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1.
Nature ; 608(7922): 353-359, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35922509

RESUMO

Regulation of transcript structure generates transcript diversity and plays an important role in human disease1-7. The advent of long-read sequencing technologies offers the opportunity to study the role of genetic variation in transcript structure8-16. In this Article, we present a large human long-read RNA-seq dataset using the Oxford Nanopore Technologies platform from 88 samples from Genotype-Tissue Expression (GTEx) tissues and cell lines, complementing the GTEx resource. We identified just over 70,000 novel transcripts for annotated genes, and validated the protein expression of 10% of novel transcripts. We developed a new computational package, LORALS, to analyse the genetic effects of rare and common variants on the transcriptome by allele-specific analysis of long reads. We characterized allele-specific expression and transcript structure events, providing new insights into the specific transcript alterations caused by common and rare genetic variants and highlighting the resolution gained from long-read data. We were able to perturb the transcript structure upon knockdown of PTBP1, an RNA binding protein that mediates splicing, thereby finding genetic regulatory effects that are modified by the cellular environment. Finally, we used this dataset to enhance variant interpretation and study rare variants leading to aberrant splicing patterns.


Assuntos
Alelos , Perfilação da Expressão Gênica , Especificidade de Órgãos , RNA-Seq , Transcriptoma , Processamento Alternativo/genética , Linhagem Celular , Conjuntos de Dados como Assunto , Genótipo , Ribonucleoproteínas Nucleares Heterogêneas/deficiência , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Especificidade de Órgãos/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/deficiência , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Reprodutibilidade dos Testes , Transcriptoma/genética
2.
Nature ; 603(7899): 124-130, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35197626

RESUMO

A hallmark pathological feature of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the depletion of RNA-binding protein TDP-43 from the nucleus of neurons in the brain and spinal cord1. A major function of TDP-43 is as a repressor of cryptic exon inclusion during RNA splicing2-4. Single nucleotide polymorphisms in UNC13A are among the strongest hits associated with FTD and ALS in human genome-wide association studies5,6, but how those variants increase risk for disease is unknown. Here we show that TDP-43 represses a cryptic exon-splicing event in UNC13A. Loss of TDP-43 from the nucleus in human brain, neuronal cell lines and motor neurons derived from induced pluripotent stem cells resulted in the inclusion of a cryptic exon in UNC13A mRNA and reduced UNC13A protein expression. The top variants associated with FTD or ALS risk in humans are located in the intron harbouring the cryptic exon, and we show that they increase UNC13A cryptic exon splicing in the face of TDP-43 dysfunction. Together, our data provide a direct functional link between one of the strongest genetic risk factors for FTD and ALS (UNC13A genetic variants), and loss of TDP-43 function.


Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Éxons/genética , Demência Frontotemporal/metabolismo , Estudo de Associação Genômica Ampla , Humanos , Neurônios Motores/patologia , Proteínas do Tecido Nervoso
3.
Nature ; 581(7809): 459-464, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32461653

RESUMO

Naturally occurring human genetic variants that are predicted to inactivate protein-coding genes provide an in vivo model of human gene inactivation that complements knockout studies in cells and model organisms. Here we report three key findings regarding the assessment of candidate drug targets using human loss-of-function variants. First, even essential genes, in which loss-of-function variants are not tolerated, can be highly successful as targets of inhibitory drugs. Second, in most genes, loss-of-function variants are sufficiently rare that genotype-based ascertainment of homozygous or compound heterozygous 'knockout' humans will await sample sizes that are approximately 1,000 times those presently available, unless recruitment focuses on consanguineous individuals. Third, automated variant annotation and filtering are powerful, but manual curation remains crucial for removing artefacts, and is a prerequisite for recall-by-genotype efforts. Our results provide a roadmap for human knockout studies and should guide the interpretation of loss-of-function variants in drug development.


