Expanding the Boundaries of RNA Sequencing as a Diagnostic Tool for Rare Mendelian Disease

Am J Hum Genet. 2019 Mar 7;104(3):466-483. doi: 10.1016/j.ajhg.2019.01.012. Epub 2019 Feb 28.


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.

Keywords: Mendelian disease; RNA-seq; diagnostics; muscular dystrophy; myotubes; transcriptomics; transdifferentiation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Adult
  • Cells, Cultured
  • Child
  • Cohort Studies
  • Female
  • Genetic Markers*
  • Genetic Variation*
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Male
  • Muscle Fibers, Skeletal / metabolism
  • Muscle Fibers, Skeletal / pathology
  • Muscular Diseases / diagnosis*
  • Muscular Diseases / genetics
  • Mutation*
  • Rare Diseases / diagnosis*
  • Rare Diseases / genetics
  • Transcriptome
  • Young Adult


  • Genetic Markers