Exome and whole-genome sequencing are becoming increasingly routine approaches in Mendelian disease diagnosis. Despite their success, the current diagnostic rate for genomic analyses across a variety of rare diseases is approximately 25 to 50%. We explore the utility of transcriptome sequencing [RNA sequencing (RNA-seq)] as a complementary diagnostic tool in a cohort of 50 patients with genetically undiagnosed rare muscle disorders. We describe an integrated approach to analyze patient muscle RNA-seq, leveraging an analysis framework focused on the detection of transcript-level changes that are unique to the patient compared to more than 180 control skeletal muscle samples. We demonstrate the power of RNA-seq to validate candidate splice-disrupting mutations and to identify splice-altering variants in both exonic and deep intronic regions, yielding an overall diagnosis rate of 35%. We also report the discovery of a highly recurrent de novo intronic mutation in COL6A1 that results in a dominantly acting splice-gain event, disrupting the critical glycine repeat motif of the triple helical domain. We identify this pathogenic variant in a total of 27 genetically unsolved patients in an external collagen VI-like dystrophy cohort, thus explaining approximately 25% of patients clinically suggestive of having collagen VI dystrophy in whom prior genetic analysis is negative. Overall, this study represents a large systematic application of transcriptome sequencing to rare disease diagnosis and highlights its utility for the detection and interpretation of variants missed by current standard diagnostic approaches

This project was supported by funding from the Broad Institute’s BroadIgnite and Broadnext10 programs. B.B.C. is supported by the NIH GM096911 training grant. T.T. is supported by the Academy of Finland, the Finnish Cultural Foundation, the Orion-Farmos Research Foundation, and the Emil Aaltonen Foundation. M.L. is supported by the Australian NHMRC (National Health and Medical Research Council) CJ Martin Fellowship, the Australian American Association Sir Keith Murdoch Fellowship, and a Muscular Dystrophy Association/American Association of Neuromuscular and Electrodiagnostic Medicine (MDA/AANEM) development grant. L.B.W., S.A.S., N.G.L., N.F.C., K.N.N., and E.C.O. are supported by the NHMRC of Australia (1080587, 1075451, 1002147, 1113531, 1022707, 1031893, and 1090428). K.J.K. is supported by a National Institute of General Medical Sciences (NIGMS) fellowship grant (F32GM115208). A.H.O.-L. is supported by an NIGMS fellowship grant (4T32GM007748). A.H.B. is supported by the NIH R01 HD075802 and R01 AR044345 and by MDA383249 from the Muscular Dystrophy Association. P.B.K., E.E., and H.K.M. are supported by NIH R01NS080929. Funding relevant to this research includes fellowship support of S.T.C. and a project grant supporting an Australian-wide program about gene discovery in inherited neuromuscular disorders performed in collaboration with D.G.M. [NHMRC APP1048816 (2013–2017) and NHMRC APP1080587 (2015–2019)]. The Broad CMG was funded by the National Human Genome Research Institute (NHGRI), the National Eye Institute, and the National Heart, Lung, and Blood Institute (NHLBI) grant UM1 HG008900 to D.G.M. and H. Rehm. The GTEx project was supported by the Common Fund of the Office of the Director of the NIH (http://commonfund.nih.gov/GTEx). Additional funds were provided by the National Cancer Institute (NCI), NHGRI, NHLBI, National Institute on Drug Abuse (NIDA), National Institute of Mental Health (NIMH), and National Institute of Neurological Disorders and Stroke (NINDS). Donors were enrolled at Biospecimen Source Sites that were funded by NCI/Science Applications International Corporation (SAIC)–Frederick Inc. (SAIC-F) subcontracts to the National Disease Research Interchange (10XS170) and the Roswell Park Cancer Institute (10XS171). The Laboratory, Data Analysis, and Coordinating Center (LDACC) was funded through a contract (HHSN268201000029C) to the Broad Institute Inc. Biorepository operations were funded through an SAIC-F subcontract to the Van Andel Institute (10ST1035). Additional data repository and project management were provided by SAIC-F (HHSN261200800001E). The Brain Bank was supported by a supplement to the University of Miami grant DA006227