A Genotype-Phenotype Correlation Study of Exon Skip-Equivalent In-Frame Deletions and Exon Skip-Amenable Out-of-Frame Deletions across the DMD Gene to Simulate the Effects of Exon-Skipping Therapies: A Meta-Analysis

Abstract

Dystrophinopathies are caused by mutations in the DMD gene. Out-of-frame deletions represent most mutational events in severe Duchenne muscular dystrophy (DMD), while in-frame deletions typically lead to milder Becker muscular dystrophy (BMD). Antisense oligonucleotide-mediated exon skipping converts an out-of-frame transcript to an in-frame one, inducing a truncated but partially functional dystrophin protein. The reading frame rule, however, has many exceptions. We thus sought to simulate clinical outcomes of exon-skipping therapies for DMD exons from clinical data of exon skip-equivalent in-frame deletions, in which the expressed quasi-dystrophins are comparable to those resulting from exon-skipping therapies. We identified a total of 1298 unique patients with exon skip-equivalent mutations in patient registries and the existing literature. We classified them into skip-equivalent deletions of each exon and statistically compared the ratio of DMD/BMD and asymptomatic individuals across the DMD gene. Our analysis identified that five exons are associated with significantly milder phenotypes than all other exons when corresponding exon skip-equivalent in-frame deletion mutations occur. Most exon skip-equivalent in-frame deletions were associated with a significantly milder phenotype compared to corresponding exon skip-amenable out-of-frame mutations. This study indicates the importance of genotype-phenotype correlation studies in the rational design of exon-skipping therapies.

Keywords: becker muscular dystrophy (BMD); duchenne muscular dystrophy (DMD); dystrophin; dystrophinopathy; exon skipping; reading frame rule; skip-equivalent deletions.

Figure 1

Flow chart showing database screening and literature search procedure to collect patients’ clinical information with confirmed DMD large deletions. N indicates the number of individuals present in each data source.

Figure 2

Clinical phenotypes of exon skip-equivalent in-frame DMD exon deletions. (A) Association between in-frame deletions starting and/or ending at each exon (exon skip-equivalent) and consequent phenotypes. Phenotypic ratios associated with in-frame deletions starting and/or ending at a given exon and all other exons were compared using Fisher’s exact test. n indicates the number of individuals with DMD (red) and milder (blue; BMD and asymptomatic) phenotypes. Green and red color indicate a significantly lower and higher incidence of DMD phenotype for a given exon, respectively, as compared to the overall incidence rate. p = p-value, as calculated by Fisher’s exact test; p* = Benjamini–Hochberg adjusted p-value. (B) Heatmap showing the relative severity of the consequence of in-frame deletions starting and/or ending at specific exons.

Figure 3

Phenotypic outcomes of exon skip-amenable mutations present in the UMD-DMD France database. (A) Distribution of phenotypes for each exon skip-amenable mutation. Phenotypic ratios associated with exon skip-amenable mutations for each exon vs. all other exons are compared using Fisher’s exact test. n indicates the number of individuals with DMD (red) and milder (blue; BMD and asymptomatic) phenotypes. Green color indicates a significantly lower incidence of DMD phenotype. p = p-value, as calculated by Fisher’s exact test; p* = Benjamini–Hochberg adjusted p-value. (B) Heatmap showing the relative severity of the consequence of out-of-frame mutations amenable to skipping of each exon.

Figure 4

Comparison of clinical phenotypes associated with out-of-frame mutations amenable to exon skipping in UMD-DMD database and in-frame exon skip-equivalent deletions of each exon to simulate the effects of exon-skipping therapies. (A) Phenotypic outcomes associated with mutations amenable to exon skipping and exon skip-equivalent in-frame deletions of each exon and their consequent phenotypes. n indicates the number of individuals with DMD (red) and milder (blue; BMD and asymptomatic) phenotypes. Asterisks indicate that exon skip-equivalent in-frame deletions are associated with a significantly milder phenotype compared to corresponding exon skip-amenable out-of-frame mutations. We compared the incidence of DMD associated with exon skip-equivalent (or group of exons, e.g., exons 3–9, and exons 45–55) and mutations amenable to skipping each exon (or group of exons, e.g., exons 3–9, and exons 45–55). The statistical significance was calculated using Fisher’s exact test. (B) Heatmap showing the relative severity of the consequence of exon skip-equivalent in-frame deletions and exon skip-amenable mutations. * p < 0.05, ** p < 0.01, *** p < 0.001

Figure 4

Comparison of clinical phenotypes associated with out-of-frame mutations amenable to exon skipping in UMD-DMD database and in-frame exon skip-equivalent deletions of each exon to simulate the effects of exon-skipping therapies. (A) Phenotypic outcomes associated with mutations amenable to exon skipping and exon skip-equivalent in-frame deletions of each exon and their consequent phenotypes. n indicates the number of individuals with DMD (red) and milder (blue; BMD and asymptomatic) phenotypes. Asterisks indicate that exon skip-equivalent in-frame deletions are associated with a significantly milder phenotype compared to corresponding exon skip-amenable out-of-frame mutations. We compared the incidence of DMD associated with exon skip-equivalent (or group of exons, e.g., exons 3–9, and exons 45–55) and mutations amenable to skipping each exon (or group of exons, e.g., exons 3–9, and exons 45–55). The statistical significance was calculated using Fisher’s exact test. (B) Heatmap showing the relative severity of the consequence of exon skip-equivalent in-frame deletions and exon skip-amenable mutations. * p < 0.05, ** p < 0.01, *** p < 0.001