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1.
Nat Commun ; 12(1): 529, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33483494

RESUMO

Aberrant splicing is a major cause of rare diseases.  However, its prediction from genome sequence alone remains in most cases inconclusive. Recently, RNA sequencing has proven to be an effective complementary avenue to detect aberrant splicing. Here, we develop FRASER, an algorithm to detect aberrant splicing from RNA sequencing data. Unlike existing methods, FRASER captures not only alternative splicing but also intron retention events. This typically doubles the number of detected aberrant events and identified a pathogenic intron retention in MCOLN1 causing mucolipidosis. FRASER automatically controls for latent confounders, which are widespread and affect sensitivity substantially. Moreover, FRASER is based on a count distribution and multiple testing correction, thus reducing the number of calls by two orders of magnitude over commonly applied z score cutoffs, with a minor loss of sensitivity. Applying FRASER to rare disease diagnostics is demonstrated by reprioritizing a pathogenic aberrant exon truncation in TAZ from a published dataset. FRASER is easy to use and freely available.


Assuntos
Algoritmos , Processamento Alternativo , Biologia Computacional/métodos , RNA-Seq/métodos , Análise de Sequência de RNA/métodos , Internet , Íntrons/genética , Software
2.
Nat Protoc ; 16(2): 1276-1296, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33462443

RESUMO

RNA sequencing (RNA-seq) has emerged as a powerful approach to discover disease-causing gene regulatory defects in individuals affected by genetically undiagnosed rare disorders. Pioneering studies have shown that RNA-seq could increase the diagnosis rates over DNA sequencing alone by 8-36%, depending on the disease entity and tissue probed. To accelerate adoption of RNA-seq by human genetics centers, detailed analysis protocols are now needed. We present a step-by-step protocol that details how to robustly detect aberrant expression levels, aberrant splicing and mono-allelic expression in RNA-seq data using dedicated statistical methods. We describe how to generate and assess quality control plots and interpret the analysis results. The protocol is based on the detection of RNA outliers pipeline (DROP), a modular computational workflow that integrates all the analysis steps, can leverage parallel computing infrastructures and generates browsable web page reports.


Assuntos
Sequência de Bases/genética , Expressão Gênica/genética , Análise de Sequência de RNA/métodos , Diagnóstico , Técnicas e Procedimentos Diagnósticos , Doença/genética , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , RNA/genética , Software , Fluxo de Trabalho
3.
Am J Hum Genet ; 103(6): 907-917, 2018 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-30503520

RESUMO

RNA sequencing (RNA-seq) is gaining popularity as a complementary assay to genome sequencing for precisely identifying the molecular causes of rare disorders. A powerful approach is to identify aberrant gene expression levels as potential pathogenic events. However, existing methods for detecting aberrant read counts in RNA-seq data either lack assessments of statistical significance, so that establishing cutoffs is arbitrary, or rely on subjective manual corrections for confounders. Here, we describe OUTRIDER (Outlier in RNA-Seq Finder), an algorithm developed to address these issues. The algorithm uses an autoencoder to model read-count expectations according to the gene covariation resulting from technical, environmental, or common genetic variations. Given these expectations, the RNA-seq read counts are assumed to follow a negative binomial distribution with a gene-specific dispersion. Outliers are then identified as read counts that significantly deviate from this distribution. The model is automatically fitted to achieve the best recall of artificially corrupted data. Precision-recall analyses using simulated outlier read counts demonstrated the importance of controlling for covariation and significance-based thresholds. OUTRIDER is open source and includes functions for filtering out genes not expressed in a dataset, for identifying outlier samples with too many aberrantly expressed genes, and for detecting aberrant gene expression on the basis of false-discovery-rate-adjusted p values. Overall, OUTRIDER provides an end-to-end solution for identifying aberrantly expressed genes and is suitable for use by rare-disease diagnostic platforms.


Assuntos
Expressão Gênica/genética , Variação Genética/genética , RNA/metabolismo , Análise de Sequência de RNA/métodos , Algoritmos , Perfilação da Expressão Gênica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos
4.
Sci Rep ; 8(1): 16719, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30425284

RESUMO

In hyper-IgE syndromes (HIES), a group of primary immunodeficiencies clinically overlapping with atopic dermatitis, early diagnosis is crucial to initiate appropriate therapy and prevent irreversible complications. Identification of underlying gene defects such as in DOCK8 and STAT3 and corresponding molecular testing has improved diagnosis. Yet, in a child and her newborn sibling with HIES phenotype molecular diagnosis was misleading. Extensive analyses driven by the clinical phenotype identified an intronic homozygous DOCK8 variant c.4626 + 76 A > G creating a novel splice site as disease-causing. While the affected newborn carrying the homozygous variant had no expression of DOCK8 protein, in the index patient molecular diagnosis was compromised due to expression of altered and wildtype DOCK8 transcripts and DOCK8 protein as well as defective STAT3 signaling. Sanger sequencing of lymphocyte subsets revealed that somatic alterations and reversions revoked the predominance of the novel over the canonical splice site in the index patient explaining DOCK8 protein expression, whereas defective STAT3 responses in the index patient were explained by a T cell phenotype skewed towards central and effector memory T cells. Hence, somatic alterations and skewed immune cell phenotypes due to selective pressure may compromise molecular diagnosis and need to be considered with unexpected clinical and molecular findings.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Íntrons/genética , Síndrome de Job/genética , Mutação , Sítios de Splice de RNA/genética , Sequência de Bases , Pré-Escolar , Biologia Computacional , Feminino , Regulação da Expressão Gênica/genética , Humanos , Lactente , Síndrome de Job/patologia , Técnicas de Diagnóstico Molecular , Gravidez , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/genética
5.
Nat Commun ; 8: 15824, 2017 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-28604674

