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1.
Nat Commun ; 11(1): 5671, 2020 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-33168804

RESUMO

To establish whether 4-nitroquinoline N-oxide-induced carcinogenesis mirrors the heterogeneity of human oral squamous cell carcinoma (OSCC), we have performed genomic analysis of mouse tongue lesions. The mutational signatures of human and mouse OSCC overlap extensively. Mutational burden is higher in moderate dysplasias and invasive SCCs than in hyperplasias and mild dysplasias, although mutations in p53, Notch1 and Fat1 occur in early lesions. Laminin-α3 mutations are associated with tumour invasiveness and Notch1 mutant tumours have an increased immune infiltrate. Computational modelling of clonal dynamics indicates that high genetic heterogeneity may be a feature of those mild dysplasias that are likely to progress to more aggressive tumours. These studies provide a foundation for exploring OSCC evolution, heterogeneity and progression.


Assuntos
Carcinoma de Células Escamosas/genética , Regulação Neoplásica da Expressão Gênica , Genômica , Neoplasias Bucais/genética , 4-Nitroquinolina-1-Óxido/efeitos adversos , Animais , Caderinas/genética , Carcinogênese/induzido quimicamente , Carcinoma de Células Escamosas/patologia , Modelos Animais de Doenças , Progressão da Doença , Exoma/genética , Genes Neoplásicos , Genes p53/genética , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Bucais/patologia , Mutação , Invasividade Neoplásica , Receptor Notch1/genética
2.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 37(11): 1205-1212, 2020 Nov 10.
Artigo em Chinês | MEDLINE | ID: mdl-33179222

RESUMO

With the rapid development and adaptation of high-throughput sequencing in clinical settings, application of exome sequencing (ES) has been gradually expanded from pediatric to prenatal diagnosis in recent years. There is an urgent need to establish criteria for clinical grade ES in order to facilitate such a complex testing. The standardization of pre- and post-test consultation, quality control for sample processing process and validation of bioinformatics data analysis, and more importantly data interpretation and reporting, as well as appropriate reporting scope, is of great importance for health care stakeholders. To achieve this, a committee composed of a wide range of healthcare professionals has proposed an ES standard for prenatal diagnosis. This has provided expert opinion on the genetic counseling and reporting standards of prenatal ES for the purpose of applying ES technology in prenatal setting.


Assuntos
Exoma , Diagnóstico Pré-Natal , Sequenciamento Completo do Exoma , Consenso , Exoma/genética , Feminino , Aconselhamento Genético , Testes Genéticos , Humanos , Gravidez
3.
Nat Commun ; 11(1): 5183, 2020 10 14.
Artigo em Inglês | MEDLINE | ID: mdl-33056981

RESUMO

Neuroblastoma is a pediatric malignancy with heterogeneous clinical outcomes. To better understand neuroblastoma pathogenesis, here we analyze whole-genome, whole-exome and/or transcriptome data from 702 neuroblastoma samples. Forty percent of samples harbor at least one recurrent driver gene alteration and most aberrations, including MYCN, ATRX, and TERT alterations, differ in frequency by age. MYCN alterations occur at median 2.3 years of age, TERT at 3.8 years, and ATRX at 5.6 years. COSMIC mutational signature 18, previously associated with reactive oxygen species, is the most common cause of driver point mutations in neuroblastoma, including most ALK and Ras-activating variants. Signature 18 appears early and is continuous throughout disease evolution. Signature 18 is enriched in neuroblastomas with MYCN amplification, 17q gain, and increased expression of mitochondrial ribosome and electron transport-associated genes. Recurrent FGFR1 variants in six patients, and ALK N-terminal structural alterations in five samples, identify additional patients potentially amenable to precision therapy.


