Evaluating Genetic Modifiers of Duchenne Muscular Dystrophy Disease Progression Using Modeling and MRI

Abstract

Background and objectives: Duchenne muscular dystrophy (DMD) is a progressive muscle degenerative disorder with a well-characterized disease phenotype but considerable interindividual heterogeneity that is not well understood. The aim of this study was to evaluate the effects of dystrophin variations and genetic modifiers of DMD on rate and age of muscle replacement by fat.

Methods: One hundred seventy-five corticosteroid treated participants from the ImagingDMD natural history study underwent repeated magnetic resonance spectroscopy (MRS) of the vastus lateralis (VL) and soleus (SOL) to determine muscle fat fraction (FF). MRS was performed annually in most instances; however, some individuals had additional visits at 3 or 6 monthss intervals. FF changes over time were modeled using nonlinear mixed effects to estimate disease trajectories based on the age that the VL or SOL reached half-maximum change in FF (mu) and the time required for FF change (sigma). Computed mu and sigma values were evaluated for dystrophin variations that have demonstrated the ability to lead to a mild phenotype as well as compared between different genetic polymorphism groups.

Results: Participants with dystrophin gene deletions amenable to exon 8 skipping (n = 4) had minimal increases in SOL FF and had an increase in VL mu value by 4.4 years compared with a reference cohort (p = 0.039). Participants with nonsense variations within exons that may produce milder phenotypes (n = 11) also had minimal increases in SOL and VL FFs. No differences in estimated FF trajectories were seen for individuals amenable to exon 44 skipping (n = 10). Modeling of the SPP1, LTBP4, and thrombospondin-1 (THBS1) genetic modifiers did not result in significant differences in muscle FF trajectories between genotype groups (p > 0.05); however, trends were noted for the polymorphisms associated with long-range regulation of LTBP4 and THBS1 that deserve further follow-up.

Discussion: The results of this study link the historically mild phenotypes seen in individuals amenable to exon 8 skipping and with certain nonsense variations with alterations in trajectories of lower extremity muscle replacement by fat.

Figure 1. Study Flow Diagram

Overall, there were 175 corticosteroid-treated participants included in this study. Of those participants, the dystrophin variation was known for 169. Exonic deletions were the most common variations in the cohort, followed by nonsense variations. N = 143 participants consented to DNA collection to evaluate genetic modifier polymorphisms. After removing individuals with exon 3–7 mutations, exon 45 deletions, and nonsense variations that can produce DMD or BMD phenotypes, there were N = 125 participants included for analysis. All of these participants were genotyped for LTBP4 and SPP1 polymorphisms while a subset with additional DNA were also genotyped for rs710160 upstream of LTBP4 and rs272577 upstream of THBS1. Abbreviations: BMD = Becker muscular dystrophy; DMD = Duchenne muscular dystrophy; THBS1 = thrombospondin-1.

Figure 2. Dystrophin Variations Associated With Slowly Progressing Individuals

When modeling disease trajectory for the entire cohort, the identified individuals had the largest mu values indicating older ages at (A) SOL and (B) VL half-maximum ΔFF. Their individual trajectories were right shifted compared with the overall cohort (black dashed line). These slowly progressing individuals had a variety of dystrophin variations including some associated with milder phenotypes and others associated with typical DMD phenotypes. (C) A T₁-weighted image of the thigh from an individual with disease progression typical of the whole cohort compared with (D) the thigh of the slow progressor with the PSC in exon 34, at the same age (∼13 years). Abbreviations: DMD = Duchenne muscular dystrophy; DEL = deletion; FF = fat fraction; PSC = premature stop codon; SOL = soleus; VL = vastus lateralis.

Figure 3. FF Modeling of Variations Amenable to Exon 8 Skipping and Nonsense Variations

Individuals with dystrophin variations amenable to exon 8 skipping had significantly slower disease progression by (A) SOL and (B) VL muscle FF modeling (blue lines) than the rest of the ImagingDMD cohort (dashed black line). Individual trajectories of muscle FF for each participant (gray lines) demonstrate almost no increase in SOL muscle FF over time and increases in VL FF that occur later than typically expected. Similarly, modeling of (C) SOL and (D) VL muscle FF trajectories in individuals with premature stop codons within exons that have been shown to potentially produce a milder phenotype showed significantly slowed disease progression. (8 Skips = individuals with variations amenable to exon 8 skipping. Abbreviations: DMD = Duchenne muscular dystrophy; FF = fat fraction; PSC = premature stop codons, SOL = soleus, VL = vastus lateralis.

Figure 4. FF Modeling of Variations Amenable to Exon 44 Skipping

(A and B) The modeled SOL and VL FF trajectories for the individuals amenable to exon 44 skipping (blue lines) did not differ significantly from modeled trajectories from the remainder of the cohort (black dashed line). Actual FF trajectories from the individual participants with variations amenable to exon 44 skipping (gray lines) showed variability; therefore, the modeled trajectories for each individual were plotted separately for the SOL (C) and VL (D). Two individuals with exon 45 deletions had trajectories that visually appeared to deviate from the rest while the remaining individuals with variations amenable to exon 44 skipping had trajectories typical of DMD. Abbrebiations: 44 skips = individuals amenable to exon 44 skipping; DMD = Duchenne muscular dystrophy; DEL = deletion; FF = fat fraction, SOL = soleus, VL = vastus lateralis.

Figure 5. Modifying Effects of Genetic Polymorphisms

(A) There was no modifying effect of SPP1 genotype SOL or VL fat fraction modeled trajectories. (B) Similarly, no significant modifying effect was observed for LTBP4 genotype. (C) For rs710160 (LTBP4) and (D) rs272579 (THBS1), there were no significant differences in mu or sigma parameters between genotype groups; however, there were small absolute changes in parameters that may warrant further investigation. In the figure, the blue trajectories indicate the genotype previously associated with older age at loss of ambulation while the red trajectories indicate the genotype previously associated with younger age at loss of ambulation. Abbrebiations: THBS1 = thrombospondin-1; VL = vastus lateralis.