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1.
Genome Res ; 29(7): 1144-1151, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31235655

RESUMO

Recent advances in DNA sequencing have expanded our understanding of the molecular basis of genetic disorders and increased the utilization of clinical genomic tests. Given the paucity of evidence to accurately classify each variant and the difficulty of experimentally evaluating its clinical significance, a large number of variants generated by clinical tests are reported as variants of unknown clinical significance. Population-scale variant databases can improve clinical interpretation. Specifically, pathogenicity prediction for novel missense variants can use features describing regional variant constraint. Constrained genomic regions are those that have an unusually low variant count in the general population. Computational methods have been introduced to capture these regions and incorporate them into pathogenicity classifiers, but these methods have yet to be compared on an independent clinical variant data set. Here, we introduce one variant data set derived from clinical sequencing panels and use it to compare the ability of different genomic constraint metrics to determine missense variant pathogenicity. This data set is compiled from 17,071 patients surveyed with clinical genomic sequencing for cardiomyopathy, epilepsy, or RASopathies. We further use this data set to demonstrate the necessity of disease-specific classifiers and to train PathoPredictor, a disease-specific ensemble classifier of pathogenicity based on regional constraint and variant-level features. PathoPredictor achieves an average precision >90% for variants from all 99 tested disease genes while approaching 100% accuracy for some genes. The accumulation of larger clinical variant training data sets can significantly enhance their performance in a disease- and gene-specific manner.

2.
Epilepsia ; 59(5): 1062-1071, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29655203

RESUMO

OBJECTIVE: We evaluated >8500 consecutive, unselected patients with epilepsy and neurodevelopmental disorders who underwent multigene panel testing to determine the average age at molecular diagnosis and diagnostic yield of 70 genes. METHODS: We reviewed molecular test results for 70 genes known to cause epilepsy and neurodevelopmental disorders using next generation sequencing (NGS) and exon-level array comparative genomic hybridization (aCGH). A positive result was defined as the presence of 1 or 2 pathogenic or likely pathogenic (P/LP) variants in a single gene, depending on the mode of inheritance of the associated disorder. RESULTS: Overall, 22 genes were found to have a high yield of positive findings by genetic testing, with SCN1A and KCNQ2 accounting for the greatest number of positive findings. In contrast, there were no positive findings in 16 genes. Most of the P/LP variants were sequence changes identified by NGS (90.9%), whereas ~9% were gross deletions or duplications detected by exon-level aCGH. The mean age of molecular diagnosis for the cohort was 5 years, 8 months (ranging from 1 week to 47 years). Recurrent P/LP variants were observed in 14 distinct genes, most commonly in MECP2, KCNQ2, SCN1A, SCN2A, STXBP1, and PRRT2. Parental testing was performed in >30% of positive cases. All variants identified in CDKL5, STXBP1, SCN8A, GABRA1, and FOXG1 were de novo, whereas 85.7% of variants in PRRT2 were inherited. SIGNIFICANCE: Using a combined approach of NGS and exon-level aCGH, testing identified a genetic etiology in 15.4% of patients in this cohort and revealed the age at molecular diagnosis for patients. Our study highlights both high- and low-yield genes associated with epilepsy and neurodevelopmental disorders, indicating which genes may be considered for molecular diagnostic testing.

3.
Genet Med ; 20(4): 403-410, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28837158

RESUMO

PurposeMosaicism probably represents an underreported cause of genetic disorders due to detection challenges during routine molecular diagnostics. The purpose of this study was to evaluate the frequency of mosaicism detected by next-generation sequencing in genes associated with epilepsy-related neurodevelopmental disorders.MethodsWe conducted a retrospective analysis of 893 probands with epilepsy who had a multigene epilepsy panel or whole-exome sequencing performed in a clinical diagnostic laboratory and were positive for a pathogenic or likely pathogenic variant in one of nine genes (CDKL5, GABRA1, GABRG2, GRIN2B, KCNQ2, MECP2, PCDH19, SCN1A, or SCN2A). Parental results were available for 395 of these probands.ResultsMosaicism was most common in the CDKL5, PCDH19, SCN2A, and SCN1A genes. Mosaicism was observed in GABRA1, GABRG2, and GRIN2B, which previously have not been reported to have mosaicism, and also in KCNQ2 and MECP2. Parental mosaicism was observed for pathogenic variants in multiple genes including KCNQ2, MECP2, SCN1A, and SCN2A.ConclusionMosaic pathogenic variants were identified frequently in nine genes associated with various neurological conditions. Given the potential clinical ramifications, our findings suggest that next-generation sequencing diagnostic methods may be utilized when testing these genes in a diagnostic laboratory.


