Practical approach to the genetic diagnosis of unsolved dystrophinopathies: a stepwise strategy in the genomic era

Abstract

ObjectiveTo investigate the diagnostic value of implementing a stepwise genetic testing strategy (SGTS) in genetically unsolved cases with dystrophinopathies.MethodsAfter routine genetic testing in 872 male patients with highly suspected dystrophinopathies, we identified 715 patients with a pathogenic DMD variant. Of the 157 patients who had no pathogenic DMD variants and underwent a muscle biopsy, 142 patients were confirmed to have other myopathies, and 15 suspected dystrophinopathies remained genetically undiagnosed. These 15 patients underwent a more comprehensive evaluation as part of the SGTS pipeline, which included the stepwise analysis of dystrophin mRNA, short-read whole-gene DMD sequencing, long-read whole-gene DMD sequencing and in silico bioinformatic analyses.ResultsSGTS successfully yielded a molecular diagnosis of dystrophinopathy in 11 of the 15 genetically unsolved cases. We identified 8 intronic and 2 complex structural variants (SVs) leading to aberrant splicing in 10 of 11 patients, of which 9 variants were novel. In one case, a molecular defect was detected on mRNA and protein level only. Aberrant splicing mechanisms included 6 pseudoexon inclusions and 4 alterations of splice sites and splicing regulatory elements. We showed for the first time the exonisation of a MER48 element as a novel pathogenic mechanism in dystrophinopathies.ConclusionOur study highlights the high diagnostic utility of implementing a SGTS pipeline in dystrophinopathies with intronic variants and complex SVs.

Long‐read whole‐genome sequencing for the genetic diagnosis of dystrophinopathies

“…Only seven cases with dystrophinopathies caused by pathogenic LINE‐1 insertions in DMD have been reported. Four of the insertions are in exons causing dystrophinopathies by exonic disruptions, 14–17 one in 5′ untranslated region of DMD causing dystrophinopathies by affecting the transcription process or the stability of mature mRNA, 18 and two in introns causing dystrophinopathies by partial exonization of themselves 7,11 . The mutational event related to LINE‐1 identified in our case is a genomic rearrangement mediated by the retrotransposition activity of two LINE‐1s in deep intronic regions.…”

“…We isolated mRNA from the remaining muscle tissue and amplified 22 overlapping cDNA fragments of the entire dystrophin mRNA using RT‐PCR (Supplementary Figure S1). 7 Gel electrophoresis analysis of the cDNA products showed that the 2nd–15th cDNA fragments were absent (Supplementary Figure S1C), suggestive of a possible skipping of exons 8/9/10 to 49/50/51. Further cDNA analysis confirmed the skipping of exons 8–51 from the mature mRNA (Supplementary Figure S1D), indicating a possible variant involving exons 8–51 at the genomic level.…”

“…Genomic DNA was isolated from peripheral blood and paired‐end short‐read whole DMD gene sequencing was performed as previously described 7 to search for variants that could cause the exon skipping. No potentially causative intronic variants were identified.…”

“…Only one pair of reads (length 150bp) were found indicating a possible inversion involving exons 8–51, but they were not sufficiently informative for further validation (Supplementary Figure S2A). We then performed the long‐read sequencing of the DMD gene (average read length 1377bp; average read depth 191X) using a previously described protocol 7 . Ten reads with an average length of 1938bp indicated a possible inversion involving exons 8–51, g.(31767103‐31767576)_(32749380‐32749897)inv (Supplementary Figure S2B), suggesting that its breakpoints would be involved in LINE‐1s as both the genomic regions chrX:32749016‐32755046 and chrX:31767278‐31768677 were classified as a LINE‐1 annotated by RepeatMasker (http://www.repeatmasker.org/cgi-bin/WEBRepeatMasker).…”

“…Finally, we identified a novel pathogenic complex SV in DMD by long‐read whole‐genome sequencing, a large‐scale inversion/deletion‐insertion rearrangement (almost 1Mb), which was mediated by long interspersed nuclear element‐1 (LINE‐1) retrotransposons. This study is a continuation of our previous work, 7 which includes a stepwise application of various genetic approaches to genetically diagnose dystrophinopathies, with the exception of the application of long‐read whole‐genome sequencing.…”