RT Journal Article SR Electronic T1 WGS and RNA Studies Diagnose Noncoding DMD Variants in Males With High Creatine Kinase JF Neurology Genetics JO Neurol Genet FD Lippincott Williams & Wilkins SP e554 DO 10.1212/NXG.0000000000000554 VO 7 IS 1 A1 Waddell, Leigh B. A1 Bryen, Samantha J. A1 Cummings, Beryl B. A1 Bournazos, Adam A1 Evesson, Frances J. A1 Joshi, Himanshu A1 Marshall, Jamie L. A1 Tukiainen, Taru A1 Valkanas, Elise A1 Weisburd, Ben A1 Sadedin, Simon A1 Davis, Mark R. A1 Faiz, Fathimath A1 Gooding, Rebecca A1 Sandaradura, Sarah A. A1 O'Grady, Gina L. A1 Tchan, Michel C. A1 Mowat, David R. A1 Oates, Emily C. A1 Farrar, Michelle A. A1 Sampaio, Hugo A1 Ma, Alan A1 Neas, Katherine A1 Wang, Min-Xia A1 Charlton, Amanda A1 Chan, Charles A1 Kenwright, Diane N. A1 Graf, Nicole A1 Arbuckle, Susan A1 Clarke, Nigel F. A1 MacArthur, Daniel G. A1 Jones, Kristi J. A1 Lek, Monkol A1 Cooper, Sandra T. YR 2021 UL http://ng.neurology.org/content/7/1/e554.abstract 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