Assuntos
Genes Essenciais/efeitos dos fármacos , Genes Essenciais/genética , Mutação com Perda de Função/genética , Terapia de Alvo Molecular , Artefatos , Automação , Consanguinidade , Éxons/genética , Mutação com Ganho de Função/genética , Frequência do Gene , Técnicas de Silenciamento de Genes , Heterozigoto , Homozigoto , Humanos , Proteína Huntingtina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Doenças Neurodegenerativas/genética , Proteínas Priônicas/genética , Reprodutibilidade dos Testes , Tamanho da Amostra , Proteínas tau/genética
4.
Nature ; 581(7809): 452-458, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32461655

RESUMO

The acceleration of DNA sequencing in samples from patients and population studies has resulted in extensive catalogues of human genetic variation, but the interpretation of rare genetic variants remains problematic. A notable example of this challenge is the existence of disruptive variants in dosage-sensitive disease genes, even in apparently healthy individuals. Here, by manual curation of putative loss-of-function (pLoF) variants in haploinsufficient disease genes in the Genome Aggregation Database (gnomAD)1, we show that one explanation for this paradox involves alternative splicing of mRNA, which allows exons of a gene to be expressed at varying levels across different cell types. Currently, no existing annotation tool systematically incorporates information about exon expression into the interpretation of variants. We develop a transcript-level annotation metric known as the 'proportion expressed across transcripts', which quantifies isoform expression for variants. We calculate this metric using 11,706 tissue samples from the Genotype Tissue Expression (GTEx) project2 and show that it can differentiate between weakly and highly evolutionarily conserved exons, a proxy for functional importance. We demonstrate that expression-based annotation selectively filters 22.8% of falsely annotated pLoF variants found in haploinsufficient disease genes in gnomAD, while removing less than 4% of high-confidence pathogenic variants in the same genes. Finally, we apply our expression filter to the analysis of de novo variants in patients with autism spectrum disorder and intellectual disability or developmental disorders to show that pLoF variants in weakly expressed regions have similar effect sizes to those of synonymous variants, whereas pLoF variants in highly expressed exons are most strongly enriched among cases. Our annotation is fast, flexible and generalizable, making it possible for any variant file to be annotated with any isoform expression dataset, and will be valuable for the genetic diagnosis of rare diseases, the analysis of rare variant burden in complex disorders, and the curation and prioritization of variants in recall-by-genotype studies.


Assuntos
Doença/genética , Haploinsuficiência/genética , Mutação com Perda de Função/genética , Anotação de Sequência Molecular , Transcrição Gênica , Transcriptoma/genética , Transtorno do Espectro Autista/genética , Conjuntos de Dados como Assunto , Deficiências do Desenvolvimento/genética , Éxons/genética , Feminino , Genótipo , Humanos , Deficiência Intelectual/genética , Masculino , Anotação de Sequência Molecular/normas , Distribuição de Poisson , RNA Mensageiro/análise , RNA Mensageiro/genética , Doenças Raras/diagnóstico , Doenças Raras/genética , Reprodutibilidade dos Testes , Sequenciamento do Exoma
5.
Nature ; 581(7809): 434-443, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32461654

RESUMO

Genetic variants that inactivate protein-coding genes are a powerful source of information about the phenotypic consequences of gene disruption: genes that are crucial for the function of an organism will be depleted of such variants in natural populations, whereas non-essential genes will tolerate their accumulation. However, predicted loss-of-function variants are enriched for annotation errors, and tend to be found at extremely low frequencies, so their analysis requires careful variant annotation and very large sample sizes1. Here we describe the aggregation of 125,748 exomes and 15,708 genomes from human sequencing studies into the Genome Aggregation Database (gnomAD). We identify 443,769 high-confidence predicted loss-of-function variants in this cohort after filtering for artefacts caused by sequencing and annotation errors. Using an improved model of human mutation rates, we classify human protein-coding genes along a spectrum that represents tolerance to inactivation, validate this classification using data from model organisms and engineered human cells, and show that it can be used to improve the power of gene discovery for both common and rare diseases.


Assuntos
Exoma/genética , Genes Essenciais/genética , Variação Genética/genética , Genoma Humano/genética , Adulto , Encéfalo/metabolismo , Doenças Cardiovasculares/genética , Estudos de Coortes , Bases de Dados Genéticas , Feminino , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Humanos , Mutação com Perda de Função/genética , Masculino , Taxa de Mutação , Pró-Proteína Convertase 9/genética , RNA Mensageiro/genética , Reprodutibilidade dos Testes , Sequenciamento do Exoma , Sequenciamento Completo do Genoma
7.
Nature ; 550(7675): 244-248, 2017 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-29022598

