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 Leigh B. Waddell A1 Samantha J. Bryen A1 Beryl B. Cummings A1 Adam Bournazos A1 Frances J. Evesson A1 Himanshu Joshi A1 Jamie L. Marshall A1 Taru Tukiainen A1 Elise Valkanas A1 Ben Weisburd A1 Simon Sadedin A1 Mark R. Davis A1 Fathimath Faiz A1 Rebecca Gooding A1 Sarah A. Sandaradura A1 Gina L. O'Grady A1 Michel C. Tchan A1 David R. Mowat A1 Emily C. Oates A1 Michelle A. Farrar A1 Hugo Sampaio A1 Alan Ma A1 Katherine Neas A1 Min-Xia Wang A1 Amanda Charlton A1 Charles Chan A1 Diane N. Kenwright A1 Nicole Graf A1 Susan Arbuckle A1 Nigel F. Clarke A1 Daniel G. MacArthur A1 Kristi J. Jones A1 Monkol Lek A1 Sandra T. Cooper 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