mutation and comutation of NF1 and KIT in mucosal melanoma.

PubMed

Hintzsche, Jennifer D; Gorden, Nicholas T; Amato, Carol M; Kim, Jihye; Wuensch, Kelsey E; Robinson, Steven E; Applegate, Allison J; Couts, Kasey L; Medina, Theresa M; Wells, Keith R; Wisell, Joshua A; McCarter, Martin D; Box, Neil F; Shellman, Yiqun G; Gonzalez, Rene C; Lewis, Karl D; Tentler, John J; Tan, Aik Choon; Robinson, William A

2017-06-01

Mucosal melanomas are a rare subtype of melanoma, arising in mucosal tissues, which have a very poor prognosis due to the lack of effective targeted therapies. This study aimed to better understand the molecular landscape of these cancers and find potential new therapeutic targets. Whole-exome sequencing was performed on mucosal melanomas from 19 patients and 135 sun-exposed cutaneous melanomas, with matched peripheral blood samples when available. Mutational profiles were compared between mucosal subgroups and sun-exposed cutaneous melanomas. Comparisons of molecular profiles identified 161 genes enriched in mucosal melanoma (P<0.05). KIT and NF1 were frequently comutated (32%) in the mucosal subgroup, with a significantly higher incidence than that in cutaneous melanoma (4%). Recurrent SF3B1 R625H/S/C mutations were identified and validated in 7 of 19 (37%) mucosal melanoma patients. Mutations in the spliceosome pathway were found to be enriched in mucosal melanomas when compared with cutaneous melanomas. Alternative splicing in four genes were observed in SF3B1-mutant samples compared with the wild-type samples. This study identified potential new therapeutic targets for mucosal melanoma, including comutation of NF1 and KIT, and recurrent R625 mutations in SF3B1. This is the first report of SF3B1 R625 mutations in vulvovaginal mucosal melanoma, with the largest whole-exome sequencing project of mucosal melanomas to date. The results here also indicated that the mutations in SF3B1 lead to alternative splicing in multiple genes. These findings expand our knowledge of this rare disease.

Investigation of Prolific Sheep from UK and Ireland for Evidence on Origin of the Mutations in BMP15 (FecXG, FecXB) and GDF9 (FecGH) in Belclare and Cambridge Sheep

PubMed Central

Mullen, Michael P.; Hanrahan, James P.; Howard, Dawn J.

2013-01-01

This paper concerns the likely origin of three mutations with large effects on ovulation rate identified in the Belclare and Cambridge sheep breeds; two in the BMP15 gene (FecXG and FecXB) and the third (FecGH) in GDF9. All three mutations segregate in Belclare sheep while one, FecXB, has not been found in the Cambridge. Both Belclare and Cambridge breeds are relatively recently developed composites that have common ancestry through the use of genetic material from the Finnish Landrace and Lleyn breeds. The development of both composites also involved major contributions from exceptionally prolific ewes screened from flocks in Ireland (Belclare) and Britain (Cambridge) during the 1960s. The objective of the current study was to establish the likely origin of the mutations (FecXG, FecXB and FecGH) through analysis of DNA from Finnish Landrace and Lleyn sheep, and Galway and Texel breeds which contributed to the development of the Belclare breed. Ewes with exceptionally high prolificacy (hyper-prolific ewes) in current flocks on Irish farms were identified to simulate the screening of ewes from Irish flocks in the 1960s. DNA was obtained from: prolific ewes in extant flocks of Lleyn sheep (n = 44) on the Lleyn peninsula in Wales; hyper-prolific ewes (n = 41); prolific Galway (n = 41) ewes; Finnish Landrace (n = 124) and Texel (n = 19) ewes. The FecXG mutation was identified in Lleyn but not in Finnish Landrace, Galway or Texel sheep; FecXB was only found among the hyper-prolific ewes. The FecGH mutation was identified in the sample of Lleyn sheep. It was concluded from these findings that the Lleyn breed was the most likely source of the FecXG and FecGH mutations in Belclare and Cambridge sheep and that the FecXB mutation came from the High Fertility line that was developed using prolific ewes selected from commercial flocks in Ireland in the 1960′s and subsequently used in the genesis of the Belclare. PMID:23301039

Spectrum of benzo[a]pyrene-induced mutations in the Pig-a gene of L5178YTk+/- cells identified with next generation sequencing.

PubMed

Revollo, Javier; Wang, Yiying; McKinzie, Page; Dad, Azra; Pearce, Mason; Heflich, Robert H; Dobrovolsky, Vasily N

2017-12-01

We used Sanger sequencing and next generation sequencing (NGS) for analysis of mutations in the endogenous X-linked Pig-a gene of clonally expanded L5178YTk +/- cells. The clones developed from single cells that were sorted on a flow cytometer based upon the expression pattern of the GPI-anchored marker, CD90, on their surface. CD90-deficient and CD90-proficient cells were sorted from untreated cultures and CD90-deficient cells were sorted from cultures treated with benzo[a]pyrene (B[a]P). Pig-a mutations were identified in all clones developed from CD90-deficient cells; no Pig-a mutations were found in clones of CD90-proficient cells. The spectrum of B[a]P-induced Pig-a mutations was dominated by basepair substitutions, small insertions and deletions at G:C, or at sequences rich in G:C content. We observed high concordance between Pig-a mutations determined by Sanger sequencing and by NGS, but NGS was able to identify mutations in samples that were difficult to analyze by Sanger sequencing (e.g., mixtures of two mutant clones). Overall, the NGS method is a cost and labor efficient high throughput approach for analysis of a large number of mutant clones. Published by Elsevier B.V.

Confirmation of Pig-a mutation in flow cytometry-identified CD48-deficient T-lymphocytes from F344 rats.

PubMed

Revollo, Javier; Pearce, Mason G; Petibone, Dayton M; Mittelstaedt, Roberta A; Dobrovolsky, Vasily N

2015-05-01

The Pig-a assay is used for monitoring somatic cell mutation in laboratory animals and humans. The assay detects haematopoietic cells deficient in glycosylphosphatidylinositol (GPI)-anchored protein surface markers using flow cytometry. However, given that synthesis of the protein markers (and the expression of their genes) is independent of the expression of the X-linked Pig-a gene and the function of its enzyme product, the deficiency of markers at the surface of the cells may be caused by a number of events (e.g. by mutation or epigenetic silencing in the marker gene itself or in any of about two dozen autosomal genes involved in the synthesis of GPI). Here we provide direct evidence that the deficiency of the GPI-anchored surface marker CD48 in rat T-cells is accompanied by mutation in the endogenous X-linked Pig-a gene. We treated male F344 rats with N-ethyl-N-nitrosourea (ENU), and established colonies from flow cytometry-identified and sorted CD48-deficient spleen T-lymphocytes. Molecular analysis confirmed that the expanded sorted cells have mutations in the Pig-a gene. The spectrum of Pig-a mutation in our model was consistent with the spectrum of ENU-induced mutation determined in other in vivo models, mostly base-pair substitutions at A:T with the mutated T on the non-transcribed strand of Pig-a genomic DNA. We also used next generation sequencing to derive a similar mutational spectrum from a pool of 64 clones developed from flow-sorted CD48-deficient lymphocytes. Our findings confirm that Pig-a assays detect what they are designed to detect-gene mutation in the Pig-a gene. © The Author 2015. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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.

