DMD genotype correlations from the Duchenne Registry: Endogenous exon skipping is a factor in prolonged ambulation for individuals with a defined mutation subtype

Abstract

Antisense oligonucleotide (AON)-mediated exon skipping is an emerging therapeutic for individuals with Duchenne muscular dystrophy (DMD). Skipping of exons adjacent to common exon deletions in DMD using AONs can produce in-frame transcripts and functional protein. Targeted skipping of DMD exons 8, 44, 45, 50, 51, 52, 53, and 55 is predicted to benefit 47% of affected individuals. We observed a correlation between mutation subgroups and age at loss of ambulation in the Duchenne Registry, a large database of phenotypic and genetic data for DMD (N = 765). Males amenable to exon 44 (N = 74) and exon 8 skipping (N = 18) showed prolonged ambulation compared to other exon skip groups and nonsense mutations (P = 0.035 and P < 0.01, respectively). In particular, exon 45 deletions were associated with prolonged age at loss of ambulation relative to the rest of the exon 44 skip amenable cohort and other DMD mutations. Exon 3-7 deletions also showed prolonged ambulation relative to all other exon 8 skippable mutations. Cultured myotubes from DMD patients with deletions of exons 3-7 or exon 45 showed higher endogenous skipping than other mutations, providing a potential biological rationale for our observations. These results highlight the utility of aggregating phenotypic and genotypic data for rare pediatric diseases to reveal progression differences, identify potentially confounding factors, and probe molecular mechanisms that may affect disease severity.

Keywords: Duchenne Registry; Duchenne muscular dystrophy; rare disease registry.

© 2018 The Authors. Human Mutation published by Wiley Periodicals, Inc.

Figures

Figure 1

Filtering steps of Duchenne Registry data for analysis in this study. Participants can respond to one or more survey modules on steroid use, genetic testing results, or muscle function. Data for individuals who responded to all three submodules were merged. We removed entries that did not include valid diagnosis, mutation type, country of residence, steroid usage, ambulatory status, and age at LOA status. Individuals amenable to exon skipping were then sorted into the predicted exon skip required to generate in‐frame DMD transcript

Figure 2

Kaplan–Meier age at LOA analysis for patients eligible for skipping therapy of exons. Delayed age at LOA was observed among individuals amenable to exon 8 skipping (P < 0.001) and exon 44 skipping (P = 0.04). Exon 51 skippable individuals had earlier age at LOA (P = 0.04). All other groups (45, 50, 52, 53, duplication and nonsense) were not significantly different and were merged. All subjects were currently using corticosteroids

Figure 3

Kaplan–Meier plots for subgroups of exon 8, 44, and exon 51 skippable mutations. (A) Exon 8 skippable patients with exon 3–7 deletions ambulated substantially longer than any other group (P = 0.0003). (B) Individuals with single exon 45 deletions ambulate longer than other exon 44 skippable subgroups or other targeted exons (P = 0.029). (C) Among exon 51 skippable subgroups, only exon 49–50 deletions show significant change in age at LOA (P = 0.008)

Figure 4

Basal levels of exon skipping are enriched in cultured myotubes derived from reprogrammed fibroblast (iDRM) from patients with del 45 mutations (exon 44 skippable) or myoblasts derived from del 3–7 (exon 8 skippable) relative to those derived from del 45–50 or del 49–50 iDRM (exon51 skippable). Experimental samples were run in triplicate and data shown reflect cumulative results of multiple experiments, with each point representing a singlet.  DMD mRNA was reverse transcribed and PCR used to detect exon 44, 51, or 8 skipped and unskipped products. Products were quantitated using a Bioanalyzer. Percentage skipped is calculated as (skipped/unskipped + skipped) × 100

Figure 5

Patient CDMD8011 (del 3–7) expresses low levels of dystrophin protein in muscle biopsy and primary myoblasts expanded and fused to myotubes in culture. (A) Dystrophin is visible at the sarcolemma in transverse sections of muscle in CDMD8011 and CDMD8006 (wild‐type) when stained with DysI (central rod domain) or Dys2 (C‐terminal). Magnification, 10×, 20× for Dys2 CDMD8011. (B) Representative images of fused myotubes showed low amounts of dystrophin in CDMD8011 and higher levels in CDMD8006. Mandys8 stains the central rod domain of DMD. Nuclei are colored blue (Dapi). Scale bar 50 μm