Integrated Genome and Transcriptome Sequencing to Solve a Neuromuscular Puzzle: Miyoshi Muscular Dystrophy and Early Onset Primary Dystonia in Siblings of the Same Family

Abstract

BackgroundNeuromuscular disorders (NMD), many of which are hereditary, affect muscular function. Due to advances in high-throughput sequencing technologies, the diagnosis of hereditary NMDs has dramatically improved in recent years.Methods and ResultsIn this study, we report an family with two siblings exhibiting two different NMD, Miyoshi muscular dystrophy (MMD) and early onset primary dystonia (EOPD). Whole exome sequencing (WES) identified a novel monoallelic frameshift deletion mutation (dysferlin: c.4404delC/p.I1469Sfs∗17) in the Dysferlin gene in the index patient who suffered from MMD. This deletion was inherited from his unaffected father and was carried by his younger sister with EOPD. However, immunostaining staining revealed an absence of dysferlin expression in the proband’s muscle tissue and thus suggested the presence of the second underlying mutant allele in dysferlin. Using integrated RNA sequencing (RNA-seq) and whole genome sequencing (WGS) of muscle tissue, a novel deep intronic mutation in dysferlin (dysferlin: c.5341-415A > G) was discovered in the index patient. This mutation caused aberrant mRNA splicing and inclusion of an additional pseudoexon (PE) which we termed PE48.1. This PE was inherited from his unaffected mother. PE48.1 inclusion altered the Dysferlin sequence, causing premature termination of translation.ConclusionUsing integrated genome and transcriptome sequencing, we discovered hereditary MMD and EOPD affecting two siblings of same family. Our results added further weight to the combined use of RNA-seq and WGS as an important method for detection of deep intronic gene mutations, and suggest that integrated sequencing assays are an effective strategy for the diagnosis of hereditary NMDs.

An in‐frame pseudoexon activation caused by a novel deep‐intronic variant in the dysferlin gene

“…To the best of our knowledge, only three deep‐intronic splice‐altering variants have been previously reported in DYSF , including c.4886 + 1249G > T in intron 44, 8 , 9 c.5341‐415A > G in intron 48, 10 and c.5668‐824C > T in intron 50. 7 The c.1397 + 649C > T in intron 15 identified in our patient is the fourth deep‐intronic DYSF variant.…”

“…Most pathogenic DYSF variants reside within canonical exons and/or flanking intronic regions and can be detected by routine exonic detection approaches. The precise detection and interpretation of pathogenic DYSF variants is sometimes challenging, as atypical pathogenic DYSF variants including noncanonical splicing site variants 4 and deep‐intronic splice‐altering variants 7 , 8 , 9 , 10 do exist in the DYSF gene. The identification and interpretation of atypical pathogenic DYSF variants require DYSF mRNA analysis and in silico bioinformatic analyses in addition to the genomic sequencing.…”

“…However, approximately 17% of patients with dysferlinopathy remained without a definite genetic diagnosis after routine approaches, as only one mutant allele was identified in them. 5 , 7 The undetected pathogenic variants in the other allele probably lie within deep‐intronic regions of DYSF , 7 , 8 , 9 , 10 of which the identification requires mRNA studies and in silico bioinformatic analyses.…”

Identification of a Novel Non-Canonical Splice-Site Variant in ABCD1

“…WGS offers advantages over WES in the detection of such intronic or intergenic mutations, and the combination of WGS and RNA-Seq is superior in the detection and analysis of non-coding variants. WGS might be optimal for further evaluation when a patient with highly suspected genetic disorder remains undiagnosed after clinical WES [ 38 , 47 ]. In our study, RNA-Seq of blood samples combined with trio-WES enabled us to identify a novel NCSS variant in ABCD1 .…”