Improving genetic diagnosis in Mendelian disease with transcriptome sequencing

Abstract

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.

Fig. 1. Experimental design and quality control

(A) Overview of the number of samples that underwent RNA-seq. We performed RNA-seq on 13 previously genetically diagnosed patients, 4 patients in whom previous genetic analysis had identified an extended splice site variant of unknown significance (VUS), 12 patients in whom genetic analysis had identified a strong candidate gene, and 34 patients with no strong candidates from previous analysis. RNA-seq enabled the diagnosis of 35% of patients overall, with the rate, shown above the bar plots, varying depending on previous evidence from genetic analysis. (B) PCA based on gene expression profiles of patient muscle samples passing quality control (n = 61) and GTEx samples of tissues that potentially contaminate muscle biopsies shows that patient samples cluster closely with GTEx skeletal muscle. (C) Overview of experimental setup and RNA-seq analyses performed. Our framework is based on identifying transcriptional aberrations that are present in patients and missing in GTEx controls. Upon ensuring that GTEx and patient RNA-seq data were comparable, we validated the capacity of RNA-seq to resolve transcriptional aberrations in previously diagnosed patients and performed analyses of aberrant splicing, allele imbalance, and variant calling in our remaining cohort of genetically undiagnosed muscle disease patients.

Fig. 2. Types of pathogenic splice aberrations discovered in patients

RNA-seq identified a range of aberrations caused by both coding and noncoding variants, such as (A) exon skipping caused by an essential splice site variant in patient D7, (B) exon extension caused by a donor +3 A>C extended splice site variant in nemaline myopathy patient C9 (where disruption of splicing at the canonical splice site results in splicing from intact GTA motifs from the intron), (C) exonic splice gain caused by a C>T donor splice site–creating variant in patient N22 with a donor +5-G sequence context, resulting in a stronger splice motif than the existing canonical splice site, and (D) intronic splice gain in patient N33 caused by a C>T donor splice site–creating deep intronic variant. Evidence for wild-type splicing in addition to the inclusion of the pseudoexon in the patient is in line with the milder Becker’s muscular dystrophy phenotype. Splice aberrations shown in (B) to (D) result in the introduction of a premature stop codon to the transcript.

Fig. 3. Identification of a recurrent splice site–creating variant in four collagen VI–related dystrophy patients

(A) Splicing-in of the pseudoexon was observed in four patients in our cohort (red) and missing in all other patients and GTEx samples (blue). (B) Inclusion of the 24–amino acid segment is caused by a C>T donor splice site–creating variant, which pairs with an AG splice acceptor site 72 bp upstream. The variant is found in a CpG nucleotide context, which likely explains its recurrent de novo status, and disrupts the Gly-X-Y repeat motifs of COL6A1. (C) The inclusion event is observable in RT-PCR amplicons from patient muscle but is found at comparatively lower levels in cultured dermal fibroblasts derived from the patients, explaining why the pathogenic event was missed in all four patients through previous fibroblast cDNA sequencing.