WGS and RNA Studies Diagnose Noncoding <i>DMD</i> Variants in Males With High Creatine Kinase
Leigh B. Waddell, Samantha J. Bryen, Beryl B. Cummings, Adam Bournazos, Frances J. Evesson, Himanshu Joshi, Jamie L. Marshall, Taru Tukiainen, Elise Valkanas, Ben Weisburd, Simon Sadedin, Mark R. Davis, Fathimath Faiz, Rebecca Gooding, Sarah A. Sandaradura, Gina O’Grady, Michel Tchan, David Mowat, Emily C. Oates, Michelle A. Farrar, Hugo Sampaio, Alan Ma, Katherine Neas, Min‐Xia Wang, Amanda Charlton, Charles Chan, Diane Kenwright, Nicole Graf, Susan Arbuckle, Nigel F. Clarke, Daniel G. MacArthur, Kristi Jones, Monkol Lek, Sandra T. Cooper
Abstract
<h3>Objective</h3> 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 <i>DMD</i> splicing in 3 families to define critical levels of wild-type dystrophin bridging clinical spectrums of Duchenne to myalgia. <h3>Methods</h3> 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 <i>DMD</i> premessenger RNA (pre-mRNA) splicing. Quantitative Western blot was used to determine levels of dystrophin, relative to control muscle. <h3>Results</h3> Splice-altering intronic single nucleotide variants or structural rearrangements in <i>DMD</i> 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 <i>DMD</i> 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. <h3>Conclusions</h3> Whole-genome sequencing relied heavily on RNA studies to identify <i>DMD</i> 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 <i>DMD</i> 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.