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1.
Gene ; 705: 109-112, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31028865

RESUMO

Variants of KCNQ4 are one of the most common causes of dominantly inherited nonsyndromic hearing loss. We investigated a consanguineous family in which two individuals had prelignual hearing loss, apparently inherited in a recessive mode. Whole-exome sequencing analyses demonstrated genetic heterogeneity as variants in two different genes segregated with the phenotype in two branches of the family. Members in one branch were homozygous for a pathogenic variant of TMC1. The other two affected individuals were homozygous for a missense pathogenic variant in KCNQ4 c.872C>T; p.(Pro291Leu). These two individuals had prelingual, progressive moderate to severe hearing loss, while a heterozygous carrier had late onset mild hearing loss. Our work demonstrates that p.Pro291L variant is semi-dominantly inherited. This is the first report of semi-dominance of a KCNQ4 variant.


Assuntos
Surdez/genética , Canais de Potássio KCNQ/genética , Mutação de Sentido Incorreto , Sequenciamento Completo do Exoma/métodos , Idade de Início , Consanguinidade , Feminino , Heterogeneidade Genética , Predisposição Genética para Doença , Humanos , Leucina/genética , Masculino , Linhagem , Prolina/genética
2.
Genet Med ; 21(4): 798-812, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30655598

RESUMO

Identifying genes and variants contributing to rare disease phenotypes and Mendelian conditions informs biology and medicine, yet potential phenotypic consequences for variation of >75% of the ~20,000 annotated genes in the human genome are lacking. Technical advances to assess rare variation genome-wide, particularly exome sequencing (ES), enabled establishment in the United States of the National Institutes of Health (NIH)-supported Centers for Mendelian Genomics (CMGs) and have facilitated collaborative studies resulting in novel "disease gene" discoveries. Pedigree-based genomic studies and rare variant analyses in families with suspected Mendelian conditions have led to the elucidation of hundreds of novel disease genes and highlighted the impact of de novo mutational events, somatic variation underlying nononcologic traits, incompletely penetrant alleles, phenotypes with high locus heterogeneity, and multilocus pathogenic variation. Herein, we highlight CMG collaborative discoveries that have contributed to understanding both rare and common diseases and discuss opportunities for future discovery in single-locus Mendelian disorder genomics. Phenotypic annotation of all human genes; development of bioinformatic tools and analytic methods; exploration of non-Mendelian modes of inheritance including reduced penetrance, multilocus variation, and oligogenic inheritance; construction of allelic series at a locus; enhanced data sharing worldwide; and integration with clinical genomics are explored. Realizing the full contribution of rare disease research to functional annotation of the human genome, and further illuminating human biology and health, will lay the foundation for the Precision Medicine Initiative.


Assuntos
Doenças Genéticas Inatas/genética , Heterogeneidade Genética , Genoma Humano/genética , Genômica/tendências , Bases de Dados Genéticas , Predisposição Genética para Doença , Humanos , National Institutes of Health (U.S.) , Linhagem , Estados Unidos , Sequenciamento Completo do Exoma/métodos
3.
J Am Coll Cardiol ; 72(6): 605-615, 2018 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-30071989

