Objective: To 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 splicing in 3 families to define critical levels of wild-type dystrophin bridging clinical spectrums of Duchenne to myalgia.
Methods: Exome, 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 premessenger RNA (pre-mRNA) splicing. Quantitative Western blot was used to determine levels of dystrophin, relative to control muscle.
Results: Splice-altering intronic single nucleotide variants or structural rearrangements in 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 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.
Conclusions: Whole-genome sequencing relied heavily on RNA studies to identify 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 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.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105888 | PMC |
http://dx.doi.org/10.1212/NXG.0000000000000554 | DOI Listing |
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