Assuntos
Epilepsia/genética , Frequência do Gene , Predisposição Genética para Doença , Variação Genética , Mosaicismo , Transtornos do Neurodesenvolvimento/genética , Alelos , Substituição de Aminoácidos , Epilepsia/diagnóstico , Estudos de Associação Genética , Testes Genéticos , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Transtornos do Neurodesenvolvimento/diagnóstico , Pais , Sequenciamento Completo do Exoma
4.
Am J Med Genet A ; 170(6): 1573-9, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27028100

RESUMO

Thanatophoric dysplasia is a type of short-limbed neonatal dwarfism that is usually lethal in the perinatal period. It is characterized by short limbs, a narrow, bell-shaped thorax, macrocephaly with a prominent forehead, and flattened vertebral bodies. These malformations result from autosomal dominant mutations in the fibroblast growth factor receptor 3 (FGFR3) gene. In this report, we describe a novel FGFR3 insertion mutation in a fetus with shortened limbs, curved femurs, and a narrow thorax. The diagnosis of thanatophoric dysplasia type 1 was suspected clinically, and FGFR3 sequencing showed a c.742_743insTGT variant, which predicts p.R248delinsLC. In vivo studies in zebrafish demonstrated that this mutation resulted in the overexpression of zebrafish Fgfr3, leading to the over-activation of downstream signaling and dorsalized embryos. To date, no insertions or deletions in FGFR3 have been reported to cause thanatophoric dysplasia types 1 or 2; therefore, this represents the first report to describe such a mutation. © 2016 Wiley Periodicals, Inc.


Assuntos
Estudos de Associação Genética , Mutagênese Insercional , Fenótipo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/deficiência , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Displasia Tanatofórica/diagnóstico , Displasia Tanatofórica/genética , Aborto Induzido , Alelos , Animais , Autopsia , Éxons , Feminino , Feto , Expressão Gênica , Genótipo , Humanos , Mutação , Gravidez , Peixe-Zebra
5.
PLoS Genet ; 11(5): e1005221, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25978409

RESUMO

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor ß (TGFß) superfamily of secreted molecules. BMPs play essential roles in multiple developmental and homeostatic processes in metazoans. Malfunction of the BMP pathway can cause a variety of diseases in humans, including cancer, skeletal disorders and cardiovascular diseases. Identification of factors that ensure proper spatiotemporal control of BMP signaling is critical for understanding how this pathway is regulated. We have used a unique and sensitive genetic screen to identify the plasma membrane-localized tetraspanin TSP-21 as a key new factor in the C. elegans BMP-like "Sma/Mab" signaling pathway that controls body size and postembryonic M lineage development. We showed that TSP-21 acts in the signal-receiving cells and genetically functions at the ligand-receptor level. We further showed that TSP-21 can associate with itself and with two additional tetraspanins, TSP-12 and TSP-14, which also promote Sma/Mab signaling. TSP-12 and TSP-14 can also associate with SMA-6, the type I receptor of the Sma/Mab pathway. Finally, we found that glycosphingolipids, major components of the tetraspanin-enriched microdomains, are required for Sma/Mab signaling. Our findings suggest that the tetraspanin-enriched membrane microdomains are important for proper BMP signaling. As tetraspanins have emerged as diagnostic and prognostic markers for tumor progression, and TSP-21, TSP-12 and TSP-14 are all conserved in humans, we speculate that abnormal BMP signaling due to altered expression or function of certain tetraspanins may be a contributing factor to cancer development.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Glicoesfingolipídeos/farmacologia , Transdução de Sinais , Tetraspaninas/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Morfogenéticas Ósseas/genética , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/antagonistas & inibidores , Proteínas de Caenorhabditis elegans/genética , Regulação da Expressão Gênica , Genes Reporter , Marcadores Genéticos , Dados de Sequência Molecular , Mutação , Fenótipo , Sensibilidade e Especificidade , Análise de Sequência de DNA , Tetraspaninas/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo
6.
Nat Commun ; 5: 4734, 2014 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-25178952

