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1.
Nat Med ; 25(6): 911-919, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31160820

RESUMO

It is estimated that 350 million individuals worldwide suffer from rare diseases, which are predominantly caused by mutation in a single gene1. The current molecular diagnostic rate is estimated at 50%, with whole-exome sequencing (WES) among the most successful approaches2-5. For patients in whom WES is uninformative, RNA sequencing (RNA-seq) has shown diagnostic utility in specific tissues and diseases6-8. This includes muscle biopsies from patients with undiagnosed rare muscle disorders6,9, and cultured fibroblasts from patients with mitochondrial disorders7. However, for many individuals, biopsies are not performed for clinical care, and tissues are difficult to access. We sought to assess the utility of RNA-seq from blood as a diagnostic tool for rare diseases of different pathophysiologies. We generated whole-blood RNA-seq from 94 individuals with undiagnosed rare diseases spanning 16 diverse disease categories. We developed a robust approach to compare data from these individuals with large sets of RNA-seq data for controls (n = 1,594 unrelated controls and n = 49 family members) and demonstrated the impacts of expression, splicing, gene and variant filtering strategies on disease gene identification. Across our cohort, we observed that RNA-seq yields a 7.5% diagnostic rate, and an additional 16.7% with improved candidate gene resolution.


Assuntos
Doenças Raras/genética , Ceramidase Ácida/genética , Estudos de Casos e Controles , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Variação Genética , Humanos , Masculino , Modelos Genéticos , Mutação , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Canais de Potássio/genética , RNA/sangue , RNA/genética , Processamento de RNA/genética , Doenças Raras/sangue , Análise de Sequência de RNA , Sequenciamento Completo do Exoma
2.
Artigo em Inglês | MEDLINE | ID: mdl-30559314

RESUMO

High-throughput sequencing has ushered in a diversity of approaches for identifying genetic variants and understanding genome structure and function. When applied to individuals with rare genetic diseases, these approaches have greatly accelerated gene discovery and patient diagnosis. Over the past decade, exome sequencing has emerged as a comprehensive and cost-effective approach to identify pathogenic variants in the protein-coding regions of the genome. However, for individuals in whom exome-sequencing fails to identify a pathogenic variant, we discuss recent advances that are helping to reduce the diagnostic gap.


Assuntos
Testes Genéticos/métodos , Doenças Raras/diagnóstico , Doenças Raras/genética , Biologia Computacional/métodos , Exoma/genética , Predisposição Genética para Doença/genética , Variação Genética/genética , Estudo de Associação Genômica Ampla , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/tendências , Humanos , Análise de Sequência de DNA/métodos , Sequenciamento Completo do Exoma/métodos
3.
Am J Hum Genet ; 102(3): 494-504, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29478781

RESUMO

ATP synthase, H+ transporting, mitochondrial F1 complex, δ subunit (ATP5F1D; formerly ATP5D) is a subunit of mitochondrial ATP synthase and plays an important role in coupling proton translocation and ATP production. Here, we describe two individuals, each with homozygous missense variants in ATP5F1D, who presented with episodic lethargy, metabolic acidosis, 3-methylglutaconic aciduria, and hyperammonemia. Subject 1, homozygous for c.245C>T (p.Pro82Leu), presented with recurrent metabolic decompensation starting in the neonatal period, and subject 2, homozygous for c.317T>G (p.Val106Gly), presented with acute encephalopathy in childhood. Cultured skin fibroblasts from these individuals exhibited impaired assembly of F1FO ATP synthase and subsequent reduced complex V activity. Cells from subject 1 also exhibited a significant decrease in mitochondrial cristae. Knockdown of Drosophila ATPsynδ, the ATP5F1D homolog, in developing eyes and brains caused a near complete loss of the fly head, a phenotype that was fully rescued by wild-type human ATP5F1D. In contrast, expression of the ATP5F1D c.245C>T and c.317T>G variants rescued the head-size phenotype but recapitulated the eye and antennae defects seen in other genetic models of mitochondrial oxidative phosphorylation deficiency. Our data establish c.245C>T (p.Pro82Leu) and c.317T>G (p.Val106Gly) in ATP5F1D as pathogenic variants leading to a Mendelian mitochondrial disease featuring episodic metabolic decompensation.

