WGS and RNA Studies Diagnose Noncoding Variants in Males With High Creatine Kinase.

Neurol Genet

Kids 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.

Published: February 2021

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 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 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 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 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 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 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.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105888PMC
http://dx.doi.org/10.1212/NXG.0000000000000554DOI Listing

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