PT - JOURNAL ARTICLE AU - Waddell, Leigh B. AU - Bryen, Samantha J. AU - Cummings, Beryl B. AU - Bournazos, Adam AU - Evesson, Frances J. AU - Joshi, Himanshu AU - Marshall, Jamie L. AU - Tukiainen, Taru AU - Valkanas, Elise AU - Weisburd, Ben AU - Sadedin, Simon AU - Davis, Mark R. AU - Faiz, Fathimath AU - Gooding, Rebecca AU - Sandaradura, Sarah A. AU - O'Grady, Gina L. AU - Tchan, Michel C. AU - Mowat, David R. AU - Oates, Emily C. AU - Farrar, Michelle A. AU - Sampaio, Hugo AU - Ma, Alan AU - Neas, Katherine AU - Wang, Min-Xia AU - Charlton, Amanda AU - Chan, Charles AU - Kenwright, Diane N. AU - Graf, Nicole AU - Arbuckle, Susan AU - Clarke, Nigel F. AU - MacArthur, Daniel G. AU - Jones, Kristi J. AU - Lek, Monkol AU - Cooper, Sandra T. TI - WGS and RNA Studies Diagnose Noncoding <em>DMD</em> Variants in Males With High Creatine Kinase AID - 10.1212/NXG.0000000000000554 DP - 2021 Feb 01 TA - Neurology Genetics PG - e554 VI - 7 IP - 1 4099 - http://ng.neurology.org/content/7/1/e554.short 4100 - http://ng.neurology.org/content/7/1/e554.full SO - Neurol Genet2021 Feb 01; 7 AB - 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 DMD 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 DMD 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 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.Conclusions Whole-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.bp=base pair; CK=creatine kinase; DMD=Duchenne muscular dystrophy; gnomAD=Genome Aggregation Database; GTEx=Genotype-Tissue Expression; IGV=Integrative Genomic Browser; MLPA=multiplex ligation-dependent probe amplification; mRNA=messenger RNA; nt=nucleotide; RNA-seq=RNA sequencing; RT-PCR=reverse transcription PCR; SNV=single nucleotide variant; WB=Western blot; WGA=wheat germ agglutinin; WT=wild type