4.
Genet Med ; 20(1): 159-163, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28640241

RESUMO

PurposeCurrent clinical genomics assays primarily utilize short-read sequencing (SRS), but SRS has limited ability to evaluate repetitive regions and structural variants. Long-read sequencing (LRS) has complementary strengths, and we aimed to determine whether LRS could offer a means to identify overlooked genetic variation in patients undiagnosed by SRS.MethodsWe performed low-coverage genome LRS to identify structural variants in a patient who presented with multiple neoplasia and cardiac myxomata, in whom the results of targeted clinical testing and genome SRS were negative.ResultsThis LRS approach yielded 6,971 deletions and 6,821 insertions > 50 bp. Filtering for variants that are absent in an unrelated control and overlap a disease gene coding exon identified three deletions and three insertions. One of these, a heterozygous 2,184 bp deletion, overlaps the first coding exon of PRKAR1A, which is implicated in autosomal dominant Carney complex. RNA sequencing demonstrated decreased PRKAR1A expression. The deletion was classified as pathogenic based on guidelines for interpretation of sequence variants.ConclusionThis first successful application of genome LRS to identify a pathogenic variant in a patient suggests that LRS has significant potential for the identification of disease-causing structural variation. Larger studies will ultimately be required to evaluate the potential clinical utility of LRS.


Assuntos
Estudos de Associação Genética , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/genética , Predisposição Genética para Doença , Variação Genética , Genoma Humano , Genômica , Análise de Sequência de DNA , Criança , Subunidade RIalfa da Proteína Quinase Dependente de AMP Cíclico/genética , Ecocardiografia , Genômica/métodos , Humanos , Masculino , Fenótipo , Análise de Sequência de DNA/métodos , Deleção de Sequência
5.
Hum Mutat ; 38(6): 611-614, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28251733

RESUMO

At least 15% of the disease-causing mutations affect mRNA splicing. Many splicing mutations are missed in a clinical setting due to limitations of in silico prediction algorithms or their location in noncoding regions. Whole-transcriptome sequencing is a promising new tool to identify these mutations; however, it will be a challenge to obtain disease-relevant tissue for RNA. Here, we describe an individual with a sporadic atypical spinal muscular atrophy, in whom clinical DNA sequencing reported one pathogenic ASAH1 mutation (c.458A>G;p.Tyr153Cys). Transcriptome sequencing on patient leukocytes identified a highly significant and atypical ASAH1 isoform not explained by c.458A>G(p<10-16 ). Subsequent Sanger-sequencing identified the splice mutation responsible for the isoform (c.504A>C;p.Lys168Asn) and provided a molecular diagnosis of autosomal-recessive spinal muscular atrophy with progressive myoclonic epilepsy. Our findings demonstrate the utility of RNA sequencing from blood to identify splice-impacting disease mutations for nonhematological conditions, providing a diagnosis for these otherwise unsolved patients.


Assuntos
Ceramidase Ácida/genética , Atrofia Muscular Espinal/sangue , Epilepsias Mioclônicas Progressivas/sangue , Processamento de RNA/genética , Ceramidase Ácida/sangue , Pré-Escolar , Humanos , Masculino , Atrofia Muscular Espinal/complicações , Atrofia Muscular Espinal/genética , Mutação , Epilepsias Mioclônicas Progressivas/complicações , Epilepsias Mioclônicas Progressivas/genética , Patologia Molecular , Análise de Sequência de DNA , Transcriptoma/genética
6.
Nat Methods ; 13(12): 1036-1042, 2016 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27798611

RESUMO

Engineering and study of protein function by directed evolution has been limited by the technical requirement to use global mutagenesis or introduce DNA libraries. Here, we develop CRISPR-X, a strategy to repurpose the somatic hypermutation machinery for protein engineering in situ. Using catalytically inactive dCas9 to recruit variants of cytidine deaminase (AID) with MS2-modified sgRNAs, we can specifically mutagenize endogenous targets with limited off-target damage. This generates diverse libraries of localized point mutations and can target multiple genomic locations simultaneously. We mutagenize GFP and select for spectrum-shifted variants, including EGFP. Additionally, we mutate the target of the cancer therapeutic bortezomib, PSMB5, and identify known and novel mutations that confer bortezomib resistance. Finally, using a hyperactive AID variant, we mutagenize loci both upstream and downstream of transcriptional start sites. These experiments illustrate a powerful approach to create complex libraries of genetic variants in native context, which is broadly applicable to investigate and improve protein function.