An inter-residue network model to identify mutational-constrained regions on the Ebola coat glycoprotein

PubMed Central

Quinlan, Devin S.; Raman, Rahul; Tharakaraman, Kannan; Subramanian, Vidya; del Hierro, Gabriella; Sasisekharan, Ram

2017-01-01

Recently, progress has been made in the development of vaccines and monoclonal antibody cocktails that target the Ebola coat glycoprotein (GP). Based on the mutation rates for Ebola virus given its natural sequence evolution, these treatment strategies are likely to impose additional selection pressure to drive acquisition of mutations in GP that escape neutralization. Given the high degree of sequence conservation among GP of Ebola viruses, it would be challenging to determine the propensity of acquiring mutations in response to vaccine or treatment with one or a cocktail of monoclonal antibodies. In this study, we analyzed the mutability of each residue using an approach that captures the structural constraints on mutability based on the extent of its inter-residue interaction network within the three-dimensional structure of the trimeric GP. This analysis showed two distinct clusters of highly networked residues along the GP1-GP2 interface, part of which overlapped with epitope surfaces of known neutralizing antibodies. This network approach also permitted us to identify additional residues in the network of the known hotspot residues of different anti-Ebola antibodies that would impact antibody-epitope interactions. PMID:28397835

KRAS Mutation as a Potential Prognostic Biomarker of Biliary Tract Cancers

PubMed Central

Yokoyama, Masaaki; Ohnishi, Hiroaki; Ohtsuka, Kouki; Matsushima, Satsuki; Ohkura, Yasuo; Furuse, Junji; Watanabe, Takashi; Mori, Toshiyuki; Sugiyama, Masanori

2016-01-01

BACKGROUND The aim of this study was to identify the unique molecular characteristics of biliary tract cancer (BTC) for the development of novel molecular-targeted therapies. MATERIALS AND METHODS We performed mutational analysis of KRAS, BRAF, PIK3CA, and FBXW7 and immunohistochemical analysis of EGFR and TP53 in 63 Japanese patients with BTC and retrospectively evaluated the association between the molecular characteristics and clinicopathological features of BTC. RESULTS KRAS mutations were identified in 9 (14%) of the 63 BTC patients; no mutations were detected within the analyzed regions of BRAF, PIK3CA, and FBXW7. EGFR overexpression was observed in 5 (8%) of the 63 tumors, while TP53 overexpression was observed in 48% (30/63) of the patients. Overall survival of patients with KRAS mutation was significantly shorter than that of patients with the wild-type KRAS gene (P = 0.005). By multivariate analysis incorporating molecular and clinicopathological features, KRAS mutations and lymph node metastasis were identified to be independently associated with shorter overall survival (KRAS, P = 0.004; lymph node metastasis, P = 0.015). CONCLUSIONS Our data suggest that KRAS mutation is a poor prognosis predictive biomarker for the survival in BTC patients. PMID:28008299

Novel mutations in the GPIHBP1 gene identified in 2 patients with recurrent acute pancreatitis.

PubMed

Ariza, María José; Martínez-Hernández, Pedro Luis; Ibarretxe, Daiana; Rabacchi, Claudio; Rioja, José; Grande-Aragón, Cristina; Plana, Nuria; Tarugi, Patrizia; Olivecrona, Gunilla; Calandra, Sebastiano; Valdivielso, Pedro

2016-01-01

Glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) has been demonstrated to be essential for the in vivo function of lipoprotein lipase (LPL), the major triglyceride (TG)-hydrolyzing enzyme involved in the intravascular lipolysis of TG-rich lipoproteins. Recently, loss-of-function mutations of GPIHBP1 have been reported as the cause of type I hyperlipoproteinemia in several patients. Two unrelated patients were referred to our Lipid Units because of a severe hypertriglyceridemia and recurrent pancreatitis. We measured LPL activity in postheparin plasma and serum ApoCII and sequenced LPL, APOC2, and GPIHBP1. The 2 patients exhibited very low LPL activity not associated with mutations in LPL gene or with ApoCII deficiency. The sequence of GPIHBP1 revealed 2 novel point mutations. One patient (proband 1) was found to be homozygous for a C>A transversion in exon 3 resulting in the conversion of threonine to lysine at position 80 (p.Thr80Lys). The other patient (proband 2) was found to be homozygous for a G>T transversion in the third base of the ATG translation initiation codon in exon 1, resulting in the conversion of methionine to isoleucine (p.Met1Ile). In conclusion, we have identified 2 novel GPIHBP1 missense mutations in 2 unrelated patients as the cause of their severe hypertriglyceridemia. Copyright © 2016 National Lipid Association. Published by Elsevier Inc. All rights reserved.

Evaluation of current prediction models for Lynch syndrome: updating the PREMM5 model to identify PMS2 mutation carriers.

PubMed

Goverde, A; Spaander, M C W; Nieboer, D; van den Ouweland, A M W; Dinjens, W N M; Dubbink, H J; Tops, C J; Ten Broeke, S W; Bruno, M J; Hofstra, R M W; Steyerberg, E W; Wagner, A

2018-07-01

Until recently, no prediction models for Lynch syndrome (LS) had been validated for PMS2 mutation carriers. We aimed to evaluate MMRpredict and PREMM5 in a clinical cohort and for PMS2 mutation carriers specifically. In a retrospective, clinic-based cohort we calculated predictions for LS according to MMRpredict and PREMM5. The area under the operator receiving characteristic curve (AUC) was compared between MMRpredict and PREMM5 for LS patients in general and for different LS genes specifically. Of 734 index patients, 83 (11%) were diagnosed with LS; 23 MLH1, 17 MSH2, 31 MSH6 and 12 PMS2 mutation carriers. Both prediction models performed well for MLH1 and MSH2 (AUC 0.80 and 0.83 for PREMM5 and 0.79 for MMRpredict) and fair for MSH6 mutation carriers (0.69 for PREMM5 and 0.66 for MMRpredict). MMRpredict performed fair for PMS2 mutation carriers (AUC 0.72), while PREMM5 failed to discriminate PMS2 mutation carriers from non-mutation carriers (AUC 0.51). The only statistically significant difference between PMS2 mutation carriers and non-mutation carriers was proximal location of colorectal cancer (77 vs. 28%, p < 0.001). Adding location of colorectal cancer to PREMM5 considerably improved the models performance for PMS2 mutation carriers (AUC 0.77) and overall (AUC 0.81 vs. 0.72). We validated these results in an external cohort of 376 colorectal cancer patients, including 158 LS patients. MMRpredict and PREMM5 cannot adequately identify PMS2 mutation carriers. Adding location of colorectal cancer to PREMM5 may improve the performance of this model, which should be validated in larger cohorts.

Association of the widespread A149P hereditary fructose intolerance mutation with newly identified sequence polymorphisms in the aldolase B gene

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

Brooks, C.C.; Tolan, D.R.

1993-04-01

Hereditary fructose intolerance (HFI) is a potentially fatal autosomal recessive disease resulting from the catalytic deficiency of fructose 1-phosphate aldolase (aldolase B) in fructose-metabolizing tissues. The A149P mutation in exon 5 of the aldolase B gene, located on chromosome 9q2l.3-q22.2, is widespread and the most common HFI mutation, accounting for 57% of HFI chromosomes. The possible origin of this mutation was studied by linkage to polymorphisms within the aldolase B gene. DNA fragments of the aldolase B gene containing the polymorphic marker loci from HFI patients homozygous for the A149P allele were amplified by PCR. Absolute linkage to a commonmore » Pvull RFLP allele was observed in 10 A149P homozygotes. In a more informative study, highly heterozygous polymorphisms were detected by direct sequence determination of a PCR-amplified aldolase B gene fragment. Two two-allele, single-base-pair polymorphisms, themselves in absolute linkage disequilibrium, in intron 8 (C at nucleotide 84 and A at nucleotide 105, or T at 84 and G at 105) of the aldolase B gene were identified. Mendelian segregation of these polymorphisms was confirmed in three families. Allele-specific oligonucleotide (ASO) hybridizations with probes for both sequence polymorphisms showed that 47% of 32 unrelated individuals were heterozygous at these loci; the calculated PIC value was .37. Finally, ASO hybridizations of PCR-amplified DNA from 15 HFI patients homozygous for the A149P allele with probes for these sequence polymorphisms revealed absolute linkage disequilibrium between the A149P mutation and the 84T/105G allele. These results are consistent with a single origin of the A149P allele and subsequent spread by genetic drift. 32 refs., 4 figs., 3 tabs.« less

Exome Sequencing Identifies a Novel Nonsense Mutation of MYO6 as the Cause of Deafness in a Brazilian Family.

PubMed

Sampaio-Silva, Juliana; Batissoco, Ana Carla; Jesus-Santos, Rafaela; Abath-Neto, Osório; Scarpelli, Luciano Cesar; Nishimura, Patricia Yoshie; Galindo, Layla Testa; Bento, Ricardo Ferreira; Oiticica, Jeanne; Lezirovitz, Karina

2018-01-01

We investigated 313 unrelated subjects who presented with hearing loss to identify the novel genetic causes of this condition in Brazil. Causative GJB2/GJB6 mutations were found in 12.7% of the patients. Among the familial cases (100/313), four were selected for exome sequencing. In one case, two novel heterozygous variants were found and were predicted to be pathogenic based on bioinformatics tools, that is, p.Ser906* (MYO6) and p.Arg42Cys (GJB3). We confirmed that this nonsense MYO6 mutation segregated with deafness in this family. Only the proband and her unaffected mother exhibited the GJB3 mutation, which is in the same amino acid of a known Erythrokeratodermia variabilis mutation. None of the patients exhibited this skin disease, but the proband exhibited a more severe hearing loss. Hence, the GJB3 mutation was considered to be a variant of uncertain significance. In conclusion, we described a novel nonsense MYO6 mutation that was responsible for the hearing loss in a Brazilian family. This mutation resides in the neck domain of myosin-VI after the motor domain. Thus, our data give further support for genotype-phenotype correlations, which state that when the motor domain of the protein is functioning, the hearing loss is milder and has a later onset. The three remaining families without mutations in the known genes suggest that there are still deafness genes to be revealed. © 2017 John Wiley & Sons Ltd/University College London.