RESUMO

BACKGROUND: Thoracic aortic aneurysms progressively enlarge and predispose to acute aortic dissections. Up to 25% of individuals with thoracic aortic disease harbor an underlying Mendelian pathogenic variant. An evidence-based strategy for selection of genes to test in hereditary thoracic aortic aneurysm and dissection (HTAAD) helps inform family screening and intervention to prevent life-threatening thoracic aortic events. OBJECTIVES: The purpose of this study was to accurately identify genes that predispose to HTAAD using the Clinical Genome Resource (ClinGen) framework. METHODS: We applied the semiquantitative ClinGen framework to assess presumed gene-disease relationships between 53 candidate genes and HTAAD. Genes were classified as causative for HTAAD if they were associated with isolated thoracic aortic disease and were clinically actionable, triggering routine aortic surveillance, intervention, and family cascade screening. All gene-disease assertions were evaluated by a pre-defined curator-expert pair and subsequently discussed with an expert panel. RESULTS: Genes were classified based on the strength of association with HTAAD into 5 categories: definitive (n = 9), strong (n = 2), moderate (n = 4), limited (n = 15), and no reported evidence (n = 23). They were further categorized by severity of associated aortic disease and risk of progression. Eleven genes in the definitive and strong groups were designated as "HTAAD genes" (category A). Eight genes were classified as unlikely to be progressive (category B) and 4 as low risk (category C). The remaining genes were recent genes with an uncertain classification or genes with no evidence of association with HTAAD. CONCLUSIONS: The ClinGen framework is useful to semiquantitatively assess the strength of gene-disease relationships for HTAAD. Gene categories resulting from the curation may inform clinical laboratories in the development, interpretation, and subsequent clinical implications of genetic testing for patients with aortic disease.

4.
Clin Cancer Res ; 24(12): 2963-2974, 2018 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-29301828

RESUMO

Purpose: The measurement of mutations in pancreatic juice samples collected from the duodenum during endoscopic ultrasound (EUS) may improve the diagnostic evaluation of patients undergoing pancreatic surveillance. Our aim was to evaluate the accuracy of using pancreatic juice mutation concentrations to predict the presence and histologic grade of neoplasia in the pancreas.Experimental Design: Digital next-generation sequencing (NGS) of pancreatic juice DNA using a targeted 12-gene panel was performed on 67 patients undergoing pancreatic evaluation during EUS, including patients with pancreatic ductal adenocarcinoma, patients who subsequently underwent pancreatic resection for precursor lesions, patients undergoing surveillance for their familial/inherited susceptibility to pancreatic cancer, and normal pancreas disease controls.Results: Patients with pancreatic cancer or high-grade dysplasia as their highest grade lesion had significantly higher pancreatic juice mutation concentrations than all other subjects (mean/SD digital NGS score; 46.6 ± 69.7 vs. 6.2 ± 11.6, P = 0.02). Pancreatic juice mutation concentrations distinguished patients with pancreatic cancer or high-grade dysplasia in their resection specimen from all other subjects with 72.2% sensitivity and 89.4% specificity [area under the curve (AUC) = 0.872]. Mutant TP53/SMAD4 concentrations could distinguish patients with pancreatic cancer or high-grade dysplasia in their resection specimen from all other subjects with 61.1% sensitivity and 95.7% specificity (AUC = 0.819). Among 31 high-risk individuals under surveillance, 2 of the 3 individuals with most abnormal pancreatic juice mutation profiles also had the most abnormalities on pancreatic imaging.Conclusions: Pancreatic juice mutation analysis using digital NGS has potential diagnostic utility in the evaluation of patients undergoing pancreatic surveillance. Clin Cancer Res; 24(12); 2963-74. ©2018 AACRSee related commentary by Lipner and Yeh, p. 2713.

5.
J Clin Oncol ; 35(30): 3382-3390, 2017 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-28767289

RESUMO

Purpose Deleterious germline mutations contribute to pancreatic cancer susceptibility and are well documented in families in which multiple members have had pancreatic cancer. Methods To define the prevalence of these germline mutations in patients with apparently sporadic pancreatic cancer, we sequenced 32 genes, including known pancreatic cancer susceptibility genes, in DNA prepared from normal tissue obtained from 854 patients with pancreatic ductal adenocarcinoma, 288 patients with other pancreatic and periampullary neoplasms, and 51 patients with non-neoplastic diseases who underwent pancreatic resection at Johns Hopkins Hospital between 2000 and 2015. Results Thirty-three (3.9%; 95% CI, 3.0% to 5.8%) of 854 patients with pancreatic cancer had a deleterious germline mutation, 31 (3.5%) of which affected known familial pancreatic cancer susceptibility genes: BRCA2 (12 patients), ATM (10 patients), BRCA1 (3 patients), PALB2 (2 patients), MLH1 (2 patients), CDKN2A (1 patient), and TP53 (1 patient). Patients with these germline mutations were younger than those without (mean ± SD, 60.8 ± 10.6 v 65.1 ± 10.5 years; P = .03). Deleterious germline mutations were also found in BUB1B (1) and BUB3 (1). Only three of these 33 patients had reported a family history of pancreatic cancer, and most did not have a cancer family history to suggest an inherited cancer syndrome. Five (1.7%) of 288 patients with other periampullary neoplasms also had a deleterious germline mutation. Conclusion Germline mutations in pancreatic cancer susceptibility genes are commonly identified in patients with pancreatic cancer without a significant family history of cancer. These deleterious pancreatic cancer susceptibility gene mutations, some of which are therapeutically targetable, will be missed if current family history guidelines are the main criteria used to determine the appropriateness of gene testing.