Assuntos
Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Evolução Molecular Direcionada/métodos , Mutação Puntual , Engenharia de Proteínas/métodos , RNA Guia/genética , Bortezomib/farmacologia , Citidina Desaminase/genética , Resistência a Medicamentos/genética , Proteínas de Fluorescência Verde/genética , Humanos , Células K562 , Levivirus/genética , Complexo de Endopeptidases do Proteassoma/genética
7.
Am J Hum Genet ; 98(1): 216-24, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26749306

RESUMO

Methods for multiple-testing correction in local expression quantitative trait locus (cis-eQTL) studies are a trade-off between statistical power and computational efficiency. Bonferroni correction, though computationally trivial, is overly conservative and fails to account for linkage disequilibrium between variants. Permutation-based methods are more powerful, though computationally far more intensive. We present an alternative correction method called eigenMT, which runs over 500 times faster than permutations and has adjusted p values that closely approximate empirical ones. To achieve this speed while also maintaining the accuracy of permutation-based methods, we estimate the effective number of independent variants tested for association with a particular gene, termed Meff, by using the eigenvalue decomposition of the genotype correlation matrix. We employ a regularized estimator of the correlation matrix to ensure Meff is robust and yields adjusted p values that closely approximate p values from permutations. Finally, using a common genotype matrix, we show that eigenMT can be applied with even greater efficiency to studies across tissues or conditions. Our method provides a simpler, more efficient approach to multiple-testing correction than existing methods and fits within existing pipelines for eQTL discovery.


Assuntos
Desequilíbrio de Ligação , Locos de Características Quantitativas , Humanos
8.
G3 (Bethesda) ; 6(2): 321-35, 2015 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-26637431

RESUMO

RNA editing is a posttranscriptional process leading to differences between genomic DNA and transcript sequences, potentially enhancing transcriptome diversity. With recent advances in high-throughput sequencing, many efforts have been made to describe mRNA editing at the transcriptome scale, especially in mammals, yielding contradictory conclusions regarding the extent of this phenomenon. We show, by detailed description of the 25 studies focusing so far on mRNA editing at the whole-transcriptome scale, that systematic sequencing artifacts are considered in most studies whereas biological replication is often neglected and multi-alignment not properly evaluated, which ultimately impairs the legitimacy of results. We recently developed a rigorous strategy to identify mRNA editing using mRNA and genomic DNA sequencing, taking into account sequencing and mapping artifacts, and biological replicates. We applied this method to screen for mRNA editing in liver and white adipose tissue from eight chickens and confirm the small extent of mRNA recoding in this species. Among the 25 unique edited sites identified, three events were previously described in mammals, attesting that this phenomenon is conserved throughout evolution. Deeper investigations on five sites revealed the impact of tissular context, genotype, age, feeding conditions, and sex on mRNA editing levels. More specifically, this analysis highlighted that the editing level at the site located on COG3 was strongly regulated by four of these factors. By comprehensively characterizing the mRNA editing landscape in chickens, our results highlight how this phenomenon is limited and suggest regulation of editing levels by various genetic and environmental factors.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Tecido Adiposo/metabolismo , Galinhas/genética , Genótipo , Fígado/metabolismo , Edição de RNA , RNA Mensageiro/genética , Proteínas Adaptadoras de Transporte Vesicular/química , Fatores Etários , Sequência de Aminoácidos , Ração Animal , Animais , Biologia Computacional/métodos , Feminino , Patrimônio Genético , Genoma , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Dados de Sequência Molecular , RNA Mensageiro/química , Reprodutibilidade dos Testes , Alinhamento de Sequência , Fatores Sexuais
10.
PLoS One ; 10(5): e0126776, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26024316

RESUMO

RNA editing results in a post-transcriptional nucleotide change in the RNA sequence that creates an alternative nucleotide not present in the DNA sequence. This leads to a diversification of transcription products with potential functional consequences. Two nucleotide substitutions are mainly described in animals, from adenosine to inosine (A-to-I) and from cytidine to uridine (C-to-U). This phenomenon is described in more details in mammals, notably since the availability of next generation sequencing technologies allowing whole genome screening of RNA-DNA differences. The number of studies recording RNA editing in other vertebrates like chicken is still limited. We chose to use high throughput sequencing technologies to search for RNA editing in chicken, and to extend the knowledge of its conservation among vertebrates. We performed sequencing of RNA and DNA from 8 embryos. Being aware of common pitfalls inherent to sequence analyses that lead to false positive discovery, we stringently filtered our datasets and found fewer than 40 reliable candidates. Conservation of particular sites of RNA editing was attested by the presence of 3 edited sites previously detected in mammals. We then characterized editing levels for selected candidates in several tissues and at different time points, from 4.5 days of embryonic development to adults, and observed a clear tissue-specificity and a gradual increase of editing level with time. By characterizing the RNA editing landscape in chicken, our results highlight the extent of evolutionary conservation of this phenomenon within vertebrates, attest to its tissue and stage specificity and provide support of the absence of non A-to-I events from the chicken transcriptome.


