Abstract
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.
Glossary
- 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
Footnotes
Go to Neurology.org/NG for full disclosures. Funding information is provided at the end of the article.
↵* Deceased.
The Article Processing Charge was funded by the authors.
Editorial, page e529
- Received May 1, 2020.
- Accepted in final form November 19, 2020.
- Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.
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