Novel mutations in CRB1 gene identified in a chinese pedigree with retinitis pigmentosa by targeted capture and next generation sequencing

PubMed Central

Lo, David; Weng, Jingning; Liu, xiaohong; Yang, Juhua; He, Fen; Wang, Yun; Liu, Xuyang

2016-01-01

PURPOSE To detect the disease-causing gene in a Chinese pedigree with autosomal-recessive retinitis pigmentosa (ARRP). METHODS All subjects in this family underwent a complete ophthalmic examination. Targeted-capture next generation sequencing (NGS) was performed on the proband to detect variants. All variants were verified in the remaining family members by PCR amplification and Sanger sequencing. RESULTS All the affected subjects in this pedigree were diagnosed with retinitis pigmentosa (RP). The compound heterozygous c.138delA (p.Asp47IlefsX24) and c.1841G>T (p.Gly614Val) mutations in the Crumbs homolog 1 (CRB1) gene were identified in all the affected patients but not in the unaffected individuals in this family. These mutations were inherited from their parents, respectively. CONCLUSION The novel compound heterozygous mutations in CRB1 were identified in a Chinese pedigree with ARRP using targeted-capture next generation sequencing. After evaluating the significant heredity and impaired protein function, the compound heterozygous c.138delA (p.Asp47IlefsX24) and c.1841G>T (p.Gly614Val) mutations are the causal genes of early onset ARRP in this pedigree. To the best of our knowledge, there is no previous report regarding the compound mutations. PMID:27806333

The Spectrum of Mutations in Progranulin

PubMed Central

Yu, Chang-En; Bird, Thomas D.; Bekris, Lynn M.; Montine, Thomas J.; Leverenz, James B.; Steinbart, Ellen; Galloway, Nichole M.; Feldman, Howard; Woltjer, Randall; Miller, Carol A.; Wood, Elisabeth McCarty; Grossman, Murray; McCluskey, Leo; Clark, Christopher M.; Neumann, Manuela; Danek, Adrian; Galasko, Douglas R.; Arnold, Steven E.; Chen-Plotkin, Alice; Karydas, Anna; Miller, Bruce L.; Trojanowski, John Q.; Lee, Virginia M.-Y.; Schellenberg, Gerard D.; Van Deerlin, Vivianna M.

2010-01-01

Background Mutation in the progranulin gene (GRN) can cause frontotemporal dementia (FTD). However, it is unclear whether some rare FTD-related GRN variants are pathogenic and whether neurodegenerative disorders other than FTD can also be caused by GRN mutations. Objectives To delineate the range of clinical presentations associated with GRN mutations and to define pathogenic candidacy of rare GRN variants. Design Case-control study. Setting Clinical and neuropathology dementia research studies at 8 academic centers. Participants Four hundred thirty-four patients with FTD, including primary progressive aphasia, semantic dementia, FTD/amyotrophic lateral sclerosis (ALS), FTD/motor neuron disease, corticobasal syndrome/corticobasal degeneration, progressive supranuclear palsy, Pick disease, dementia lacking distinctive histopathology, and pathologically confirmed cases of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U); and 111 non-FTD cases (controls) in which TDP-43 deposits were a prominent neuropathological feature, including subjects with ALS, Guam ALS and/or parkinsonism dementia complex, Guam dementia, Alzheimer disease, multiple system atrophy, and argyrophilic grain disease. Main Outcome Measures Variants detected on sequencing of all 13 GRN exons and at least 80 base pairs of flanking introns, and their pathogenic candidacy determined by in silico and ex vivo splicing assays. Results We identified 58 genetic variants that included 26 previously unknown changes. Twenty-four variants appeared to be pathogenic, including 8 novel mutations. The frequency of GRN mutations was 6.9% (30 of 434) of all FTD-spectrum cases, 21.4% (9 of 42) of cases with a pathological diagnosis of FTLD-U, 16.0% (28 of 175) of FTD-spectrum cases with a family history of a similar neurodegenerative disease, and 56.2% (9 of 16) of cases of FTLD-U with a family history. Conclusions Pathogenic mutations were found only in FTD-spectrum cases and not in other

TP53 mutation-correlated genes predict the risk of tumor relapse and identify MPS1 as a potential therapeutic kinase in TP53-mutated breast cancers.

PubMed

Győrffy, Balázs; Bottai, Giulia; Lehmann-Che, Jacqueline; Kéri, György; Orfi, László; Iwamoto, Takayuki; Desmedt, Christine; Bianchini, Giampaolo; Turner, Nicholas C; de Thè, Hugues; André, Fabrice; Sotiriou, Christos; Hortobagyi, Gabriel N; Di Leo, Angelo; Pusztai, Lajos; Santarpia, Libero

2014-05-01

Breast cancers (BC) carry a complex set of gene mutations that can influence their gene expression and clinical behavior. We aimed to identify genes driven by the TP53 mutation status and assess their clinical relevance in estrogen receptor (ER)-positive and ER-negative BC, and their potential as targets for patients with TP53 mutated tumors. Separate ROC analyses of each gene expression according to TP53 mutation status were performed. The prognostic value of genes with the highest AUC were assessed in a large dataset of untreated, and neoadjuvant chemotherapy treated patients. The mitotic checkpoint gene MPS1 was the most significant gene correlated with TP53 status, and the most significant prognostic marker in all ER-positive BC datasets. MPS1 retained its prognostic value independently from the type of treatment administered. The biological functions of MPS1 were investigated in different BC cell lines. We also assessed the effects of a potent small molecule inhibitor of MPS1, SP600125, alone and in combination with chemotherapy. Consistent with the gene expression profiling and siRNA assays, the inhibition of MPS1 by SP600125 led to a reduction in cell viability and a significant increase in cell death, selectively in TP53-mutated BC cells. Furthermore, the chemical inhibition of MPS1 sensitized BC cells to conventional chemotherapy, particularly taxanes. Our results collectively demonstrate that TP53-correlated kinase MPS1, is a potential therapeutic target in BC patients with TP53 mutated tumors, and that SP600125 warrant further development in future clinical trials. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Analysis of Hungarian patients with Rett syndrome phenotype for MECP2, CDKL5 and FOXG1 gene mutations.

PubMed

Hadzsiev, Kinga; Polgar, Noemi; Bene, Judit; Komlosi, Katalin; Karteszi, Judit; Hollody, Katalin; Kosztolanyi, Gyorgy; Renieri, Alessandra; Melegh, Bela

2011-03-01

Rett syndrome (RTT) is characterized by a relatively specific clinical phenotype. We screened 152 individuals with RTT phenotype. A total of 22 different known MECP2 mutations were identified in 42 subjects (27.6%). Of the 22 mutations, we identified 7 (31.8%) frameshift-causing deletions, 4 (18.2%) nonsense, 10 (45.5%) missense mutations and one insertion (4.5%). The most frequent pathologic changes were: p.Thr158Met (14.2%) and p.Arg133Cys (11.9%) missense, and p.Arg255Stop (9.5%) and p.Arg294Stop (9.5%) nonsense mutations. We also detected the c.925C >T (p.Arg309Trp) mutation in an affected patient, whose role in RTT pathogenesis is still unknown. Patients without detectable MECP2 defects were screened for mutations of cyclin-dependent kinase-like 5 (CDKL5) gene, responsible for the early-onset variant of RTT. We discovered two novel mutations: c.607G >T resulting in a termination codon at aa203, disrupting the catalytic domain, and c.1708G >T leading to a stop at aa570 of the C terminus. Both patients with CDKL5 mutation presented therapy-resistant epilepsy and a phenotype fitting with the diagnosis of early-onset variant of RTT. No FOXG1 mutation was detected in any of the remaining patients. A total of 110 (72.5%) patients remained without molecular genetic diagnosis that necessitates further search for novel gene mutations in this phenotype. Our results also suggest the need of screening for CDKL5 mutations in patients with Rett phenotype tested negative for MECP2 mutations.