RESUMO

X chromosome inactivation (XCI) silences transcription from one of the two X chromosomes in female mammalian cells to balance expression dosage between XX females and XY males. XCI is, however, incomplete in humans: up to one-third of X-chromosomal genes are expressed from both the active and inactive X chromosomes (Xa and Xi, respectively) in female cells, with the degree of 'escape' from inactivation varying between genes and individuals. The extent to which XCI is shared between cells and tissues remains poorly characterized, as does the degree to which incomplete XCI manifests as detectable sex differences in gene expression and phenotypic traits. Here we describe a systematic survey of XCI, integrating over 5,500 transcriptomes from 449 individuals spanning 29 tissues from GTEx (v6p release) and 940 single-cell transcriptomes, combined with genomic sequence data. We show that XCI at 683 X-chromosomal genes is generally uniform across human tissues, but identify examples of heterogeneity between tissues, individuals and cells. We show that incomplete XCI affects at least 23% of X-chromosomal genes, identify seven genes that escape XCI with support from multiple lines of evidence and demonstrate that escape from XCI results in sex biases in gene expression, establishing incomplete XCI as a mechanism that is likely to introduce phenotypic diversity. Overall, this updated catalogue of XCI across human tissues helps to increase our understanding of the extent and impact of the incompleteness in the maintenance of XCI.


Assuntos
Especificidade de Órgãos/genética , Análise de Célula Única , Inativação do Cromossomo X/genética , Cromossomos Humanos X/genética , Feminino , Genes Ligados ao Cromossomo X/genética , Genoma Humano/genética , Genômica , Humanos , Masculino , Fenótipo , Análise de Sequência de RNA , Transcriptoma/genética
8.
Genome Res ; 29(1): 53-63, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30552105

RESUMO

The evolutionary history of a gene helps predict its function and relationship to phenotypic traits. Although sequence conservation is commonly used to decipher gene function and assess medical relevance, methods for functional inference from comparative expression data are lacking. Here, we use RNA-seq across seven tissues from 17 mammalian species to show that expression evolution across mammals is accurately modeled by the Ornstein-Uhlenbeck process, a commonly proposed model of continuous trait evolution. We apply this model to identify expression pathways under neutral, stabilizing, and directional selection. We further demonstrate novel applications of this model to quantify the extent of stabilizing selection on a gene's expression, parameterize the distribution of each gene's optimal expression level, and detect deleterious expression levels in expression data from individual patients. Our work provides a statistical framework for interpreting expression data across species and in disease.


Assuntos
Evolução Molecular , Regulação da Expressão Gênica/fisiologia , Modelos Genéticos , Animais , Cães , Coelhos
11.
Nature ; 536(7616): 285-91, 2016 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-27535533

RESUMO

Large-scale reference data sets of human genetic variation are critical for the medical and functional interpretation of DNA sequence changes. Here we describe the aggregation and analysis of high-quality exome (protein-coding region) DNA sequence data for 60,706 individuals of diverse ancestries generated as part of the Exome Aggregation Consortium (ExAC). This catalogue of human genetic diversity contains an average of one variant every eight bases of the exome, and provides direct evidence for the presence of widespread mutational recurrence. We have used this catalogue to calculate objective metrics of pathogenicity for sequence variants, and to identify genes subject to strong selection against various classes of mutation; identifying 3,230 genes with near-complete depletion of predicted protein-truncating variants, with 72% of these genes having no currently established human disease phenotype. Finally, we demonstrate that these data can be used for the efficient filtering of candidate disease-causing variants, and for the discovery of human 'knockout' variants in protein-coding genes.


Assuntos
Exoma/genética , Variação Genética/genética , Análise Mutacional de DNA , Conjuntos de Dados como Assunto , Humanos , Fenótipo , Proteoma/genética , Doenças Raras/genética , Tamanho da Amostra
14.
Am J Hum Genet ; 103(6): 930-947, 2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30503522

RESUMO

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 7 out of 1,000,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole-exome sequencing (WES). We identified relevant rare and predicted damaging mutations for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and located in 1 of 19 previously reported ribosomal protein (RP)-encoding genes. Using exon coverage estimates, we identified and validated 31 deletions in RP genes. We also observed an enrichment for extended splice site mutations and validated their diverse effects using RNA sequencing in cell lines obtained from individuals with DBA. Leveraging the size of our cohort, we observed robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. We further identified rare mutations in seven previously unreported RP genes that may cause DBA, as well as several distinct disorders that appear to phenocopy DBA, including nine individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain >5% of DBA-affected case subjects. Overall, this report should inform not only clinical practice for DBA-affected individuals, but also the design and analysis of rare variant studies for heterogeneous Mendelian disorders.


