RT Journal Article
SR Electronic
T1 WGS and RNA Studies Diagnose Noncoding <em>DMD</em> Variants in Males With High Creatine Kinase
JF Neurology Genetics
JO Neurol Genet
FD Lippincott Williams &amp; 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