Assuntos
Carcinoma Ductal Pancreático/genética , Predisposição Genética para Doença/genética , Mutação em Linhagem Germinativa , Neoplasias Pancreáticas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Ductal Pancreático/epidemiologia , Carcinoma Ductal Pancreático/terapia , Feminino , Frequência do Gene , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/epidemiologia , Neoplasias Pancreáticas/terapia , Prevalência , Análise de Sequência de DNA , Estados Unidos/epidemiologia
6.
Curr Protoc Hum Genet ; 92: 18.10.1-18.10.25, 2017 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-28075488

RESUMO

This unit describes a technique for generating exome-enriched sequencing libraries using DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples. Utilizing commercially available kits, we present a low-input FFPE workflow starting with 50 ng of DNA. This procedure includes a repair step to address damage caused by FFPE preservation that improves sequence quality. Subsequently, libraries undergo an in-solution-targeted selection for exons, followed by sequencing using the Illumina next-generation short-read sequencing platform. © 2017 by John Wiley & Sons, Inc.


Assuntos
DNA/genética , Exoma/genética , Formaldeído , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Inclusão em Parafina , Fixação de Tecidos , Sequenciamento Completo do Exoma , Humanos , Parafina
8.
Am J Hum Genet ; 97(2): 199-215, 2015 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-26166479

RESUMO

Discovering the genetic basis of a Mendelian phenotype establishes a causal link between genotype and phenotype, making possible carrier and population screening and direct diagnosis. Such discoveries also contribute to our knowledge of gene function, gene regulation, development, and biological mechanisms that can be used for developing new therapeutics. As of February 2015, 2,937 genes underlying 4,163 Mendelian phenotypes have been discovered, but the genes underlying ∼50% (i.e., 3,152) of all known Mendelian phenotypes are still unknown, and many more Mendelian conditions have yet to be recognized. This is a formidable gap in biomedical knowledge. Accordingly, in December 2011, the NIH established the Centers for Mendelian Genomics (CMGs) to provide the collaborative framework and infrastructure necessary for undertaking large-scale whole-exome sequencing and discovery of the genetic variants responsible for Mendelian phenotypes. In partnership with 529 investigators from 261 institutions in 36 countries, the CMGs assessed 18,863 samples from 8,838 families representing 579 known and 470 novel Mendelian phenotypes as of January 2015. This collaborative effort has identified 956 genes, including 375 not previously associated with human health, that underlie a Mendelian phenotype. These results provide insight into study design and analytical strategies, identify novel mechanisms of disease, and reveal the extensive clinical variability of Mendelian phenotypes. Discovering the gene underlying every Mendelian phenotype will require tackling challenges such as worldwide ascertainment and phenotypic characterization of families affected by Mendelian conditions, improvement in sequencing and analytical techniques, and pervasive sharing of phenotypic and genomic data among researchers, clinicians, and families.