RESUMO

Across a variety of Mendelian disorders, ∼50-75% of patients do not receive a genetic diagnosis by exome sequencing indicating disease-causing variants in non-coding regions. Although genome sequencing in principle reveals all genetic variants, their sizeable number and poorer annotation make prioritization challenging. Here, we demonstrate the power of transcriptome sequencing to molecularly diagnose 10% (5 of 48) of mitochondriopathy patients and identify candidate genes for the remainder. We find a median of one aberrantly expressed gene, five aberrant splicing events and six mono-allelically expressed rare variants in patient-derived fibroblasts and establish disease-causing roles for each kind. Private exons often arise from cryptic splice sites providing an important clue for variant prioritization. One such event is found in the complex I assembly factor TIMMDC1 establishing a novel disease-associated gene. In conclusion, our study expands the diagnostic tools for detecting non-exonic variants and provides examples of intronic loss-of-function variants with pathological relevance.


Assuntos
Perfilação da Expressão Gênica , Doenças Mitocondriais/genética , Análise de Sequência de RNA , Técnicas e Procedimentos Diagnósticos , Humanos , Processamento de RNA
6.
Nat Genet ; 49(5): 742-752, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28369036

RESUMO

We identify SMARCD2 (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily D, member 2), also known as BAF60b (BRG1/Brahma-associated factor 60b), as a critical regulator of myeloid differentiation in humans, mice, and zebrafish. Studying patients from three unrelated pedigrees characterized by neutropenia, specific granule deficiency, myelodysplasia with excess of blast cells, and various developmental aberrations, we identified three homozygous loss-of-function mutations in SMARCD2. Using mice and zebrafish as model systems, we showed that SMARCD2 controls early steps in the differentiation of myeloid-erythroid progenitor cells. In vitro, SMARCD2 interacts with the transcription factor CEBPɛ and controls expression of neutrophil proteins stored in specific granules. Defective expression of SMARCD2 leads to transcriptional and chromatin changes in acute myeloid leukemia (AML) human promyelocytic cells. In summary, SMARCD2 is a key factor controlling myelopoiesis and is a potential tumor suppressor in leukemia.


Assuntos
Diferenciação Celular/genética , Redes Reguladoras de Genes , Neutrófilos/metabolismo , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona , Análise Mutacional de DNA , Saúde da Família , Feminino , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Linhagem , Peixe-Zebra
7.
Am J Hum Genet ; 100(1): 151-159, 2017 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-27989324

RESUMO

MDH2 encodes mitochondrial malate dehydrogenase (MDH), which is essential for the conversion of malate to oxaloacetate as part of the proper functioning of the Krebs cycle. We report bi-allelic pathogenic mutations in MDH2 in three unrelated subjects presenting with early-onset generalized hypotonia, psychomotor delay, refractory epilepsy, and elevated lactate in the blood and cerebrospinal fluid. Functional studies in fibroblasts from affected subjects showed both an apparently complete loss of MDH2 levels and MDH2 enzymatic activity close to null. Metabolomics analyses demonstrated a significant concomitant accumulation of the MDH substrate, malate, and fumarate, its immediate precursor in the Krebs cycle, in affected subjects' fibroblasts. Lentiviral complementation with wild-type MDH2 cDNA restored MDH2 levels and mitochondrial MDH activity. Additionally, introduction of the three missense mutations from the affected subjects into Saccharomyces cerevisiae provided functional evidence to support their pathogenicity. Disruption of the Krebs cycle is a hallmark of cancer, and MDH2 has been recently identified as a novel pheochromocytoma and paraganglioma susceptibility gene. We show that loss-of-function mutations in MDH2 are also associated with severe neurological clinical presentations in children.


Assuntos
Encefalopatias/genética , Ciclo do Ácido Cítrico , Malato Desidrogenase/genética , Mutação , Idade de Início , Alelos , Sequência de Aminoácidos , Criança , Pré-Escolar , Ciclo do Ácido Cítrico/genética , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Fumaratos/metabolismo , Teste de Complementação Genética , Humanos , Lactente , Recém-Nascido , Malato Desidrogenase/química , Malato Desidrogenase/metabolismo , Malatos/metabolismo , Masculino , Metabolômica , Modelos Moleculares
8.
Biomed Res Int ; 2013: 398968, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23971032

RESUMO

We report the release of PredictProtein for the Debian operating system and derivatives, such as Ubuntu, Bio-Linux, and Cloud BioLinux. The PredictProtein suite is available as a standard set of open source Debian packages. The release covers the most popular prediction methods from the Rost Lab, including methods for the prediction of secondary structure and solvent accessibility (profphd), nuclear localization signals (predictnls), and intrinsically disordered regions (norsnet). We also present two case studies that successfully utilize PredictProtein packages for high performance computing in the cloud: the first analyzes protein disorder for whole organisms, and the second analyzes the effect of all possible single sequence variants in protein coding regions of the human genome.


Assuntos
Internet , Modelos Químicos , Modelos Genéticos , Modelos Moleculares , Linguagens de Programação , Proteínas , Software , Sequência de Aminoácidos , Sequência de Bases , Simulação por Computador , Mineração de Dados/métodos , Bases de Dados de Proteínas , Dados de Sequência Molecular , Proteínas/química , Proteínas/genética , Proteínas/ultraestrutura , Análise de Sequência de Proteína/métodos , Relação Estrutura-Atividade
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