Assuntos
Anemia de Diamond-Blackfan/genética , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , Exoma/genética , Éxons/genética , Feminino , Deleção de Genes , Estudos de Associação Genética/métodos , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Mutação/genética , Fenótipo , Proteínas Ribossômicas/genética , Ribossomos/genética , Análise de Sequência de RNA/métodos , Sequenciamento do Exoma/métodos
15.
Am J Hum Genet ; 99(5): 1086-1105, 2016 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-27745833

RESUMO

This study establishes PYROXD1 variants as a cause of early-onset myopathy and uses biospecimens and cell lines, yeast, and zebrafish models to elucidate the fundamental role of PYROXD1 in skeletal muscle. Exome sequencing identified recessive variants in PYROXD1 in nine probands from five families. Affected individuals presented in infancy or childhood with slowly progressive proximal and distal weakness, facial weakness, nasal speech, swallowing difficulties, and normal to moderately elevated creatine kinase. Distinctive histopathology showed abundant internalized nuclei, myofibrillar disorganization, desmin-positive inclusions, and thickened Z-bands. PYROXD1 is a nuclear-cytoplasmic pyridine nucleotide-disulphide reductase (PNDR). PNDRs are flavoproteins (FAD-binding) and catalyze pyridine-nucleotide-dependent (NAD/NADH) reduction of thiol residues in other proteins. Complementation experiments in yeast lacking glutathione reductase glr1 show that human PYROXD1 has reductase activity that is strongly impaired by the disease-associated missense mutations. Immunolocalization studies in human muscle and zebrafish myofibers demonstrate that PYROXD1 localizes to the nucleus and to striated sarcomeric compartments. Zebrafish with ryroxD1 knock-down recapitulate features of PYROXD1 myopathy with sarcomeric disorganization, myofibrillar aggregates, and marked swimming defect. We characterize variants in the oxidoreductase PYROXD1 as a cause of early-onset myopathy with distinctive histopathology and introduce altered redox regulation as a primary cause of congenital muscle disease.


Assuntos
Núcleo Celular/genética , Miopatias Distais/genética , Variação Genética , Miopatias Congênitas Estruturais/genética , Oxirredutases/genética , Sequência de Aminoácidos , Animais , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Estudos de Coortes , Creatina Quinase/genética , Creatina Quinase/metabolismo , Citoplasma/metabolismo , Miopatias Distais/patologia , Proteína Semelhante a ELAV 4/genética , Proteína Semelhante a ELAV 4/metabolismo , Feminino , Flavoproteínas/metabolismo , Deleção de Genes , Estudo de Associação Genômica Ampla , Glutationa Redutase/genética , Glutationa Redutase/metabolismo , Células HEK293 , Humanos , Masculino , Músculo Esquelético/patologia , Mutação de Sentido Incorreto , Miopatias Congênitas Estruturais/patologia , Oxirredutases/metabolismo , Linhagem , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Peixe-Zebra/genética
16.
Mol Genet Metab ; 126(1): 77-82, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30558828

RESUMO

BACKGROUND: In almost half of patients with acute liver failure the cause is unknown, making targeted treatment and decisions about liver transplantation a challenge. Monogenic disorders may contribute to a significant proportion of these undiagnosed patients, and so the incorporation of technologies such as next generation sequencing (NGS) in the clinic could aid in providing a definitive diagnosis. However, this technology may present a major challenge in interpretation of sequence variants, particularly those in non-coding regions. RESULTS: In this report we describe a case of Infantile liver failure syndrome 2 (ILFS2; MIM 616483) due to novel bi-allelic variants in the NBAS gene. A missense variant NM_015909.3(NBAS):c.2617C > T, NP_056993.2(NBAS):p.(Arg873Trp) was identified by whole genome sequencing (WGS). By combining WGS and reverse transcription-polymerase chain reaction (RT-PCR) we were able to identify a novel deep intronic variant, NM_015909.3(NBAS):c.2423 + 404G > C, leading to the inclusion of a pseudo-exon. This mechanism has not been described previously in this syndrome. CONCLUSIONS: This study highlights the utility of analyzing NGS data in conjunction with investigating complementary DNA (cDNA) using techniques such as RT-PCR for detection of variants that otherwise would be likely to be missed in common NGS bioinformatic analysis pipelines. Combining these approaches, particularly when the phenotype match is strong, could lead to an increase in the diagnostic yield in acute liver failure and thus aid in targeted treatment, accurate genetic counseling and restoration of reproductive confidence.


