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Published on: May 24, 2016
WGS and RNA Studies Diagnose Noncoding DMD Variants in Males With High Creatine Kinase
Leigh B Waddell1, Samantha J Bryen1, Beryl B Cummings1
1Kids Neuroscience Centre (L.B.W., S.J.B., A.B., F.J.E., H.J., S.A.S., G.L.O., E.C.O., N.F.C., K.J.J., S.T.C.), Kids Research Institute, The Children's Hospital at Westmead, New South Wales, Australia; Discipline of Child and Adolescent Health (L.B.W., S.J.B., A.B., F.J.E., S.A.S., G.L.O., E.C.O., N.F.C., K.J.J., S.T.C.), Faculty of Medicine and Health, The University of Sydney, Westmead, New South Wales, Australia; Analytic and Translational Genetics Unit (B.B.C., J.L.M., T.T., E.V., D.G.M., M.L.), Massachusetts General Hospital, Boston; Medical and Population Genetics (B.B.C., J.L.M., T.T., E.V., B.W., S.S., D.G.M., M.L.), and Center for Mendelian Genomics (B.B.C., J.L.M., E.V., B.W., S.S., D.G.M., M.L.), Broad Institute of MIT & Harvard, Cambridge, MA; Functional Neuromics (F.J.E., S.T.C.), Children's Medical Research Institute, Westmead, New South Wales, Australia; Murdoch Children's Research Institute (S.S.), Parkville, Victoria, Australia; Department of Diagnostic Genomics (M.R.D., F.F., R.G.), PathWest Laboratory Medicine WA, Nedlands, Australia; Department of Clinical Genetics (S.A.S., A.M., K.J.J.), Children's Hospital at Westmead, New South Wales, Australia; Department of Genetic Medicine (M.C.T.), Westmead Hospital, New South Wales, Australia; Discipline of Genomic Medicine (M.C.T., A.M.), Sydney Medical School, The University of Sydney, New South Wales, Australia; Centre for Clinical Genetics (D.R.M.), Sydney Children's Hospital, Randwick, New South Wales, Australia; School of Women's and Children's Health (D.R.M., M.A.F.), UNSW Medicine, UNSW Sydney, Australia; Department of Neurology (M.A.F., H.S.), Sydney Children's Hospital, Randwick, New South Wales, Australia; Department of Clinical Genetics (A.M.), Nepean Hospital, Sydney, Australia; Genetic Health Service NZ (K.N.), Wellington, New Zealand; Neurology Laboratory (M.-X.W.), Royal Prince Alfred Hospital, Camperdown, New South Wales, Australia; Central Clinical School (M.-X.W.), Faculty of Medicine and Health, The University of Sydney, Camperdown, New South Wales, Australia; Anatomic Pathology (A.C., C.C., N.G., S.A.), The Children's Hospital at Westmead, New South Wales, Australia; Anatomic Pathologist (D.N.K.), Department of Pathology and Molecular Medicine, University of Otago, Wellington, New Zealand; and Harvard Medical School (D.G.M.), Boston, MA.
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.
