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Am J Hum Genet ; 104(3): 466-483, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30827497

RESUMO

Gene-panel and whole-exome analyses are now standard methodologies for mutation detection in Mendelian disease. However, the diagnostic yield achieved is at best 50%, leaving the genetic basis for disease unsolved in many individuals. New approaches are thus needed to narrow the diagnostic gap. Whole-genome sequencing is one potential strategy, but it currently has variant-interpretation challenges, particularly for non-coding changes. In this study we focus on transcriptome analysis, specifically total RNA sequencing (RNA-seq), by using monogenetic neuromuscular disorders as proof of principle. We examined a cohort of 25 exome and/or panel "negative" cases and provided genetic resolution in 36% (9/25). Causative mutations were identified in coding and non-coding exons, as well as in intronic regions, and the mutational pathomechanisms included transcriptional repression, exon skipping, and intron inclusion. We address a key barrier of transcriptome-based diagnostics: the need for source material with disease-representative expression patterns. We establish that blood-based RNA-seq is not adequate for neuromuscular diagnostics, whereas myotubes generated by transdifferentiation from an individual's fibroblasts accurately reflect the muscle transcriptome and faithfully reveal disease-causing mutations. Our work confirms that RNA-seq can greatly improve diagnostic yield in genetically unresolved cases of Mendelian disease, defines strengths and challenges of the technology, and demonstrates the suitability of cell models for RNA-based diagnostics. Our data set the stage for development of RNA-seq as a powerful clinical diagnostic tool that can be applied to the large population of individuals with undiagnosed, rare diseases and provide a framework for establishing minimally invasive strategies for doing so.


Assuntos
Marcadores Genéticos , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Doenças Musculares/diagnóstico , Mutação , Doenças Raras/diagnóstico , Adolescente , Adulto , Células Cultivadas , Criança , Estudos de Coortes , Feminino , Humanos , Masculino , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/patologia , Doenças Musculares/genética , Doenças Raras/genética , Transcriptoma , Adulto Jovem
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