Alternative splicing dysregulation across tissue and therapeutic approaches in a mouse model of myotonic dystrophy type 1

Abstract

Myotonic dystrophy type 1 (DM1), the leading cause of adult-onset muscular dystrophy, is caused by a CTG repeat expansion. Expression of the repeat causes widespread alternative splicing (AS) defects and downstream pathogenesis, including significant skeletal muscle impacts. The HSA ^LR mouse model plays a significant role in therapeutic development. This mouse model features a transgene composed of approximately 220 interrupted CTG repeats, which results in skeletal muscle pathology that mirrors DM1. To better understand this model and the growing number of therapeutic approaches developed with it, we performed a meta-analysis of publicly available RNA sequencing data for AS changes across three widely examined skeletal muscles: quadriceps, gastrocnemius, and tibialis anterior. Our analysis demonstrated that transgene expression correlated with the extent of splicing dysregulation across these muscles from gastrocnemius (highest), quadriceps (medium), to tibialis anterior (lowest). We identified 95 splicing events consistently dysregulated across all examined datasets. Comparison of splicing rescue across seven therapeutic approaches showed a range of rescue across the 95 splicing events from the three muscle groups. This analysis contributes to our understanding of the HSA ^LR model and the growing number of therapeutic approaches currently in preclinical development for DM1.

Keywords: DM1; HSALR; MT: Bioinformatics; RNA sequencing; RNA-seq; alternative splicing; therapeutics.

Graphical abstract

Figure 1

Tissue-specific differences in transgene expression and AS dysregulation in the HSA^LR mouse model (A) ACTA1 transgene expression in gastrocnemius (GA, blue, squares), quadriceps (Q, green, diamonds), and TA (orange, circles) tissues from HSA^LR mice from each RNA-seq dataset. Data normalized to Gtf2b mRNA. Bars represent mean ± SD of biological replicates. (B) ACTA1 transgene expression grouped by tissue type and statistically compared. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001. (C) Absolute inclusion level difference (|Δ Inclusion|) of 95 common and dysregulated (FDR ≤0.05; |Δ Inclusion| ≥ 0.1) AS events among all datasets. (D) Positive correlation of |Δ Inclusion| to ACTA1 expression normalized to Gtf2b (Pearson r = 0.7127; p = 0.0138).

Figure 2

Tissue-specific differences in AS for key events in DM1 and the HSA^LR mouse model (A) Atp2a1 exon 22, (B) Clasp1 exon 20, (C) Neb exon 149, (D) Clcn1 exon 7a, (E) Nfix exon 7, and Mbnl1 exon 5 (F) mis-splicing events display tissue-specific differences in PSI. WT samples are colored in gray; untreated HSA^LR mice are colored and shaped by tissue type where gastrocnemius (GA) are blue squares, quadriceps (Q) are green diamonds, and TA are orange circles. Black whiskers depict mean ± SD for sample replicates.

Figure 3

Treatment effects of published therapeutics in the HSA^LR model (A) Distribution of percent rescue scores among the dysregulated AS events common to all datasets. PSI values for WT, HSA^LR, and treated-HSA^LR groups are shown for the individual events (B) Atp2a1 exon 22, (C) Clcn1 exon 7a, and (D) Mbnl1 exon 5. Black whiskers depict mean ± SD for sample replicates.