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