Cantú Syndrome Resulting from Activating Mutation in the KCNJ8 Gene

PubMed Central

Cooper, Paige E.; Reutter, Heiko; Woelfle, Joachim; Engels, Hartmut; Grange, Dorothy K.; van Haaften, Gijs; van Bon, Bregje W.; Hoischen, Alexander; Nichols, Colin G.

2014-01-01

ATP-sensitive potassium (KATP) channels, composed of inward-rectifying potassium channel subunits (Kir6.1 and Kir6.2, encoded by KCNJ8 and KCNJ11, respectively) and regulatory sulfonylurea receptor (SUR1 and SUR2, encoded by ABCC8 and ABCC9, respectively), couple metabolism to excitability in multiple tissues. Mutations in ABCC9 cause Cantú syndrome, a distinct multi-organ disease, potentially via enhanced KATP channel activity. We screened KCNJ8 in an ABCC9 mutation-negative patient who also exhibited clinical hallmarks of Cantú syndrome (hypertrichosis, macrosomia, macrocephaly, coarse facial appearance, cardiomegaly, and skeletal abnormalities). We identified a de novo missense mutation encoding Kir6.1[p.Cys176Ser] in the patient. Kir6.1[p.Cys176Ser] channels exhibited markedly higher activity than wild-type channels, as a result of reduced ATP sensitivity, whether co-expressed with SUR1 or SUR2A subunits. Our results identify a novel causal gene in Cantú syndrome, but also demonstrate that the cardinal features of the disease result from gain of KATP channel function, not from Kir6-independent SUR2 function. PMID:24700710

Cantú syndrome resulting from activating mutation in the KCNJ8 gene.

PubMed

Cooper, Paige E; Reutter, Heiko; Woelfle, Joachim; Engels, Hartmut; Grange, Dorothy K; van Haaften, Gijs; van Bon, Bregje W; Hoischen, Alexander; Nichols, Colin G

2014-07-01

ATP-sensitive potassium (KATP ) channels, composed of inward-rectifying potassium channel subunits (Kir6.1 and Kir6.2, encoded by KCNJ8 and KCNJ11, respectively) and regulatory sulfonylurea receptor (SUR1 and SUR2, encoded by ABCC8 and ABCC9, respectively), couple metabolism to excitability in multiple tissues. Mutations in ABCC9 cause Cantú syndrome (CS), a distinct multiorgan disease, potentially via enhanced KATP channel activity. We screened KCNJ8 in an ABCC9 mutation-negative patient who also exhibited clinical hallmarks of CS (hypertrichosis, macrosomia, macrocephaly, coarse facial appearance, cardiomegaly, and skeletal abnormalities). We identified a de novo missense mutation encoding Kir6.1[p.Cys176Ser] in the patient. Kir6.1[p.Cys176Ser] channels exhibited markedly higher activity than wild-type channels, as a result of reduced ATP sensitivity, whether coexpressed with SUR1 or SUR2A subunits. Our results identify a novel causal gene in CS, but also demonstrate that the cardinal features of the disease result from gain of KATP channel function, not from a Kir6-independent SUR2 function. © 2014 WILEY PERIODICALS, INC.

Whole-exome sequencing identifies recurrent SF3B1 R625 mutation and comutation of NF1 and KIT in mucosal melanoma

PubMed Central

Hintzsche, Jennifer D.; Gorden, Nicholas T.; Amato, Carol M.; Kim, Jihye; Wuensch, Kelsey E.; Robinson, Steven E.; Applegate, Allison J.; Couts, Kasey L.; Medina, Theresa M.; Wells, Keith R.; Wisell, Joshua A.; McCarter, Martin D.; Box, Neil F.; Shellman, Yiqun G.; Gonzalez, Rene C.; Lewis, Karl D.; Tentler, John J.

2017-01-01

Mucosal melanomas are a rare subtype of melanoma, arising in mucosal tissues, which have a very poor prognosis due to the lack of effective targeted therapies. This study aimed to better understand the molecular landscape of these cancers and find potential new therapeutic targets. Whole-exome sequencing was performed on mucosal melanomas from 19 patients and 135 sun-exposed cutaneous melanomas, with matched peripheral blood samples when available. Mutational profiles were compared between mucosal subgroups and sun-exposed cutaneous melanomas. Comparisons of molecular profiles identified 161 genes enriched in mucosal melanoma (P<0.05). KIT and NF1 were frequently comutated (32%) in the mucosal subgroup, with a significantly higher incidence than that in cutaneous melanoma (4%). Recurrent SF3B1 R625H/S/C mutations were identified and validated in 7 of 19 (37%) mucosal melanoma patients. Mutations in the spliceosome pathway were found to be enriched in mucosal melanomas when compared with cutaneous melanomas. Alternative splicing in four genes were observed in SF3B1-mutant samples compared with the wild-type samples. This study identified potential new therapeutic targets for mucosal melanoma, including comutation of NF1 and KIT, and recurrent R625 mutations in SF3B1. This is the first report of SF3B1 R625 mutations in vulvovaginal mucosal melanoma, with the largest whole-exome sequencing project of mucosal melanomas to date. The results here also indicated that the mutations in SF3B1 lead to alternative splicing in multiple genes. These findings expand our knowledge of this rare disease. PMID:28296713

Comparison of Thoracic Radiotherapy Efficacy Between Patients With and Without EGFR-mutated Lung Adenocarcinoma.

PubMed

Li, Ming-Hsien; Tsai, Jo-Ting; Ting, Lai-Lei; Lin, Jang-Chun; Liu, Yu-Chang

2018-01-01

To investigate the association between tumor response to thoracic radiotherapy and epidermal growth factor receptor (EGFR) mutation status in patients with lung adenocarcinoma, we collected 48 patients treated between January 2010 and December 2013. Of the 18 patients with EGFR mutation, 15 (83.3%) had a single mutation, and three (16.7%) had double mutation. Different EGFR mutation subtypes exhibited different responses to radiotherapy. The identified double EGFR mutations were associated with reduction of residual tumor burden (RTB) after radiotherapy. In univariate analysis, EGFR mutations in exon 18, 20, and 21 and double EGFR mutation were significant factors predicting RTB. In multivariate analysis, exon 20 mutation was the only significant factor. Patients with EGFR mutation seemed to have longer mean overall survival (OS) compared to the group with wild-type EGFR (31.1 vs. 26.6 months, p=0.49). The median and mean OS in patients with double EGFR mutation vs. wild-type EGFR were 20.1 vs. 16.9 months and 28.9 vs. 26.6 months, respectively. Further studies with larger sample size are warranted to clarify the association of EGFR mutation status with the lung tumor response after radiotherapy. Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Frontotemporal dementia with the C9ORF72 hexanucleotide repeat expansion: clinical, neuroanatomical and neuropathological features

PubMed Central

Mahoney, Colin J.; Beck, Jon; Rohrer, Jonathan D.; Lashley, Tammaryn; Mok, Kin; Shakespeare, Tim; Yeatman, Tom; Warrington, Elizabeth K.; Schott, Jonathan M.; Fox, Nick C.; Rossor, Martin N.; Hardy, John; Collinge, John; Revesz, Tamas; Mead, Simon

2012-01-01

An expanded hexanucleotide repeat in the C9ORF72 gene has recently been identified as a major cause of familial frontotemporal lobar degeneration and motor neuron disease, including cases previously identified as linked to chromosome 9. Here we present a detailed retrospective clinical, neuroimaging and histopathological analysis of a C9ORF72 mutation case series in relation to other forms of genetically determined frontotemporal lobar degeneration ascertained at a specialist centre. Eighteen probands (19 cases in total) were identified, representing 35% of frontotemporal lobar degeneration cases with identified mutations, 36% of cases with clinical evidence of motor neuron disease and 7% of the entire cohort. Thirty-three per cent of these C9ORF72 cases had no identified relevant family history. Families showed wide variation in clinical onset (43–68 years) and duration (1.7–22 years). The most common presenting syndrome (comprising a half of cases) was behavioural variant frontotemporal dementia, however, there was substantial clinical heterogeneity across the C9ORF72 mutation cohort. Sixty per cent of cases developed clinical features consistent with motor neuron disease during the period of follow-up. Anxiety and agitation and memory impairment were prominent features (between a half to two-thirds of cases), and dominant parietal dysfunction was also frequent. Affected individuals showed variable magnetic resonance imaging findings; however, relative to healthy controls, the group as a whole showed extensive thinning of frontal, temporal and parietal cortices, subcortical grey matter atrophy including thalamus and cerebellum and involvement of long intrahemispheric, commissural and corticospinal tracts. The neuroimaging profile of the C9ORF72 expansion was significantly more symmetrical than progranulin mutations with significantly less temporal lobe involvement than microtubule-associated protein tau mutations. Neuropathological examination in six cases

A Presenilin-1 Mutation Identified in Familial Alzheimer Disease with Cotton Wool Plaques Causes a Nearly Complete Loss of γ-Secretase Activity*

PubMed Central

Heilig, Elizabeth A.; Xia, Weiming; Shen, Jie; Kelleher, Raymond J.