Assuntos
Doenças Genéticas Inatas/genética , Genética Médica/métodos , Genética Médica/tendências , Fenótipo , Proteínas/genética , Humanos
9.
Am J Med Genet A ; 167A(2): 271-81, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25394726

RESUMO

Lateral meningocele syndrome (LMS, OMIM%130720), also known as Lehman syndrome, is a very rare skeletal disorder with facial anomalies, hypotonia and meningocele-related neurologic dysfunction. The characteristic lateral meningoceles represent the severe end of the dural ectasia spectrum and are typically most severe in the lower spine. Facial features of LMS include hypertelorism and telecanthus, high arched eyebrows, ptosis, midfacial hypoplasia, micrognathia, high and narrow palate, low-set ears and a hypotonic appearance. Hyperextensibility, hernias and scoliosis reflect a connective tissue abnormality, and aortic dilation, a high-pitched nasal voice, wormian bones and osteolysis may be present. Lateral meningocele syndrome has phenotypic overlap with Hajdu-Cheney syndrome. We performed exome resequencing in five unrelated individuals with LMS and identified heterozygous truncating NOTCH3 mutations. In an additional unrelated individual Sanger sequencing revealed a deleterious variant in the same exon 33. In total, five novel de novo NOTCH3 mutations were identified in six unrelated patients. One had a 26 bp deletion (c.6461_6486del, p.G2154fsTer78), two carried the same single base pair insertion (c.6692_93insC, p.P2231fsTer11), and three individuals had a nonsense point mutation at c.6247A > T (pK2083*), c.6663C > G (p.Y2221*) or c.6732C > A, (p.Y2244*). All mutations cluster into the last coding exon, resulting in premature termination of the protein and truncation of the negative regulatory proline-glutamate-serine-threonine rich PEST domain. Our results suggest that mutant mRNA products escape nonsense mediated decay. The truncated NOTCH3 may cause gain-of-function through decreased clearance of the active intracellular product, resembling NOTCH2 mutations in the clinically related Hajdu-Cheney syndrome and contrasting the NOTCH3 missense mutations causing CADASIL.


Assuntos
Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Éxons , Meningocele/diagnóstico , Meningocele/genética , Mutação , Receptores Notch/genética , Criança , Pré-Escolar , Análise Mutacional de DNA , Exoma , Facies , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Imagem por Ressonância Magnética , Masculino , Fenótipo , Receptor Notch3 , Adulto Jovem
10.
Proc Natl Acad Sci U S A ; 110(6): E507-16, 2013 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-23341629

RESUMO

The microRNA-183/96/182 cluster is highly expressed in the retina and other sensory organs. To uncover its in vivo functions in the retina, we generated a knockout mouse model, designated "miR-183C(GT/GT)," using a gene-trap embryonic stem cell clone. We provide evidence that inactivation of the cluster results in early-onset and progressive synaptic defects of the photoreceptors, leading to abnormalities of scotopic and photopic electroretinograms with decreased b-wave amplitude as the primary defect and progressive retinal degeneration. In addition, inactivation of the miR-183/96/182 cluster resulted in global changes in retinal gene expression, with enrichment of genes important for synaptogenesis, synaptic transmission, photoreceptor morphogenesis, and phototransduction, suggesting that the miR-183/96/182 cluster plays important roles in postnatal functional differentiation and synaptic connectivity of photoreceptors.


Assuntos
MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Degeneração Retiniana/genética , Animais , Modelos Animais de Doenças , Íntrons , Luz/efeitos adversos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Família Multigênica , Neurogênese/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Retina/lesões , Retina/metabolismo , Retina/efeitos da radiação , Degeneração Retiniana/patologia , Degeneração Retiniana/fisiopatologia , Órgãos dos Sentidos/metabolismo , Sinapses/genética , Sinapses/metabolismo , Transmissão Sináptica/genética , Síndrome , Visão Ocular/genética
11.
Am J Med Genet A ; 152A(3): 646-52, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20186779

RESUMO

Mutations in miRNA genes have been implicated in hearing loss in human families and mice. It is also possible that mutations in miRNA binding sites of inner ear targets alter gene expression levels and lead to hearing loss. To investigate these possibilities we screened predicted target genes of the miR-183 miRNA cluster known to be expressed in the inner ear sensory epithelium. In one Iranian family segregating autosomal recessive non-syndromic hearing loss (ARNSHL), we identified a homozygous variant in a predicted miR-96/182 binding site in the 3'UTR of the RDX (DFNB24) gene. However, in vitro functional studies showed that this site is not a functional target for miR-96/182. We extended our study to include the miR-183 genes themselves and 24 additional predicted target genes of the miRNA-183 cluster. Screening these miRNAs and target sequences in numerous families segregating either autosomal dominant non-syndromic deafness (ADNSHL) or ARNSHL did not identify any potential deafness-causing mutations. These results suggest that mutations disrupting gene regulation by the miR-183 cluster are not a common cause of human hearing loss.


