WGS and RNA Studies Diagnose Noncoding DMD Variants in Males With High Creatine Kinase

Abstract

ObjectiveTo describe the diagnostic utility of whole-genome sequencing and RNA studies in boys with suspected dystrophinopathy, for whom multiplex ligation-dependent probe amplification and exomic parallel sequencing failed to yield a genetic diagnosis, and to use remnant normal DMD splicing in 3 families to define critical levels of wild-type dystrophin bridging clinical spectrums of Duchenne to myalgia.MethodsExome, genome, and/or muscle RNA sequencing was performed for 7 males with elevated creatine kinase. PCR of muscle-derived complementary DNA (cDNA) studied consequences for DMD premessenger RNA (pre-mRNA) splicing. Quantitative Western blot was used to determine levels of dystrophin, relative to control muscle.ResultsSplice-altering intronic single nucleotide variants or structural rearrangements in DMD were identified in all 7 families. Four individuals, with abnormal splicing causing a premature stop codon and nonsense-mediated decay, expressed remnant levels of normally spliced DMD mRNA. Quantitative Western blot enabled correlation of wild-type dystrophin and clinical severity, with 0%–5% dystrophin conferring a Duchenne phenotype, 10% ± 2% a Becker phenotype, and 15% ± 2% dystrophin associated with myalgia without manifesting weakness.ConclusionsWhole-genome sequencing relied heavily on RNA studies to identify DMD splice-altering variants. Short-read RNA sequencing was regularly confounded by the effectiveness of nonsense-mediated mRNA decay and low read depth of the giant DMD mRNA. PCR of muscle cDNA provided a simple, yet informative approach. Highly relevant to genetic therapies for dystrophinopathies, our data align strongly with previous studies of mutant dystrophin in Becker muscular dystrophy, with the collective conclusion that a fractional increase in levels of normal dystrophin between 5% and 20% is clinically significant.

MINTIE: identifying novel structural and splice variants in transcriptomes using RNA-seq data

“…However, two other sequences were found to correspond to regions 650 bp upstream (2 split-reads) and 70 kb downstream (3 split-reads), suggesting multiple potential fusion transcripts. We confirmed the presence of this rearrangement with the original authors through private correspondence, who independently discovered it using whole genome sequencing and recently reported it in Waddell et al [ 45 ].

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MINTIE: identifying novel structural and splice variants in transcriptomes using RNA-seq data

“…However, two other sequences were found to correspond to regions 650bp upstream (2 split-reads) and 70kb downstream (3 split-reads), suggesting multiple potential fusion transcripts. We confirmed the presence of this rearrangement with the original authors through private correspondence, who independently discovered it using whole genome sequencing and recently reported it in Waddell et al, 2021 45 .…”

Molecular Diagnosis in 100% of Dystrophinopathies

“…4,5 Waddel et al using skeletal muscle biopsies reveals that normal dystrophin level ;15 ± 2% correlates to the milder spectrum of dystrophinopathies (i.e., myalgia without overt weakness), ;10 ± 2% levels of dystrophin predicts a Becker muscular dystrophy phenotype with mild weakness and cardiac involvement, and 0%-5% levels of dystrophin results in severe Becker muscular dystrophy or DMD. 2 These predictions are in line with early studies in which level of dystrophin 29%-57% of normal was shown to be sufficient to avoid muscle weakness in X-linked dilated cardiomyopathy families. 6 The novelty in the approach by Waddel et al is the estimate of normal dystrophin vs the amount of internally deleted dystrophin able to modulate the phenotype.…”

“…After being the first muscular dystrophy in which the disease gene has been identified, it is now the first muscular dystrophy to be treated, in some cases, with personalized, dystrophin-restoring therapy. 1 In spite of these tremendous advancements in the treatment of DMD, in this issue of Neurology ® Genetics, Waddel et al 2 represent the challenges in achieving a genetic diagnosis in a cohort of male patients with elevated serum creatine kinase, dystrophin protein studies suggesting an underlying dystrophinopathy, yet normal multiplex ligation-dependent probe amplification and exomic sequencing.…”

“…1,3 Under these premises, the achievement of the molecular diagnosis in DMD is of a foremost relevance. The experimental approach used by Waddel et al 2 demonstrates also the relevance of muscle biopsy in selected cases of unsolved muscular dystrophy. DMD mRNA isolated from skeletal muscle tissue will allow the localization of sequence anomalies in the transcript pointing to deep intronic causative mutations.…”