Assuntos
Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica , Neuroblastoma/genética , Adolescente , Adulto , Fatores Etários , Quinase do Linfoma Anaplásico/genética , Criança , Pré-Escolar , Estudos de Coortes , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Conjuntos de Dados como Assunto , Transporte de Elétrons/genética , Exoma/genética , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Ribossomos Mitocondriais , Mutação , Neuroblastoma/patologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Proteínas Ribossômicas/genética , Transcriptoma/genética , Sequenciamento Completo do Genoma , Adulto Jovem
4.
Lancet Neurol ; 19(11): 908-918, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33098801

RESUMO

BACKGROUND: Dystonia is a clinically and genetically heterogeneous condition that occurs in isolation (isolated dystonia), in combination with other movement disorders (combined dystonia), or in the context of multisymptomatic phenotypes (isolated or combined dystonia with other neurological involvement). However, our understanding of its aetiology is still incomplete. We aimed to elucidate the monogenic causes for the major clinical categories of dystonia. METHODS: For this exome-wide sequencing study, study participants were identified at 33 movement-disorder and neuropaediatric specialty centres in Austria, Czech Republic, France, Germany, Poland, Slovakia, and Switzerland. Each individual with dystonia was diagnosed in accordance with the dystonia consensus definition. Index cases were eligible for this study if they had no previous genetic diagnosis and no indication of an acquired cause of their illness. The second criterion was not applied to a subset of participants with a working clinical diagnosis of dystonic cerebral palsy. Genomic DNA was extracted from blood of participants and whole-exome sequenced. To find causative variants in known disorder-associated genes, all variants were filtered, and unreported variants were classified according to American College of Medical Genetics and Genomics guidelines. All considered variants were reviewed in expert round-table sessions to validate their clinical significance. Variants that survived filtering and interpretation procedures were defined as diagnostic variants. In the cases that went undiagnosed, candidate dystonia-causing genes were prioritised in a stepwise workflow. FINDINGS: We sequenced the exomes of 764 individuals with dystonia and 346 healthy parents who were recruited between June 1, 2015, and July 31, 2019. We identified causative or probable causative variants in 135 (19%) of 728 families, involving 78 distinct monogenic disorders. We observed a larger proportion of individuals with diagnostic variants in those with dystonia (either isolated or combined) with coexisting non-movement disorder-related neurological symptoms (100 [45%] of 222; excepting cases with evidence of perinatal brain injury) than in those with combined (19 [19%] of 98) or isolated (16 [4%] of 388) dystonia. Across all categories of dystonia, 104 (65%) of the 160 detected variants affected genes which are associated with neurodevelopmental disorders. We found diagnostic variants in 11 genes not previously linked to dystonia, and propose a predictive clinical score that could guide the implementation of exome sequencing in routine diagnostics. In cases without perinatal sentinel events, genomic alterations contributed substantively to the diagnosis of dystonic cerebral palsy. In 15 families, we delineated 12 candidate genes. These include IMPDH2, encoding a key purine biosynthetic enzyme, for which robust evidence existed for its involvement in a neurodevelopmental disorder with dystonia. We identified six variants in IMPDH2, collected from four independent cohorts, that were predicted to be deleterious de-novo variants and expected to result in deregulation of purine metabolism. INTERPRETATION: In this study, we have determined the role of monogenic variants across the range of dystonic disorders, providing guidance for the introduction of personalised care strategies and fostering follow-up pathophysiological explorations. FUNDING: Else Kröner-Fresenius-Stiftung, Technische Universität München, Helmholtz Zentrum München, Medizinische Universität Innsbruck, Charles University in Prague, Czech Ministry of Education, the Slovak Grant and Development Agency, the Slovak Research and Grant Agency.


Assuntos
Distonia/diagnóstico , Distonia/genética , Exoma/genética , Variação Genética/genética , Sequenciamento Completo do Exoma/métodos , Adolescente , Criança , Pré-Escolar , Distonia/epidemiologia , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Linhagem , Adulto Jovem
5.
PLoS Genet ; 16(9): e1009010, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32956375