Assuntos
Surdez/genética , MicroRNAs/genética , Mutação , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Sítios de Ligação/genética , Proteínas do Citoesqueleto/genética , Primers do DNA/genética , Feminino , Genes Dominantes , Genes Recessivos , Homozigoto , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Família Multigênica , Linhagem , Homologia de Sequência do Ácido Nucleico
12.
Cancer Biol Ther ; 6(8): 1284-8, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17660710

RESUMO

MicroRNAs (miRNAs), an important class of small regulatory molecules for gene expression, are transcribed by RNA polymerase II. But little is known about the mechanisms that control miRNA expression. Comparing miRNA expression profiles between colon cancer cell line HCT 116 and its derivative, DNA methyltransferase 1 and 3b (DNMT1 and DNMT3b) double knockout cell line, we found that the expression of about 10% miRNAs was regulated by DNA methylation. In addition, neither 5-aza-2'-deoxycytidine treatment nor deletion of DNMT1 alone recapitulated miRNA expression profile seen in the double knockout cell line, suggesting that miRNA expression was tightly controlled by DNA methylation and partial methylation reduction was not sufficient for miRNA reexpression. We also found that HOXA3 and HOXD10 were putative targets of mir-10a, one of the differentially expressed miRNAs that is located in HOX gene cluster.


Assuntos
Neoplasias do Colo/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , MicroRNAs/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/metabolismo , Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Fatores de Transcrição/genética
13.
J Biol Chem ; 282(34): 25053-66, 2007 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-17597072

RESUMO

Although microRNAs (miRNAs) provide a newly recognized level of regulation of gene expression, the miRNA transcriptome of the retina and the contributions of miRNAs to retinal development and function are largely unknown. To begin to understand the functions of miRNAs in retina, we compared miRNA expression profiles in adult mouse retina, brain, and heart by microarray analysis. Our results show that at least 78 miRNAs are expressed in adult mouse retina, 21 of which are potentially retina-specific. Among these, we identified a polycistronic, sensory organ-specific paralogous miRNA cluster that includes miR-96, miR-182, and miR-183 on mouse chromosome 6qA3 with conservation of synteny to human chromosome 7q32.2. In situ hybridization showed that members of this cluster are expressed in photoreceptors, retinal bipolar and amacrine cells. Consistent with their genomic organization, these miRNAs have a similar expression pattern during development with abundance increasing postnatally and peaking in adult retina. Target prediction and in vitro functional studies showed that MITF, a transcription factor required for the establishment and maintenance of retinal pigmented epithelium, is a direct target of miR-96 and miR-182. Additionally, to identify miRNAs potentially involved in circadian rhythm regulation of the retina, we performed miRNA expression profiling with retinal RNA harvested at noon (Zeitgeber time 5) and midnight (Zeitgeber time 17) and identified a subgroup of 12 miRNAs, including members of the miR-183/96/182 cluster with diurnal variation in expression pattern. Our results suggest that miR-96 and miR-182 are involved in circadian rhythm regulation, perhaps by modulating the expression of adenylyl cyclase VI (ADCY6).


Assuntos
Regulação da Expressão Gênica , MicroRNAs , Retina/metabolismo , Adenilil Ciclases/metabolismo , Animais , Sequência de Bases , Ritmo Circadiano , Perfilação da Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Células Receptoras Sensoriais/metabolismo , Homologia de Sequência do Ácido Nucleico , Distribuição Tecidual , Transcrição Genética
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