2010-01-01

Mutations in presenilin-1 and presenilin-2 (PS1 and PS2) are the most common cause of familial Alzheimer disease. PS1 and PS2 are the presumptive catalytic components of the multisubunit γ-secretase complex, which proteolyzes a number of type I transmembrane proteins, including the amyloid precursor protein (APP) and Notch. APP processing by γ-secretase produces β-amyloid peptides (Aβ40 and Aβ42) that accumulate in the Alzheimer disease brain. Here we identify a pathogenic L435F mutation in PS1 in two affected siblings with early-onset familial Alzheimer disease characterized by deposition of cerebral cotton wool plaques. The L435F mutation resides in a conserved C-terminal PAL sequence implicated in active site conformation and catalytic activity. The impact of PS1 mutations in and around the PAL motif on γ-secretase activity was assessed by expression of mutant PS1 in mouse embryo fibroblasts lacking endogenous PS1 and PS2. Surprisingly, the L435F mutation caused a nearly complete loss of γ-secretase activity, including >90% reductions in the generation of Aβ40, Aβ42, and the APP and Notch intracellular domains. Two nonpathogenic PS1 mutations, P433L and L435R, caused essentially complete loss of γ-secretase activity, whereas two previously identified pathogenic PS1 mutations, P436Q and P436S, caused partial loss of function with substantial reductions in production of Aβ40, Aβ42, and the APP and Notch intracellular domains. These results argue against overproduction of Aβ42 as an essential property of presenilin proteins bearing pathogenic mutations. Rather, our findings provide support for the hypothesis that pathogenic mutations cause a general loss of presenilin function. PMID:20460383

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

The Prevalence of JAK2, MPL, and CALR Mutations in Chinese Patients With BCR-ABL1-Negative Myeloproliferative Neoplasms.

PubMed

Lin, Yani; Liu, Enbin; Sun, Qi; Ma, Jiao; Li, QingHua; Cao, Zeng; Wang, Jun; Jia, Yujiao; Zhang, Hongju; Song, Zhen; Ai, Xiaofei; Shi, Lihui; Feng, Xiaofang; Li, Chenwei; Wang, Jianxiang; Ru, Kun

2015-07-01

To evaluate the mutation frequency of JAK2 V617F, JAK2 exon 12, MPL exon 10, and CALR exon 9 and the value of the combined tests in the diagnosis of BCR-ABL1-negative myeloproliferative neoplasms (MPNs). In the current study, mutations of JAK2 V617F, JAK2 exon 12, MPL exon 10, and CALR exon 9 were analyzed in 929 Chinese patients with BCR-ABL1-negative MPN, including 234 cases of polycythemia vera (PV), 428 ETs, 187 PMFs, and 80 unclassifiable MPNs (MPN-Us). Our result showed that the positive rate of any of four mutations in patients with PV, ET, PMF, and MPN-U was 89.3%, 83.4%, 87.2%, and 77.5%, respectively, which significantly improved the diagnostic rate, especially in ET and PMF. Meanwhile, we also found that the patients without any of four mutations were younger than those with one or more mutations. Unexpectedly, the coexistence of JAK2 V617F and CALR exon 9 was identified in six (0.6%) patients, and JAK2 V617F and MPL exon 10 were present simultaneously in two (0.2%) patients. In addition, we also identified several novel mutation types in CALR exon 9. The combined genetic tests of JAK2 V617F, JAK2 exon 12, MPL exon 10, and CALR exon 9 help improve the diagnostic rate for BCR-ABL1-negative MPN. Copyright© by the American Society for Clinical Pathology.

A screen to identify Drosophila genes required for integrin-mediated adhesion.

PubMed Central

Walsh, E P; Brown, N H

1998-01-01

Drosophila integrins have essential adhesive roles during development, including adhesion between the two wing surfaces. Most position-specific integrin mutations cause lethality, and clones of homozygous mutant cells in the wing do not adhere to the apposing surface, causing blisters. We have used FLP-FRT induced mitotic recombination to generate clones of randomly induced mutations in the F1 generation and screened for mutations that cause wing blisters. This phenotype is highly selective, since only 14 lethal complementation groups were identified in screens of the five major chromosome arms. Of the loci identified, 3 are PS integrin genes, 2 are blistered and bloated, and the remaining 9 appear to be newly characterized loci. All 11 nonintegrin loci are required on both sides of the wing, in contrast to integrin alpha subunit genes. Mutations in 8 loci only disrupt adhesion in the wing, similar to integrin mutations, while mutations in the 3 other loci cause additional wing defects. Mutations in 4 loci, like the strongest integrin mutations, cause a "tail-up" embryonic lethal phenotype, and mutant alleles of 1 of these loci strongly enhance an integrin mutation. Thus several of these loci are good candidates for genes encoding cytoplasmic proteins required for integrin function. PMID:9755209

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.

Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch.

PubMed

Oakes, Benjamin L; Nadler, Dana C; Flamholz, Avi; Fellmann, Christof; Staahl, Brett T; Doudna, Jennifer A; Savage, David F

2016-06-01

The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disruption of the enzyme's binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the estrogen receptor-α ligand-binding domain. This protein showed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9.

FLG mutations in ichthyosis vulgaris and atopic eczema: spectrum of mutations and population genetics.

PubMed

Akiyama, M

2010-03-01

Filaggrin is a key protein involved in skin barrier function. Mutations in the gene encoding filaggrin (FLG) have been identified as the cause of ichthyosis vulgaris and have been shown to be major predisposing factors for atopic eczema (AE), initially in European populations. Subsequently, FLG mutations were identified in Japanese, Chinese, Taiwanese and Korean populations. It was demonstrated that FLG mutations are closely associated with AE in the Japanese population. Notably, the same FLG mutations identified in the European population were rarely found in Asians. These results exemplify differences in filaggrin population genetics between Europe and Asia. For mutation screening, background information needs to be obtained on prevalent FLG mutations for each geographical population. It is therefore important to establish the global population genetics maps for FLG mutations. Mutations at any site within FLG, even mutations in C-terminal imperfect filaggrin repeats, cause significant reductions in amounts of profilaggrin/filaggrin peptide in patient epidermis as the C-terminal region is essential for proper processing of profilaggrin into filaggrin. Thus, no genotype-phenotype correlation has been observed in patients with FLG mutations. A restoration of the barrier function seems a feasible and promising strategy for treatment and prevention in individuals with filaggrin deficiency.

Variation of p53 mutational spectra between carcinoma of the upper and lower respiratory tract.

PubMed

Law, J C; Whiteside, T L; Gollin, S M; Weissfeld, J; El-Ashmawy, L; Srivastava, S; Landreneau, R J; Johnson, J T; Ferrell, R E

1995-07-01

Mutations of the p53 tumor suppressor gene are the most common genetic alterations associated with human cancer. Tumor-associated p53 mutations often show characteristic tissue-specific profiles which may infer environmentally induced mutational mechanisms. The p53 mutational frequency and spectrum were determined for 95 carcinomas of the upper and lower respiratory tract (32 lung and 63 upper respiratory tract). Mutations were identified at a frequency of 30% in upper respiratory tract (URT) tumors and 31% in lung tumors. All 29 identified mutations were single-base substitutions. Comparison of the frequency of specific base substitutions between lung and URT showed a striking difference. Transitions occurred at a frequency of 68% in URT, but only 30% in lung. Mutations involving G:C-->A:T transitions, which are commonly reported in gastric and esophageal tumors, were the most frequently identified alteration in URT (11/19). Mutations involving G:C-->T:A transversions, which were relatively common in lung tumors (3/10) and are representative of tobacco smoke-induced mutations were rare in URT tumors (1/19). Interestingly, G:C-->A:T mutations at CpG sites, which are characteristic of endogenous processes, were observed frequently in URT tumors (9/19) but only rarely in lung tumors (1/10), suggesting that both endogenous and exogenous factors are responsible for the observed differences in mutational spectra between the upper and lower respiratory systems.