Assuntos
Galinhas/genética , Genoma , Edição de RNA , Animais , Embrião de Galinha , Biologia Computacional , DNA/química , Evolução Molecular , Sequenciamento de Nucleotídeos em Larga Escala , RNA/química , Análise de Sequência de DNA , Análise de Sequência de RNA
11.
Nucleic Acids Res ; 42(6): 3768-82, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24452801

RESUMO

Genomic imprinting is an epigenetic mechanism by which alleles of some specific genes are expressed in a parent-of-origin manner. It has been observed in mammals and marsupials, but not in birds. Until now, only a few genes orthologous to mammalian imprinted ones have been analyzed in chicken and did not demonstrate any evidence of imprinting in this species. However, several published observations such as imprinted-like QTL in poultry or reciprocal effects keep the question open. Our main objective was thus to screen the entire chicken genome for parental-allele-specific differential expression on whole embryonic transcriptomes, using high-throughput sequencing. To identify the parental origin of each observed haplotype, two chicken experimental populations were used, as inbred and as genetically distant as possible. Two families were produced from two reciprocal crosses. Transcripts from 20 embryos were sequenced using NGS technology, producing ∼200 Gb of sequences. This allowed the detection of 79 potentially imprinted SNPs, through an analysis method that we validated by detecting imprinting from mouse data already published. However, out of 23 candidates tested by pyrosequencing, none could be confirmed. These results come together, without a priori, with previous statements and phylogenetic considerations assessing the absence of genomic imprinting in chicken.


Assuntos
Galinhas/genética , Impressão Genômica , Transcriptoma , Animais , Embrião de Galinha , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Camundongos , Camundongos Endogâmicos DBA , Polimorfismo de Nucleotídeo Único , Análise de Sequência de RNA
12.
Genet Sel Evol ; 45: 16, 2013 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-23758635

RESUMO

Little is known about epigenetic mechanisms in birds with the exception of the phenomenon of dosage compensation of sex chromosomes, although such mechanisms could be involved in the phenotypic variability of birds, as in several livestock species. This paper reviews the literature on epigenetic mechanisms that could contribute significantly to trait variability in birds, and compares the results to the existing knowledge of epigenetic mechanisms in mammals. The main issues addressed in this paper are: (1) Does genomic imprinting exist in birds? (2) How does the embryonic environment influence the adult phenotype in avian species? (3) Does the embryonic environment have an impact on phenotypic variability across several successive generations? The potential for epigenetic studies to improve the performance of individual animals through the implementation of limited changes in breeding conditions or the addition of new parameters in selection models is still an open question.


Assuntos
Aves/genética , Epigênese Genética , Fenótipo , Animais , Evolução Biológica , Meio Ambiente , Feminino , Interação Gene-Ambiente , Impressão Genômica , Masculino
13.
BMC Genomics ; 13: 551, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-23066875

RESUMO

BACKGROUND: As for other non-model species, genetic analyses in quail will benefit greatly from a higher marker density, now attainable thanks to the evolution of sequencing and genotyping technologies. Our objective was to obtain the first genome wide panel of Japanese quail SNP (Single Nucleotide Polymorphism) and to use it for the fine mapping of a QTL for a fear-related behaviour, namely tonic immobility, previously localized on Coturnix japonica chromosome 1. To this aim, two reduced representations of the genome were analysed through high-throughput 454 sequencing: AFLP (Amplified Fragment Length Polymorphism) fragments as representatives of genomic DNA, and EST (Expressed Sequence Tag) as representatives of the transcriptome. RESULTS: The sequencing runs produced 399,189 and 1,106,762 sequence reads from cDNA and genomic fragments, respectively. They covered over 434 Mb of sequence in total and allowed us to detect 17,433 putative SNP. Among them, 384 were used to genotype two Advanced Intercross Lines (AIL) obtained from three quail lines differing for duration of tonic immobility. Despite the absence of genotyping for founder individuals in the analysis, the previously identified candidate region on chromosome 1 was refined and led to the identification of a candidate gene. CONCLUSIONS: These data confirm the efficiency of transcript and AFLP-sequencing for SNP discovery in a non-model species, and its application to the fine mapping of a complex trait. Our results reveal a significant association of duration of tonic immobility with a genomic region comprising the DMD (dystrophin) gene. Further characterization of this candidate gene is needed to decipher its putative role in tonic immobility in Coturnix.


Assuntos
Proteínas Aviárias/genética , Mapeamento Cromossômico , Coturnix/genética , Distrofina/genética , Estudos de Associação Genética , Genoma , Resposta de Imobilidade Tônica , Análise do Polimorfismo de Comprimento de Fragmentos Amplificados , Animais , Galinhas/genética , Cromossomos , Cruzamentos Genéticos , Etiquetas de Sequências Expressas , Feminino , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Fenótipo , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Transcriptoma
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