RESUMO

Essential tremor (ET) is the most common adult-onset movement disorder. In the present study, we performed whole exome sequencing of a large ET-affected family (10 affected and 6 un-affected family members) and identified a TUB p.V431I variant (rs75594955) segregating in a manner consistent with autosomal-dominant inheritance. Subsequent targeted re-sequencing of TUB in 820 unrelated individuals with sporadic ET and 630 controls revealed significant enrichment of rare nonsynonymous TUB variants (e.g. rs75594955: p.V431I, rs1241709665: p.Ile20Phe, rs55648406: p.Arg49Gln) in the ET cohort (SKAT-O test p-value = 6.20e-08). TUB encodes a transcription factor predominantly expressed in neuronal cells and has been previously implicated in obesity. ChIP-seq analyses of the TUB transcription factor across different regions of the mouse brain revealed that TUB regulates the pathways responsible for neurotransmitter production as well thyroid hormone signaling. Together, these results support the association of rare variants in TUB with ET.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Tremor Essencial/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Sequenciamento de Cromatina por Imunoprecipitação/métodos , Estudos de Coortes , Exoma/genética , Família , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Linhagem , Polimorfismo de Nucleotídeo Único/genética , Fatores de Transcrição/genética , Sequenciamento Completo do Exoma/métodos
6.
Nat Commun ; 11(1): 4748, 2020 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-32958763

RESUMO

The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts.


Assuntos
Genoma Humano/genética , Mutação , Neoplasias/genética , Composição de Bases , DNA Intergênico , Bases de Dados Genéticas , Exoma/genética , Éxons , Humanos , Estudos Retrospectivos , Sequenciamento Completo do Exoma , Sequenciamento Completo do Genoma
7.
Nat Commun ; 11(1): 4085, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32796935

RESUMO

Intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs) are non-invasive neoplasms that are often observed in association with invasive pancreatic cancers, but their origins and evolutionary relationships are poorly understood. In this study, we analyze 148 samples from IPMNs, MCNs, and small associated invasive carcinomas from 18 patients using whole exome or targeted sequencing. Using evolutionary analyses, we establish that both IPMNs and MCNs are direct precursors to pancreatic cancer. Mutations in SMAD4 and TGFBR2 are frequently restricted to invasive carcinoma, while RNF43 alterations are largely in non-invasive lesions. Genomic analyses suggest an average window of over three years between the development of high-grade dysplasia and pancreatic cancer. Taken together, these data establish non-invasive IPMNs and MCNs as origins of invasive pancreatic cancer, identifying potential drivers of invasion, highlighting the complex clonal dynamics prior to malignant transformation, and providing opportunities for early detection and intervention.


Assuntos
Progressão da Doença , Genômica , Cisto Pancreático/genética , Neoplasias Pancreáticas/genética , Carcinogênese/genética , Transformação Celular Neoplásica/genética , Exoma/genética , Dosagem de Genes , Humanos , Mutação , Cisto Pancreático/patologia , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Proteína Smad4/genética
8.
Am J Hum Genet ; 107(3): 403-417, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32755546

RESUMO

Human Phenotype Ontology (HPO)-based analysis has become standard for genomic diagnostics of rare diseases. Current algorithms use a variety of semantic and statistical approaches to prioritize the typically long lists of genes with candidate pathogenic variants. These algorithms do not provide robust estimates of the strength of the predictions beyond the placement in a ranked list, nor do they provide measures of how much any individual phenotypic observation has contributed to the prioritization result. However, given that the overall success rate of genomic diagnostics is only around 25%-50% or less in many cohorts, a good ranking cannot be taken to imply that the gene or disease at rank one is necessarily a good candidate. Here, we present an approach to genomic diagnostics that exploits the likelihood ratio (LR) framework to provide an estimate of (1) the posttest probability of candidate diagnoses, (2) the LR for each observed HPO phenotype, and (3) the predicted pathogenicity of observed genotypes. LIkelihood Ratio Interpretation of Clinical AbnormaLities (LIRICAL) placed the correct diagnosis within the first three ranks in 92.9% of 384 case reports comprising 262 Mendelian diseases, and the correct diagnosis had a mean posttest probability of 67.3%. Simulations show that LIRICAL is robust to many typically encountered forms of genomic and phenomic noise. In summary, LIRICAL provides accurate, clinically interpretable results for phenotype-driven genomic diagnostics.