Exome Capture and Massively Parallel Sequencing Identifies a Novel HPSE2 Mutation in a Saudi Arabian Child with Ochoa (Urofacial) Syndrome

PubMed Central

Al Badr, Wisam; Al Bader, Suha; Otto, Edgar; Hildebrandt, Friedhelm; Ackley, Todd; Peng, Weiping; Xu, Jishu; Li, Jun; Owens, Kailey M.; Bloom, David; Innis, Jeffrey W.

2011-01-01

We describe a child of Middle Eastern descent by first-cousin mating with idiopathic neurogenic bladder and high grade vesicoureteral reflux at 1 year of age, whose characteristic facial grimace led to the diagnosis of Ochoa (Urofacial) syndrome at age 5 years. We used homozygosity mapping, exome capture and paired end sequencing to identify the disease causing mutation in the proband. We reviewed the literature with respect to the urologic manifestations of Ochoa syndrome. A large region of marker homozygosity was observed at 10q24, consistent with known autosomal recessive inheritance, family consanguinity and previous genetic mapping in other families with Ochoa syndrome. A homozygous mutation was identified in the proband in HPSE2: c.1374_1378delTGTGC, a deletion of 5 nucleotides in exon 10 that is predicted to lead to a frameshift followed by replacement of 132 C-terminal amino acids with 153 novel amino acids (p.Ala458Alafsdel132ins153). This mutation is novel relative to very recently published mutations in HPSE2 in other families. Early intervention and recognition of Ochoa syndrome with control of risk factors and close surveillance will decrease complications and renal failure. PMID:21450525

Use of CBL exon 8 and 9 mutations in diagnosis of myeloproliferative neoplasms and myelodysplastic/myeloproliferative disorders: an analysis of 636 cases

PubMed Central

Schnittger, Susanne; Bacher, Ulrike; Alpermann, Tamara; Reiter, Andreas; Ulke, Madlen; Dicker, Frank; Eder, Christiane; Kohlmann, Alexander; Grossmann, Vera; Kowarsch, Andreas; Kern, Wolfgang; Haferlach, Claudia; Haferlach, Torsten

2012-01-01

We analyzed 636 patients with diverse myeloproliferative neoplasms or myelodysplastic/myeloproliferative neoplasms for mutations of the Casitas B-cell lymphoma gene (CBLmut) in exons 8 and 9 and performed correlations to other genetic alterations. CBLmut were detected in 63 of 636 (9.9%) of these selected patients. CBLmut were more frequent in myelodysplastic/myeloproliferative neoplasms than myeloproliferative neoplasms (51 of 328, 15.5% vs. 12 of 291, 4.1%; P<0.001). Frequency was 48 of 278 (17.3%) in chronic myelomonocytic leukemia and 3 of 33 (9.1%) in unclassifiable myelodysplastic/myeloproliferative neoplasms. CBLmut was not detected in polycythemia vera, primary myelofibrosis, essential thrombocythemia, or refractory anemia with ring sideroblasts and marked thrombocytosis. CBLmut were underrepresented in JAK2V617F mutated as compared to JAK2V617wt cases (P<0.001), and mutually exclusive of JAK2exon12mut and MPLW515mut. CBLmut were associated with monosomy 7 (P=0.008) and TET2mut (P=0.003). In chronic myelomonocytic leukemia, CBLmut had no significant impact on survival outcomes. Therefore, CBLmut are frequent in chronic myelomonocytic leukemia, absent in classical myeloproliferative neoplasms, and are only exceptionally found in coincidence with JAK-STAT pathway activating mutations. PMID:22733026

Frequent genes in rare diseases: panel-based next generation sequencing to disclose causal mutations in hereditary neuropathies.

PubMed

Dohrn, Maike F; Glöckle, Nicola; Mulahasanovic, Lejla; Heller, Corina; Mohr, Julia; Bauer, Christine; Riesch, Erik; Becker, Andrea; Battke, Florian; Hörtnagel, Konstanze; Hornemann, Thorsten; Suriyanarayanan, Saranya; Blankenburg, Markus; Schulz, Jörg B; Claeys, Kristl G; Gess, Burkhard; Katona, Istvan; Ferbert, Andreas; Vittore, Debora; Grimm, Alexander; Wolking, Stefan; Schöls, Ludger; Lerche, Holger; Korenke, G Christoph; Fischer, Dirk; Schrank, Bertold; Kotzaeridou, Urania; Kurlemann, Gerhard; Dräger, Bianca; Schirmacher, Anja; Young, Peter; Schlotter-Weigel, Beate; Biskup, Saskia

2017-12-01

Hereditary neuropathies comprise a wide variety of chronic diseases associated to more than 80 genes identified to date. We herein examined 612 index patients with either a Charcot-Marie-Tooth phenotype, hereditary sensory neuropathy, familial amyloid neuropathy, or small fiber neuropathy using a customized multigene panel based on the next generation sequencing technique. In 121 cases (19.8%), we identified at least one putative pathogenic mutation. Of these, 54.4% showed an autosomal dominant, 33.9% an autosomal recessive, and 11.6% an X-linked inheritance. The most frequently affected genes were PMP22 (16.4%), GJB1 (10.7%), MPZ, and SH3TC2 (both 9.9%), and MFN2 (8.3%). We further detected likely or known pathogenic variants in HINT1, HSPB1, NEFL, PRX, IGHMBP2, NDRG1, TTR, EGR2, FIG4, GDAP1, LMNA, LRSAM1, POLG, TRPV4, AARS, BIC2, DHTKD1, FGD4, HK1, INF2, KIF5A, PDK3, REEP1, SBF1, SBF2, SCN9A, and SPTLC2 with a declining frequency. Thirty-four novel variants were considered likely pathogenic not having previously been described in association with any disorder in the literature. In one patient, two homozygous mutations in HK1 were detected in the multigene panel, but not by whole exome sequencing. A novel missense mutation in KIF5A was considered pathogenic because of the highly compatible phenotype. In one patient, the plasma sphingolipid profile could functionally prove the pathogenicity of a mutation in SPTLC2. One pathogenic mutation in MPZ was identified after being previously missed by Sanger sequencing. We conclude that panel based next generation sequencing is a useful, time- and cost-effective approach to assist clinicians in identifying the correct diagnosis and enable causative treatment considerations. © 2017 International Society for Neurochemistry.

GAMES identifies and annotates mutations in next-generation sequencing projects.

PubMed

Sana, Maria Elena; Iascone, Maria; Marchetti, Daniela; Palatini, Jeff; Galasso, Marco; Volinia, Stefano

2011-01-01

Next-generation sequencing (NGS) methods have the potential for changing the landscape of biomedical science, but at the same time pose several problems in analysis and interpretation. Currently, there are many commercial and public software packages that analyze NGS data. However, the limitations of these applications include output which is insufficiently annotated and of difficult functional comprehension to end users. We developed GAMES (Genomic Analysis of Mutations Extracted by Sequencing), a pipeline aiming to serve as an efficient middleman between data deluge and investigators. GAMES attains multiple levels of filtering and annotation, such as aligning the reads to a reference genome, performing quality control and mutational analysis, integrating results with genome annotations and sorting each mismatch/deletion according to a range of parameters. Variations are matched to known polymorphisms. The prediction of functional mutations is achieved by using different approaches. Overall GAMES enables an effective complexity reduction in large-scale DNA-sequencing projects. GAMES is available free of charge to academic users and may be obtained from http://aqua.unife.it/GAMES.

A Novel GRN Mutation (GRN c.708+6_+9delTGAG) in Frontotemporal Lobar Degeneration with TDP-43-positive Inclusions: Clinicopathologic Report of 6 Cases

PubMed Central

Bit-Ivan, Esther N.; Suh, Eunran; Shim, H-S; Weintraub, Sandra; Hyman, Bradley T.; Arnold, Steven E.; McCarty-Wood, Elisabeth; Van Deerlin, Viviana M.; Schneider, Julie A.; Trojanowski, John Q.; Frosch, Matthew P.; Baker, Matt C.; Rademakers, Rosa; Mesulam, Marsel; Bigio, Eileen H.