Assuntos
Variação Genética , Íntrons , Falência Hepática Aguda/genética , Proteínas de Neoplasias/genética , Alelos , Criança , Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Falência Hepática Aguda/diagnóstico , Transplante de Fígado , Mutação , Fenótipo , Sequenciamento Completo do Genoma
17.
Nucleic Acids Res ; 45(D1): D840-D845, 2017 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-27899611

RESUMO

Worldwide, hundreds of thousands of humans have had their genomes or exomes sequenced, and access to the resulting data sets can provide valuable information for variant interpretation and understanding gene function. Here, we present a lightweight, flexible browser framework to display large population datasets of genetic variation. We demonstrate its use for exome sequence data from 60 706 individuals in the Exome Aggregation Consortium (ExAC). The ExAC browser provides gene- and transcript-centric displays of variation, a critical view for clinical applications. Additionally, we provide a variant display, which includes population frequency and functional annotation data as well as short read support for the called variant. This browser is open-source, freely available at http://exac.broadinstitute.org, and has already been used extensively by clinical laboratories worldwide.


Assuntos
Biologia Computacional/métodos , Bases de Dados Genéticas , Exoma , Genômica/métodos , Navegador , Estudo de Associação Genômica Ampla/métodos , Humanos , Software , Interface Usuário-Computador
18.
Hum Mutat ; 39(3): 383-388, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29266598

RESUMO

A male neonate presented with severe weakness, hypotonia, contractures and congenital scoliosis. Skeletal muscle specimens showed marked atrophy and degeneration of fast fibers with striking nemaline rods and hypertrophy of slow fibers that were ultrastructurally normal. A neuromuscular gene panel identified a homozygous essential splice variant in TNNT3 (chr11:1956150G > A, NM_006757.3:c.681+1G > A). TNNT3 encodes skeletal troponin-Tfast and is associated with autosomal dominant distal arthrogryposis. TNNT3 has not previously been associated with nemaline myopathy (NM), a rare congenital myopathy linked to defects in proteins associated with thin filament structure and regulation. cDNA studies confirmed pathogenic consequences of the splice variant, eliciting exon-skipping and intron retention events leading to a frameshift. Western blot showed deficiency of troponin-Tfast protein with secondary loss of troponin-Ifast . We establish a homozygous splice variant in TNNT3 as the likely cause of severe congenital NM with distal arthrogryposis, characterized by specific involvement of Type-2 fibers and deficiency of troponin-Tfast .


Assuntos
Artrogripose/complicações , Artrogripose/genética , Genes Recessivos , Miopatias da Nemalina/complicações , Miopatias da Nemalina/genética , Splicing de RNA/genética , Troponina T/genética , Humanos , Lactente , Recém-Nascido , Masculino , Miopatias da Nemalina/patologia , Sítios de Splice de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
19.
Hum Mutat ; 38(5): 517-523, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28229513

RESUMO

The clinical interpretation of genetic variants has come to rely heavily on reference population databases such as the Exome Aggregation Consortium (ExAC) database. Pathogenic variants in genes associated with severe, pediatric-onset, highly penetrant, autosomal dominant conditions are assumed to be absent or rare in these databases. Exome sequencing of a 6-year-old female patient with seizures, developmental delay, dysmorphic features, and failure to thrive identified an ASXL1 variant previously reported as causative of Bohring-Opitz syndrome (BOS). Surprisingly, the variant was observed seven times in the ExAC database, presumably in individuals without BOS. Although the BOS phenotype fit, the presence of the variant in reference population databases introduced ambiguity in result interpretation. Review of the literature revealed that acquired somatic mosaicism of ASXL1 variants (including pathogenic variants) during hematopoietic clonal expansion can occur with aging in healthy individuals. We examined all ASXL1 truncating variants in the ExAC database and determined most are likely somatic. Failure to consider somatic mosaicism may lead to the inaccurate assumption that conditions like BOS have reduced penetrance, or the misclassification of potentially pathogenic variants.


Assuntos
Craniossinostoses/diagnóstico , Craniossinostoses/genética , Estudos de Associação Genética , Mutação em Linhagem Germinativa , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Mutação , Proteínas Repressoras/genética , Idoso , Idoso de 80 Anos ou mais , Alelos , Substituição de Aminoácidos , Pré-Escolar , Bases de Dados Genéticas , Fácies , Feminino , Estudos de Associação Genética/métodos , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Fenótipo
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