Assuntos
Biologia Computacional , Bases de Dados Genéticas , Genômica , Doenças Raras/diagnóstico , Algoritmos , Exoma/genética , Humanos , Fenótipo , Doenças Raras/genética , Software
9.
Nat Med ; 26(9): 1392-1397, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32778825

RESUMO

Public health newborn screening (NBS) programs provide population-scale ascertainment of rare, treatable conditions that require urgent intervention. Tandem mass spectrometry (MS/MS) is currently used to screen newborns for a panel of rare inborn errors of metabolism (IEMs)1-4. The NBSeq project evaluated whole-exome sequencing (WES) as an innovative methodology for NBS. We obtained archived residual dried blood spots and data for nearly all IEM cases from the 4.5 million infants born in California between mid-2005 and 2013 and from some infants who screened positive by MS/MS, but were unaffected upon follow-up testing. WES had an overall sensitivity of 88% and specificity of 98.4%, compared to 99.0% and 99.8%, respectively for MS/MS, although effectiveness varied among individual IEMs. Thus, WES alone was insufficiently sensitive or specific to be a primary screen for most NBS IEMs. However, as a secondary test for infants with abnormal MS/MS screens, WES could reduce false-positive results, facilitate timely case resolution and in some instances even suggest more appropriate or specific diagnosis than that initially obtained. This study represents the largest, to date, sequencing effort of an entire population of IEM-affected cases, allowing unbiased assessment of current capabilities of WES as a tool for population screening.


Assuntos
Exoma/genética , Erros Inatos do Metabolismo/diagnóstico , Erros Inatos do Metabolismo/genética , Triagem Neonatal/métodos , Sequenciamento Completo do Exoma/métodos , Testes Genéticos , Humanos , Recém-Nascido , Erros Inatos do Metabolismo/epidemiologia , Espectrometria de Massas em Tandem
10.
Nat Med ; 26(9): 1375-1379, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32778826

RESUMO

The promise of precision medicine lies in data diversity. More than the sheer size of biomedical data, it is the layering of multiple data modalities, offering complementary perspectives, that is thought to enable the identification of patient subgroups with shared pathophysiology. In the present study, we use autism to test this notion. By combining healthcare claims, electronic health records, familial whole-exome sequences and neurodevelopmental gene expression patterns, we identified a subgroup of patients with dyslipidemia-associated autism.


Assuntos
Transtorno Autístico/diagnóstico , Dislipidemias/diagnóstico , Medicina de Precisão/métodos , Transtorno Autístico/genética , Transtorno Autístico/patologia , Dislipidemias/genética , Dislipidemias/patologia , Registros Eletrônicos de Saúde , Exoma/genética , Feminino , Predisposição Genética para Doença/genética , Humanos , Lipídeos/sangue , Masculino , Técnicas de Diagnóstico Molecular , Sequenciamento Completo do Exoma
11.
Am J Hum Genet ; 107(2): 330-341, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32619401

RESUMO

Sperm malformation is a direct factor for male infertility. Multiple morphological abnormalities of the flagella (MMAF), a severe form of asthenoteratozoospermia, are characterized by immotile spermatozoa with malformed and/or absent flagella in the ejaculate. Previous studies indicated genetic heterogeneity in MMAF. To further define genetic factors underlying MMAF, we performed whole-exome sequencing in a cohort of 90 Chinese MMAF-affected men. Two cases (2.2%) were identified as carrying bi-allelic missense DNAH8 variants, variants which were either absent or rare in the control human population and were predicted to be deleterious by multiple bioinformatic tools. Re-analysis of exome data from a second cohort of 167 MMAF-affected men from France, Iran, and North Africa permitted the identification of an additional male carrying a DNAH8 homozygous frameshift variant. DNAH8 encodes a dynein axonemal heavy-chain component that is expressed preferentially in the testis. Hematoxylin-eosin staining and electron microscopy analyses of the spermatozoa from men harboring bi-allelic DNAH8 variants showed a highly aberrant morphology and ultrastructure of the sperm flagella. Immunofluorescence assays performed on the spermatozoa from men harboring bi-allelic DNAH8 variants revealed the absent or markedly reduced staining of DNAH8 and its associated protein DNAH17. Dnah8-knockout male mice also presented typical MMAF phenotypes and sterility. Interestingly, intracytoplasmic sperm injections using the spermatozoa from Dnah8-knockout male mice resulted in good pregnancy outcomes. Collectively, our experimental observations from humans and mice demonstrate that DNAH8 is essential for sperm flagellar formation and that bi-allelic deleterious DNAH8 variants lead to male infertility with MMAF.