2014-01-01

Understanding of frontotemporal lobar degeneration (FTLD), the underlying pathology that is most often linked to the clinical diagnosis of frontotemporal dementia (FTD), is rapidly increasing. Mutations in 7 known genes (MAPT, GRN, C9orf72, VCP, CHMP2B, and rarely TARDBP and FUS) are associated with FTD and the pathologic classification of FTLD has recently been modified to reflect these discoveries. Mutations in one of these genes (GRN), which encodes progranulin, have been implicated in up to one quarter of FTLD cases with TAR DNA-binding protein 43-positive inclusions (FTLD-TDP); there currently are more than 60 known pathogenic mutations of the gene. We present the clinical, pathologic, and genetic findings of 6 cases from 4 families, 5 of which were shown to have a novel GRN c.708+6_+9delTGAG mutation. PMID:24709683

Alteration of DNA binding, dimerization, and nuclear translocation of SHOX homeodomain mutations identified in idiopathic short stature and Leri-Weill dyschondrosteosis.

PubMed

Schneider, Katja U; Marchini, Antonio; Sabherwal, Nitin; Röth, Ralph; Niesler, Beate; Marttila, Tiina; Blaschke, Rüdiger J; Lawson, Margaret; Dumic, Miroslav; Rappold, Gudrun

2005-07-01

Haploinsufficiency of the short stature homeobox gene SHOX has been found in patients with idiopathic short stature (ISS) and Leri-Weill dyschondrosteosis (LWD). In addition to complete gene deletions and nonsense mutations, several missense mutations have been identified in both patient groups, leading to amino acid substitutions in the SHOX protein. The majority of missense mutations were found to accumulate in the region encoding the highly conserved homeodomain of the paired-like type. In this report, we investigated nine different amino acid exchanges in the homeodomain of SHOX patients with ISS and LWD. We were able show that these mutations cause an alteration of the biological function of SHOX by loss of DNA binding, reduced dimerization ability, and/or impaired nuclear translocation. Additionally, one of the mutations (c.458G>T, p.R153L) is defective in transcriptional activation even though it is still able to bind to DNA, dimerize, and translocate to the nucleus. Thus, we demonstrate that single missense mutations in the homeodomain fundamentally impair SHOX key functions, thereby leading to the phenotype observed in patients with LWD and ISS.

Whole exome sequencing identifies a POLRID mutation segregating in a father and two daughters with findings of Klippel-Feil and Treacher Collins syndromes.

PubMed

Giampietro, Philip F; Armstrong, Linlea; Stoddard, Alex; Blank, Robert D; Livingston, Janet; Raggio, Cathy L; Rasmussen, Kristen; Pickart, Michael; Lorier, Rachel; Turner, Amy; Sund, Sarah; Sobrera, Nara; Neptune, Enid; Sweetser, David; Santiago-Cornier, Alberto; Broeckel, Ulrich

2015-01-01

We report on a father and his two daughters diagnosed with Klippel-Feil syndrome (KFS) but with craniofacial differences (zygomatic and mandibular hypoplasia and cleft palate) and external ear abnormalities suggestive of Treacher Collins syndrome (TCS). The diagnosis of KFS was favored, given that the neck anomalies were the predominant manifestations, and that the diagnosis predated later recognition of the association between spinal segmentation abnormalities and TCS. Genetic heterogeneity and the rarity of large families with KFS have limited the ability to identify mutations by traditional methods. Whole exome sequencing identified a nonsynonymous mutation in POLR1D (subunit of RNA polymerase I and II): exon2:c.T332C:p.L111P. Mutations in POLR1D are present in about 5% of individuals diagnosed with TCS. We propose that this mutation is causal in this family, suggesting a pathogenetic link between KFS and TCS. © 2014 Wiley Periodicals, Inc.

Sodium Channel Mutations and Susceptibility to Heart Failure and Atrial Fibrillation

PubMed Central

Olson, Timothy M.; Michels, Virginia V.; Ballew, Jeffrey D.; Reyna, Sandra P.; Karst, Margaret L.; Herron, Kathleen J.; Horton, Steven C.; Rodeheffer, Richard J.; Anderson, Jeffrey L.

2007-01-01

Context Dilated cardiomyopathy (DCM), a genetically heterogeneous disorder, causes heart failure and rhythm disturbances. The majority of identified DCM genes encode structural proteins of the contractile apparatus and cytoskeleton. Recently, genetic defects in calcium and potassium regulation have been discovered in patients with DCM, implicating an alternative disease mechanism. The full spectrum of genetic defects in DCM, however, has not been established. Objectives To identify a novel gene for DCM at a previously mapped locus, define the spectrum of mutations in this gene within a DCM cohort, and determine the frequency of DCM among relatives inheriting a mutation in this gene. Design, Setting, and Participants Refined mapping of a DCM locus on chromosome 3p in a multigenerational family and mutation scanning in 156 unrelated pro-bands with DCM, prospectively identified at the Mayo Clinic between 1987 and 2004. Relatives underwent screening echocardiography and electrocardiography and DNA sample procurement. Main Outcome Measure Correlation of identified mutations with cardiac phenotype. Results Refined locus mapping revealed SCN5A, encoding the cardiac sodium channel, as a candidate gene. Mutation scans identified a missense mutation (D1275N) that cosegregated with an age-dependent, variably expressed phenotype of DCM, atrial fibrillation, impaired automaticity, and conduction delay. In the DCM cohort, additional missense (T220I, R814W, D1595H) and truncation (2550-2551insTG) SCN5A mutations, segregating with cardiac disease or arising de novo, were discovered in unrelated probands. Among individuals with an SCN5A mutation 27% had early features of DCM (mean age at diagnosis, 20.3 years), 38% had DCM (mean age at diagnosis, 47.9 years), and 43% had atrial fibrillation (mean age at diagnosis, 27.8 years). Conclusions Heritable SCN5A defects are associated with susceptibility to early-onset DCM and atrial fibrillation. Similar or even identical mutations may

[Mutation analysis of beta myosin heavy chain gene in hypertrophic cardiomyopathy families].

PubMed

Fan, Xin-ping; Yang, Zhong-wei; Feng, Xiu-li; Yang, Fu-hui; Xiao, Bai; Liang, Yan

2011-08-01

To detect the gene mutations of beta-myosin heavy chain gene (MYH7) in Chinese pedigrees with hypertrophic cardiomyopathy (HCM), and to analyze the correlation between the genotype and phenotype. Exons 3, 5, 7-9, 11-16 and 18-23 of the MYH7 gene were amplified with PCR in three Chinese pedigrees with HCM. The products were sequenced. Sequence alignment between the detected and the standard sequences was performed. A missense mutation of Thr441Met in exon 14 was identified in a pedigree, which was not detected in the controls. Several synonymous mutations of MYH7 gene were detected in the three pedigrees. The mutation of Thr441Met, located in the actin binding domain of the globular head, was first identified in Chinese. It probably caused HCM. HCM is a heterogeneous disease. Many factors are involved in the process of its occurrence and development.

Two novel AGXT mutations identified in primary hyperoxaluria type-1 and distinct morphological and structural difference in kidney stones

PubMed Central

Wang, Cui; Lu, Jingru; Lang, Yanhua; Liu, Ting; Wang, Xiaoling; Zhao, Xiangzhong; Shao, Leping

2016-01-01

Primary hyperoxaluria type 1 (PH1) is a rare genetic disease characterized by excessive oxalate accumulation in plasma and urine, resulting in various phenotypes because of allelic and clinical heterogeneity. This study aimed to detect disease-associated genetic mutations in three PH1 patients in a Chinese family. All AGXT exons and 3 common polymorphisms which might synergistically interact with mutations, including P11L, I340 M and IVSI+74 bp were analyzed by direct sequencing in all family members. It demonstrated that in each of three patients, a previously reported nonsense mutation p.R333* was in cis with a novel missense mutation p.M49L in the minor allele characterized by the polymorphism of 74-bp duplication in intron 1, while the other novel missense mutation p.N72I was in trans with both p.R333* and P.M49L in the major allele. Kidney stones from two sibling patients were also observed though stereomicroscopic examination and scanning electron microscopy. Distinct morphological and inner-structure differences in calculi were noticed, suggesting clinical heterozygosity of PH1 to a certain extent. In brief, two novel missense mutations were identified probably in association with PH1, a finding which should provide an accurate tool for prenatal diagnosis, genetic counseling and screening for potential presymptomatic individuals. PMID:27644547

Two novel AGXT mutations identified in primary hyperoxaluria type-1 and distinct morphological and structural difference in kidney stones.

