RNAseq analysis for the diagnosis of muscular dystrophy

Abstract

The precise genetic cause remains elusive in nearly 50% of patients with presumed neurogenetic disease, representing a significant barrier for clinical care. This is despite significant advances in clinical genetic diagnostics, including the application of whole-exome sequencing and next-generation sequencing-based gene panels. In this study, we identify a deep intronic mutation in the DMD gene in a patient with muscular dystrophy using both conventional and RNAseq-based transcriptome analyses. The implications of our data are that noncoding mutations likely comprise an important source of unresolved genetic disease and that RNAseq is a powerful platform for detecting such mutations.

Figure 1

Deep intronic mutation in the DMD gene as a cause of muscular dystrophy. (A–C) Diagnostic muscle biopsy results. (A) Hematoxylin and Eosin staining revealed a typical dystrophic pattern (areas of fibrosis, fatty infiltrate, and degenerating and regenerating fibers). (B) IHC for dystrophin showing absent expression, with the exception of some revertant fibers (arrow). (C) Inmunohistochemistry (IHC) for a‐sarcoglycan showing a normal staining pattern. (D) RT‐PCR analysis using cDNA from patient muscle and overlapping primer sets that span multiple DMD exons. The primer sets including both exons 37 and 38 yielded larger than expected bands (arrows). (E) Sanger sequencing of an RT‐PCR fragment with exons 37/38 revealed a 51 base pair insertion of intron 37 sequence. (F) Schematic of the mutation and its consequences.

Figure 2

RNA‐seq analysis of muscle from a patient with an intronic DMD mutation. (A) Pairwise correlation heatmap of RNA‐seq samples based on the Pearson correlation of log gene expression values for all genes. Gene expression (counts) was determined and normalized by effective library size using DESeq2. (B) Counts for the top four downregulated genes in DMD patient versus controls as ranked by significance. Bar plots show counts for the patient (blue) and sample means for controls (red). Error bars represent 95% confidence intervals. Circles represent counts for individual samples. (C) Per‐exon read counts and differential exon usage for DMD in patient versus control samples. The novel DMD exon was detected as the most differently expressed exon between the patient and controls (E049 at chrX:32366809‐32366856; marked by arrow). Additional novel exons were detected (E050, E090, E091) but likely represent transcriptional noise. (D) UCSC genome browser screenshot of raw RNA‐seq signal normalized to library size at the DMD locus corresponding to the shaded region in (C) (upper panel). A zoom‐in of the boxed region including the novel DMD exon is shown in the lower panel (novel exon marked by arrow).

Figure 3

RNA‐seq as a tool for mutation detection in muscular dystrophy. (A) RNA‐seq reads for the DMD transcript from the patient and a representative control. Note the existence of an aberrant transcript fragment in all reads from the DMD patient (arrow). (B) Examination at the resolution of the individual base pair of the aberrant transcript fragment. Direct evaluation reveals the causative sequence variant in the patient (box). (C) exon usage data plot (normalized as reads per million) for four large, muscle‐specific transcripts. Note that there is adequate coverage of these large transcripts throughout the gene. The exception is DMD, which shows uniformly low expression in the patient sample (control in blue, patient in red).