Assuntos
Anormalidades Múltiplas/genética , Dineínas do Axonema/genética , Flagelos/genética , Variação Genética/genética , Infertilidade Masculina/genética , Cauda do Espermatozoide/patologia , Alelos , Animais , Estudos de Coortes , Exoma/genética , Feminino , Homozigoto , Humanos , Masculino , Camundongos , Camundongos Knockout , Espermatozoides/anormalidades , Testículo/anormalidades , Sequenciamento Completo do Exoma/métodos
12.
Am J Hum Genet ; 107(2): 251-264, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32640185

RESUMO

Applying exome sequencing to populations with unique genetic architecture has the potential to reveal novel genes and variants associated with traits and diseases. We sequenced and analyzed the exomes of 6,716 individuals from a Southwestern American Indian (SWAI) population with well-characterized metabolic traits. We found that the SWAI population has distinct allelic architecture compared to populations of European and East Asian ancestry, and there were many predicted loss-of-function (pLOF) and nonsynonymous variants that were highly enriched or private in the SWAI population. We used pLOF and nonsynonymous variants in the SWAI population to evaluate gene-burden associations of candidate genes from European genome-wide association studies (GWASs) for type 2 diabetes, body mass index, and four major plasma lipids. We found 19 significant gene-burden associations for 11 genes, providing additional evidence for prioritizing candidate effector genes of GWAS signals. Interestingly, these associations were mainly driven by pLOF and nonsynonymous variants that are unique or highly enriched in the SWAI population. Particularly, we found four pLOF or nonsynonymous variants in APOB, APOE, PCSK9, and TM6SF2 that are private or enriched in the SWAI population and associated with low-density lipoprotein (LDL) cholesterol levels. Their large estimated effects on LDL cholesterol levels suggest strong impacts on protein function and potential clinical implications of these variants in cardiovascular health. In summary, our study illustrates the utility and potential of exome sequencing in genetically unique populations, such as the SWAI population, to prioritize candidate effector genes within GWAS loci and to find additional variants in known disease genes with potential clinical impact.


Assuntos
Exoma/genética , Predisposição Genética para Doença/genética , Índios Norte-Americanos/genética , Polimorfismo de Nucleotídeo Único/genética , Alelos , Índice de Massa Corporal , Feminino , Genética Populacional/métodos , Estudo de Associação Genômica Ampla/métodos , Humanos , Masculino , Fenótipo , Sudoeste dos Estados Unidos
13.
Clin Chem ; 66(1): 199-206, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32609854

RESUMO

BACKGROUND: Exome sequencing has become a commonly used clinical diagnostic test. Multiple studies have examined the diagnostic utility and individual laboratory performance of exome testing; however, no previous study has surveyed and compared the data quality from multiple clinical laboratories. METHODS: We examined sequencing data from 36 clinical exome tests from 3 clinical laboratories. Exome data were compared in terms of overall characteristics and coverage of specific genes and nucleotide positions. The sets of genes examined included genes in Consensus Coding Sequence (CCDS) (n = 17723), a subset of genes clinically relevant to epilepsy (n = 108), and genes that are recommended for reporting of secondary findings (n = 57; excludes X-linked genes). RESULTS: The average exome nucleotide coverage (≥20×) of each laboratory varied at 96.49% (CV = 3%), 96.54% (CV = 1%), and 91.68% (CV = 4%), for laboratories A, B, and C, respectively. For CCDS genes, the average number of completely covered genes varied at 12184 (CV = 29%), 11687 (CV = 13%), and 5989 (CV = 37%), for laboratories A, B, and C, respectively. With smaller subsets of genes related to epilepsy and secondary findings, the CV revealed low consistency, with a maximum CV seen in laboratory C for both epilepsy genes (CV = 60%) and secondary findings genes (CV = 71%). CONCLUSIONS: Poor consistency in complete gene coverage was seen in the clinical exome laboratories surveyed. The degree of consistency varied widely between the laboratories.