PubMed

Wang, Cui; Lu, Jingru; Lang, Yanhua; Liu, Ting; Wang, Xiaoling; Zhao, Xiangzhong; Shao, Leping

2016-09-20

Primary hyperoxaluria type 1 (PH1) is a rare genetic disease characterized by excessive oxalate accumulation in plasma and urine, resulting in various phenotypes because of allelic and clinical heterogeneity. This study aimed to detect disease-associated genetic mutations in three PH1 patients in a Chinese family. All AGXT exons and 3 common polymorphisms which might synergistically interact with mutations, including P11L, I340 M and IVSI+74 bp were analyzed by direct sequencing in all family members. It demonstrated that in each of three patients, a previously reported nonsense mutation p.R333(*) was in cis with a novel missense mutation p.M49L in the minor allele characterized by the polymorphism of 74-bp duplication in intron 1, while the other novel missense mutation p.N72I was in trans with both p.R333(*) and P.M49L in the major allele. Kidney stones from two sibling patients were also observed though stereomicroscopic examination and scanning electron microscopy. Distinct morphological and inner-structure differences in calculi were noticed, suggesting clinical heterozygosity of PH1 to a certain extent. In brief, two novel missense mutations were identified probably in association with PH1, a finding which should provide an accurate tool for prenatal diagnosis, genetic counseling and screening for potential presymptomatic individuals.

[Frequency of the most common mutations of the CFTR gene in peruvian patients with cystic fibrosis using the ARMS-PCR technique].

PubMed

Aquino, Ruth; Protzel, Ana; Rivera, Juan; Abarca, Hugo; Dueñas, Milagros; Nestarez, Cecilia; Purizaga, Nestor; Diringer, Benoit

2017-01-01

To determine the frequency of the ten most common mutations of the CFTR gene reported in Latin Americausing amplification-refractory mutation system-polymerase chain reaction (ARMS-PCR) in patients with cystic fibrosis (CF) in two referral hospitals in Peru during the year 2014. The frequency of the ten most common mutations of the CFTR gene was assessed in patients of the Hospital Nacional Edgardo Rebagliati Martins and the Instituto Nacional de Salud del Niño, both located in Lima, Peru. Blood samples were collected from 36 patients with CF, and the ARMS-PCR technique was used to determine the presence of these mutations. The study group included 73.5% of patients with a known diagnosis of CF in the country when the study was carried out. ARMS-PCR allowed three of the mutations to be identified in a combined 30.6% of the alleles from patients with CF, and 64.9% of the mutated alleles were not identified. The mutations found were p.Phe508del (22,2%), p.Gly542* (6,9%), and p.Arg1162* (1,4%). There is significant variability in both the frequency and type of mutations present in our study population and in what has been reported in other Latin American countries. It is necessary to perform studies that use complete sequencing technology for the CFTR gene to identify other mutations present in our population.

Identification of 5 novel mutations in the AGXT gene.

PubMed

Basmaison, O; Rolland, M O; Cochat, P; Bozon, D

2000-06-01

In order to identify additional genotypes in primary hyperoxaluria type 1, we sequenced the AGXT genes of 9 patients. We report 5 new mutations. Three are splice-site mutations situated at the end of intron 4 and 8 (647-1G>A, 969-1G>C, 969-3C>G), one is a missense mutation in exon 5 (D183N), and one is a short duplication in exon 2 (349ins7). Their consequence is always a lack of enzymatic activity of the Alanine-Glyoxylate Aminotransferase (AGT); for 4 of them, we were able to deduce that they were associated to the absence of AGT protein. These mutations are rare, as they have been found on one allele in our study (except 969-3C>G present in 2 unrelated families), and have not been previously reported.

Mutations during the Adaptation of H9N2 Avian Influenza Virus to the Respiratory Epithelium of Pigs Enhance Sialic Acid Binding Activity and Virulence in Mice.

PubMed

Yang, W; Punyadarsaniya, D; Lambertz, R L O; Lee, D C C; Liang, C H; Höper, D; Leist, S R; Hernández-Cáceres, A; Stech, J; Beer, M; Wu, C Y; Wong, C H; Schughart, K; Meng, F; Herrler, G

2017-04-15

The natural reservoir for influenza viruses is waterfowl, and from there they succeeded in crossing the barrier to different mammalian species. We analyzed the adaptation of avian influenza viruses to a mammalian host by passaging an H9N2 strain three times in differentiated swine airway epithelial cells. Using precision-cut slices from the porcine lung to passage the parental virus, isolates from each of the three passages (P1 to P3) were characterized by assessing growth curves and ciliostatic effects. The only difference noted was an increased growth kinetics of the P3 virus. Sequence analysis revealed four mutations: one each in the PB2 and NS1 proteins and two in the HA protein. The HA mutations, A190V and T212I, were characterized by generating recombinant viruses containing either one or both amino acid exchanges. Whereas the parental virus recognized α2,3-linked sialic acids preferentially, the HA190 mutant bound to a broad spectrum of glycans with α2,6/8/9-linked sialic acids. The HA212 mutant alone differed only slightly from the parental virus; however, the combination of both mutations (HA190+HA212) increased the binding affinity to those glycans recognized by the HA190 mutant. Remarkably, only the HA double mutant showed a significantly increased pathogenicity in mice. In contrast, none of those mutations affected the ciliary activity of the epithelial cells which is characteristic for virulent swine influenza viruses. Taken together, our results indicate that shifts in the HA receptor affinity are just an early adaptation step of avian H9N2 strains; further mutational changes may be required to become virulent for pigs. IMPORTANCE Swine play an important role in the interspecies transmission of influenza viruses. Avian influenza A viruses (IAV) of the H9N2 subtype have successfully infected hosts from different species but have not established a stable lineage. We have analyzed the adaptation of IAV-H9N2 virus to target cells of a new host by

Whole-exome sequencing, without prior linkage, identifies a mutation in LAMB3 as a cause of dominant hypoplastic amelogenesis imperfecta.

PubMed

Poulter, James A; El-Sayed, Walid; Shore, Roger C; Kirkham, Jennifer; Inglehearn, Chris F; Mighell, Alan J

2014-01-01

The conventional approach to identifying the defective gene in a family with an inherited disease is to find the disease locus through family studies. However, the rapid development and decreasing cost of next generation sequencing facilitates a more direct approach. Here, we report the identification of a frameshift mutation in LAMB3 as a cause of dominant hypoplastic amelogenesis imperfecta (AI). Whole-exome sequencing of three affected family members and subsequent filtering of shared variants, without prior genetic linkage, sufficed to identify the pathogenic variant. Simultaneous analysis of multiple family members confirms segregation, enhancing the power to filter the genetic variation found and leading to rapid identification of the pathogenic variant. LAMB3 encodes a subunit of Laminin-5, one of a family of basement membrane proteins with essential functions in cell growth, movement and adhesion. Homozygous LAMB3 mutations cause junctional epidermolysis bullosa (JEB) and enamel defects are seen in JEB cases. However, to our knowledge, this is the first report of dominant AI due to a LAMB3 mutation in the absence of JEB.

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

Somatic CALR Mutations in Myeloproliferative Neoplasms with Nonmutated JAK2

PubMed Central

Baxter, E.J.; Nice, F.L.; Gundem, G.; Wedge, D.C.; Avezov, E.; Li, J.; Kollmann, K.; Kent, D.G.; Aziz, A.; Godfrey, A.L.; Hinton, J.; Martincorena, I.; Van Loo, P.; Jones, A.V.; Guglielmelli, P.; Tarpey, P.; Harding, H.P.; Fitzpatrick, J.D.; Goudie, C.T.; Ortmann, C.A.; Loughran, S.J.; Raine, K.; Jones, D.R.; Butler, A.P.; Teague, J.W.; O’Meara, S.; McLaren, S.; Bianchi, M.; Silber, Y.; Dimitropoulou, D.; Bloxham, D.; Mudie, L.; Maddison, M.; Robinson, B.; Keohane, C.; Maclean, C.; Hill, K.; Orchard, K.; Tauro, S.; Du, M.-Q.; Greaves, M.; Bowen, D.; Huntly, B.J.P.; Harrison, C.N.; Cross, N.C.P.; Ron, D.; Vannucchi, A.M.; Papaemmanuil, E.; Campbell, P.J.; Green, A.R.

2014-01-01

BACKGROUND Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. METHODS We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry. RESULTS Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients. CONCLUSIONS Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with

Exome Sequencing Identifies a REEP1 Mutation Involved in Distal Hereditary Motor Neuropathy Type V