RESUMO

Animals need to sense and react to potentially dangerous environments. TRP ion channels participate in nociception, presumably via Ca(2+) influx, in most animal species. However, the relationship between ion permeation and animals' nocifensive behaviour is unknown. Here we use an invertebrate animal model with relevance for mammalian pain. We analyse the putative selectivity filter of OSM-9, a TRPV channel, in osmotic avoidance behaviour of Caenorhabditis elegans. Using mutagenized OSM-9 expressed in the head nociceptor neuron, ASH, we study nocifensive behaviour and Ca(2+) influx. Within the selectivity filter, M(601)-F(609), Y604G strongly reduces avoidance behaviour and eliminates Ca(2+) transients. Y604F also abolishes Ca(2+) transients in ASH, while sustaining avoidance behaviour, yet it disrupts behavioral plasticity. Homology modelling of the OSM-9 pore suggests that Y(604) may assume a scaffolding role. Thus, aromatic residues in the OSM-9 selectivity filter are critical for pain behaviour and ion permeation. These findings have relevance for understanding evolutionary roots of mammalian nociception.


Assuntos
Aprendizagem da Esquiva/fisiologia , Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/fisiologia , Cálcio/metabolismo , Proteínas do Tecido Nervoso/química , Nociceptividade/fisiologia , Nociceptores/metabolismo , Canais de Cátion TRPV/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sinalização do Cálcio , Expressão Gênica , Transporte de Íons , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Nociceptores/citologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo
7.
Am J Med Genet A ; 164A(9): 2391-7, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24924585

RESUMO

Cenani-Lenz syndrome (CLS) is an autosomal recessive skeletal dysplasia that results in malformations of the distal limb, renal anomalies, and characteristic facies. In 2010, this condition was found to be caused by mutations in LRP4, a member of the low-density lipoprotein family of receptors. LRP4 has been shown to antagonize LRP5/LRP6 activation of WNT and ß-catenin signaling. Loss of LRP4 function leads to excessive Wnt and ß-catenin signaling in the limb bud, which causes abnormal limb development. The large majority of patients with CLS reported in the literature have splicing and missense mutations, which result in syndactyly, oligodactyly, and minor renal malformations. More recently, a patient with CLS has been identified with a homozygous nonsense mutation and a more severe presentation of findings typically associated with this condition. Here we present two sibling fetuses with a prenatal lethal presentation of mesomelic limb reductions, oligosyndactyly, genitourinary malformation and compound heterozygosity for two novel truncating mutations in LRP4. These findings lend further support to the CLS genotype-phenotype correlation presented in recent publications.


Assuntos
Feto/anormalidades , Proteínas Relacionadas a Receptor de LDL/genética , Mutação/genética , Sindactilia/genética , Evolução Fatal , Feminino , Feto/diagnóstico por imagem , Humanos , Masculino , Linhagem , Mudanças Depois da Morte , Radiografia , Irmãos
9.
Development ; 133(15): 2887-96, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16790477

RESUMO

In C. elegans, the Sma/Mab TGFbeta signaling pathway regulates body size and male tail patterning. SMA-9, the C. elegans homolog of Schnurri, has been shown to function as a downstream component to mediate the Sma/Mab TGFbeta signaling pathway in these processes. We have discovered a new role for SMA-9 in dorsoventral patterning of the C. elegans post-embryonic mesoderm, the M lineage. In addition to a small body size, sma-9 mutant animals exhibit a dorsal-to-ventral fate transformation within the M lineage. This M lineage defect of sma-9 mutants is unique in that animals carrying mutations in all other known components of the TGFbeta pathway exhibit no M lineage defects. Surprisingly, mutations in the core components of the Sma/Mab TGFbeta signaling pathway suppressed the M lineage defects of sma-9 mutants without suppressing their body size defects. We show that this suppression specifically happens within the M lineage. Our studies have uncovered an unexpected role of SMA-9 in antagonizing the TGFbeta signaling pathway during mesodermal patterning, suggesting a novel mode of function for the SMA-9/Schnurri family of proteins.


Assuntos
Proteínas de Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/fisiologia , Embrião não Mamífero/fisiologia , Mesoderma/fisiologia , Fatores de Transcrição/fisiologia , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Animais Geneticamente Modificados , Padronização Corporal , Tamanho Corporal , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Deleção de Genes , Transdução de Sinais , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Dedos de Zinco
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