Assuntos
Exoma/genética , Proteína BRCA1/genética , Epilepsia/genética , Epilepsia/patologia , Éxons , Guias como Assunto , Humanos , Laboratórios Hospitalares/normas , Proteína 1 Homóloga a MutL/genética , Sequenciamento Completo do Exoma
14.
Proc Natl Acad Sci U S A ; 117(32): 19367-19375, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719112

RESUMO

Whole-exome sequencing (WES) has facilitated the discovery of genetic lesions underlying monogenic disorders. Incomplete penetrance and variable expressivity suggest a contribution of additional genetic lesions to clinical manifestations and outcome. Some monogenic disorders may therefore actually be digenic. However, only a few digenic disorders have been reported, all discovered by candidate gene approaches applied to at least one locus. We propose here a two-locus genome-wide test for detecting digenic inheritance in WES data. This approach uses the gene as the unit of analysis and tests all pairs of genes to detect pairwise gene × gene interactions underlying disease. It is a case-only method, which has several advantages over classic case-control tests, in particular by avoiding recruitment of controls. Our simulation studies based on real WES data identified two major sources of type I error inflation in this case-only test: linkage disequilibrium and population stratification. Both were corrected by specific procedures. Moreover, our case-only approach is more powerful than the corresponding case-control test for detecting digenic interactions in various population stratification scenarios. Finally, we confirmed the potential of our unbiased, genome-wide approach by successfully identifying a previously reported digenic lesion in patients with craniosynostosis. Our case-only test is a powerful and timely tool for detecting digenic inheritance in WES data from patients.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Herança Multifatorial , Sequenciamento Completo do Exoma/métodos , Craniossinostoses/genética , Epistasia Genética , Exoma/genética , Ligação Genética , Variação Genética , Estudo de Associação Genômica Ampla , Humanos , Modelos Genéticos
15.
PLoS One ; 15(6): e0234246, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32502225

RESUMO

INTRODUCTION: Esophageal atresia with or without tracheoesophageal fistula (EA/TEF) occurs approximately 1 in 3.500 live births representing the most common malformation of the upper digestive tract. Only half a century ago, EA/TEF was fatal among affected newborns suggesting that the steady birth prevalence might in parts be due to mutational de novo events in genes involved in foregut development. METHODS: To identify mutational de novo events in EA/TEF patients, we surveyed the exome of 30 case-parent trios. Identified and confirmed de novo variants were prioritized using in silico prediction tools. To investigate the embryonic role of genes harboring prioritized de novo variants we performed targeted analysis of mouse transcriptome data of esophageal tissue obtained at the embryonic day (E) E8.5, E12.5, and postnatal. RESULTS: In total we prioritized 14 novel de novo variants in 14 different genes (APOL2, EEF1D, CHD7, FANCB, GGT6, KIAA0556, NFX1, NPR2, PIGC, SLC5A2, TANC2, TRPS1, UBA3, and ZFHX3) and eight rare de novo variants in eight additional genes (CELSR1, CLP1, GPR133, HPS3, MTA3, PLEC, STAB1, and PPIP5K2). Through personal communication during the project, we identified an additional EA/TEF case-parent trio with a rare de novo variant in ZFHX3. In silico prediction analysis of the identified variants and comparative analysis of mouse transcriptome data of esophageal tissue obtained at E8.5, E12.5, and postnatal prioritized CHD7, TRPS1, and ZFHX3 as EA/TEF candidate genes. Re-sequencing of ZFHX3 in additional 192 EA/TEF patients did not identify further putative EA/TEF-associated variants. CONCLUSION: Our study suggests that rare mutational de novo events in genes involved in foregut development contribute to the development of EA/TEF.


Assuntos
DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos/metabolismo , Atresia Esofágica/genética , Exoma/genética , Perfilação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas Repressoras/genética , Fístula Traqueoesofágica/genética , Animais , Humanos , Camundongos , Sequenciamento Completo do Exoma
16.
BMC Med Genet ; 21(1): 136, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32590954

RESUMO

BACKGROUND: Alkuraya-Kucinskas syndrome is an autosomal recessive disorder characterized by brain abnormalities associated with cerebral parenchymal underdevelopment, arthrogryposis, club foot and global developmental delay. KIAA1109, a functionally uncharacterized gene is identified as the molecular cause for Alkuraya-Kucinskas syndrome. Most of the reported mutations in KIAA1109 gene result in premature termination of pregnancies or neonatal deaths while a few mutations have been reported in surviving patients with global developmental delay and intellectual disability. To our knowledge, only three surviving patients from two families have been reported with missense variants in KIAA1109. In this study, we describe four surviving patients from two related families (a multiplex family) with global developmental delay and mild to severe intellectual disability with no other systemic manifestations. There were no miscarriages or neonatal deaths reported in these families. METHODS: X-chromosome exome panel sequencing was carried out in one patient and whole exome sequencing was carried out on the remaining three affected individuals and the unaffected father of the index family. Data analysis was carried out followed by variant filtering and segregation analysis. Sanger sequencing was carried out to validate the segregation of mutation in all four affected siblings and unaffected parents from both families. RESULTS: A novel homozygous missense mutation in a conserved region of KIAA1109 protein was identified. Sanger sequencing confirmed the segregation of mutation in both families in an autosomal recessive fashion. CONCLUSION: Our study is the second study reporting a KIAA1109 variant in surviving patients with Alkuraya-Kucinskas syndrome. Our study expands the spectrum of phenotypic features and mutations associated with Alkuraya-Kucinskas syndrome.


Assuntos
Anormalidades Múltiplas/genética , Mutação/genética , Proteínas/genética , Adolescente , Adulto , Sequência de Bases , Criança , Sequência Conservada , Exoma/genética , Feminino , Humanos , Cariótipo , Masculino , Linhagem , Fenótipo , Análise de Sobrevida , Síndrome , Adulto Jovem
17.
Hum Genet ; 139(11): 1429-1441, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32488467

RESUMO

Autozygosity-driven exome analysis has been shown effective for identification of genes underlying recessive diseases especially in countries of the so-called Greater Middle East (GME), where high consanguinity unravels the phenotypic effects of recessive alleles and large family sizes facilitate homozygosity mapping. In Italy, as in most European countries, consanguinity is estimated low. Nonetheless, consanguineous Italian families are not uncommon in publications of genetic findings and are often key to new associations of genes with rare diseases. We collected 52 patients from 47 consanguineous families with suspected recessive diseases, 29 originated in GME countries and 18 of Italian descent. We performed autozygosity-driven exome analysis by detecting long runs of homozygosity (ROHs > 1.5 Mb) and by prioritizing candidate clinical variants within. We identified a pathogenic synonymous variant that had been previously missed in NARS2 and we increased an initial high diagnostic rate (47%) to 55% by matchmaking our candidate genes and including in the analysis shorter ROHs that may also happen to be autozygous. GME and Italian families contributed to diagnostic yield comparably. We found no significant difference either in the extension of the autozygous genome, or in the distribution of candidate clinical variants between GME and Italian families, while we showed that the average autozygous genome was larger and the mean number of candidate clinical variants was significantly higher (p = 0.003) in mutation-positive than in mutation-negative individuals, suggesting that these features influence the likelihood that the disease is autozygosity-related. We highlight the utility of autozygosity-driven genomic analysis also in countries and/or communities, where consanguinity is not widespread cultural tradition.


Assuntos
Testes Genéticos/métodos , Genoma Humano/genética , Mapeamento Cromossômico/métodos , Consanguinidade , Exoma/genética , Família , Feminino , Genes Recessivos/genética , Humanos , Itália , Masculino , Oriente Médio , Mutação/genética , Linhagem
18.
Hum Genet ; 139(11): 1443-1454, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32514796

RESUMO

Dilated cardiomyopathy (DCM) belongs to the most frequent forms of cardiomyopathy mainly characterized by cardiac dilatation and reduced systolic function. Although most cases of DCM are classified as sporadic, 20-30% of cases show a heritable pattern. Familial forms of DCM are genetically heterogeneous, and mutations in several genes have been identified that most commonly play a role in cytoskeleton and sarcomere-associated processes. Still, a large number of familial cases remain unsolved. Here, we report five individuals from three independent families who presented with severe dilated cardiomyopathy during the neonatal period. Using whole-exome sequencing (WES), we identified causative, compound heterozygous missense variants in RPL3L (ribosomal protein L3-like) in all the affected individuals. The identified variants co-segregated with the disease in each of the three families and were absent or very rare in the human population, in line with an autosomal recessive inheritance pattern. They are located within the conserved RPL3 domain of the protein and were classified as deleterious by several in silico prediction software applications. RPL3L is one of the four non-canonical riboprotein genes and it encodes the 60S ribosomal protein L3-like protein that is highly expressed only in cardiac and skeletal muscle. Three-dimensional homology modeling and in silico analysis of the affected residues in RPL3L indicate that the identified changes specifically alter the interaction of RPL3L with the RNA components of the 60S ribosomal subunit and thus destabilize its binding to the 60S subunit. In conclusion, we report that bi-allelic pathogenic variants in RPL3L are causative of an early-onset, severe neonatal form of dilated cardiomyopathy, and we show for the first time that cytoplasmic ribosomal proteins are involved in the pathogenesis of non-syndromic cardiomyopathies.


Assuntos
Cardiomiopatia Dilatada/genética , Mutação de Sentido Incorreto/genética , Proteínas Ribossômicas/genética , Ribossomos/genética , Alelos , Exoma/genética , Feminino , Coração/fisiopatologia , Humanos , Lactente , Recém-Nascido , Masculino , Músculo Esquelético/fisiopatologia , Linhagem , Fenótipo , RNA/genética
19.
Hum Genet ; 139(11): 1471-1483, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32583022

RESUMO

Human growth is a complex trait determined by genetic factors in combination with external stimuli, including environment, nutrition and hormonal status. In the past, several genome-wide association studies (GWAS) have collectively identified hundreds of genetic variants having a putative effect on determining adult height in different worldwide populations. Theoretically, a valuable approach to better understand the mechanisms of complex traits as adult height is to study a population exhibiting extreme stature phenotypes, such as African Baka Pygmies. After phenotypic characterization, we sequenced the whole exomes of a cohort of Baka Pygmies and their non-Pygmies Bantu neighbors to highlight genetic variants associated with the reduced stature. Whole exome data analysis revealed 29 single nucleotide polymorphisms (SNPs) significantly associated with the reduced height in the Baka group. Among these variants, we focused on SNP rs7629425, located in the 5'-UTR of the Hyaluronidase-2 (HYAL2) gene. The frequency of the alternative allele was significantly increased compared to African and non-African populations. In vitro luciferase assay showed significant differences in transcription modulation by rs7629425 C/T alleles. In conclusion, our results suggested that the HYAL2 gene variants may play a role in the etiology of short stature in Baka Pygmies population.


Assuntos
Grupo com Ancestrais do Continente Africano/genética , Moléculas de Adesão Celular/genética , Proteínas Ligadas por GPI/genética , Transtornos do Crescimento/genética , Hialuronoglucosaminidase/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Alelos , Estatura/genética , Exoma/genética , Feminino , Estudo de Associação Genômica Ampla/métodos , Humanos , Masculino
20.
Sci Rep ; 10(1): 9424, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32523024

RESUMO

The exome contains many obscure regions difficult to explore with current short-read sequencing methods. Repetitious genomic regions prevent the unique alignment of reads, which is essential for the identification of clinically-relevant genetic variants. Long-read technologies attempt to resolve multiple-mapping regions, but they still produce many sequencing errors. Thus, a new approach is required to enlighten the obscure regions of the genome and rescue variants that would be otherwise neglected. This work aims to improve the alignment of multiple-mapping reads through the extension of the standard DNA fragment size. As Illumina can sequence fragments up to 550 bp, we tested different DNA fragment lengths using four major commercial WES platforms and found that longer DNA fragments achieved a higher genotypability. This metric, which indicates base calling calculated by combining depth of coverage with the confidence of read alignment, increased from hundreds to thousands of genes, including several associated with clinical phenotypes. While depth of coverage has been considered crucial for the assessment of WES performance, we demonstrated that genotypability has a greater impact in revealing obscure regions, with ~1% increase in variant calling in respect to shorter DNA fragments. Results confirmed that this approach enlightened many regions previously not explored.


Assuntos
DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Algoritmos , Exoma/genética , Genoma Humano/genética , Genômica/métodos , Genótipo , Humanos , Análise de Sequência de DNA/métodos
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