Skeletal muscle regeneration in facioscapulohumeral muscular dystrophy is correlated with pathological severity

Figure 1. The Myogenesis score is higher in DMD. (A) A box plot shows that the Myogenesis score is higher in RNA-seq data from healthy control myotubes than from healthy control myoblasts. (B) Box plot showing that the Myogenesis score is also elevated in transcriptomic data from FSHD1 myotubes compared to FSHD1 myoblasts. The box represents the interquartile range (IQR), with the median indicated by a line. Whiskers denote min [1.5*IQR, max (observed value)]. ‘o’ represents data points greater than 1.5 IQR from the median, n = 15 control, and n = 18 FSHD, myoblasts (MBs) and myotube (MTs) datasets. P values are given. (C) A forest plot shows that the Myogenesis score is elevated across all four published DMD muscle biopsy data sets and on meta-analysis (n = 61 DMD and n = 47 control muscle biopsies). The differential scores (DMD score minus control score) alongside 95% confidence intervals are provided. For single studies, a two-tailed Wilcoxon U-test was performed to assess significance, where an asterisk denotes P < 0.05, two asterisks denote P < 0.005, three asterisks denote P < 0.0005 and four asterisks denote P < 0.00005. A Fisher’s combined test was employed for overall assessment…

Figure 2. The Myogenesis score is elevated in FSHD muscle biopsies. (A) A forest plot shows that the Myogenesis score is elevated in FSHD muscle biopsies compared to relevant controls in four of seven independent published studies and on meta-analysis across the seven FSHD muscle biopsy data sets (n = 130 FSHD and n = 98 control muscle biopsies). The differential scores (FSHD score minus control score) alongside 95% confidence intervals are provided. For single studies, a two-tailed Wilcoxon U-test was performed to assess significance, where asterisks denote P < 0.05. A Fisher’s combined test was employed for overall assessment, where hash indicates P = 3.23 × 10−7. (B) A ROC curve compares the discriminatory power of the Myogenesis score across the pooled seven FSHD muscle biopsy datasets. The AUC is 0.761, demonstrating that the Myogenesis score is a good discriminator of FSHD status. (C) Box plot illustrates that the Myogenesis score is significantly lower in RNA-seq data from FSHD myoblasts compared to control myoblasts. (D) Box plot shows that the Myogenesis score is not significantly different between FSHD and control myotubes. Boxes represent the IQR, with the median indicated by a…

Figure 3. DM2 muscle biopsies have a higher Myogenesis score than controls. (A) A forest plot reveals that the Myogenesis score is elevated in three of five published DM2 muscle biopsy data sets and on meta-analysis (n = 52 DM2 and n = 35 control muscle biopsies). The differential scores (DM2 score minus control score) alongside 95% confidence intervals are provided. For single studies, a two-tailed Wilcoxon U-test was performed to assess significance, while a Fisher’s combined test was employed for overall assessment: asterisks denote P < 0.05. (B) A ROC curve compares the discriminatory power of the Myogenesis score across the pooled five DM2 muscle biopsy datasets. The AUC is 0.859, demonstrating that the Myogenesis score is a very good discriminator of DM2 status.

Figure 4. Regenerating muscle fibres are present in most FSHD muscle biopsies. (A) Histopathological section from a quadriceps biopsy from a FSHD patient immunolabelled for Novocastra NCL-MHCd (Clone RNMy2/9D2). Regenerating myofibres containing Dev MyHC are highlighted by arrows or a blue box. (B) Adjacent section to that in (A) stained with H&E with the same Dev MyHC-containing regenerating muscle fibres again highlighted. (C) Area delimited by the blue box in (A) at higher magnification to better show the two Dev MyHC-containing regenerating muscle fibres. (D) Area delimited by blue box from (B) shows that the Dev MyHC-containing muscle fibres have histological feature associated with regeneration, including more basophilic stippling in the cytoplasm due to the increased RNA content and big, plump, less dense myonuclei, associated with a more active nucleus. Area delimited by white dashed line encompasses a necrotic muscle fibre undergoing phagocytosis, as the cytoplasm appears fragmented, without the usual uniform pink eosinophilic staining. (E) Dev MyHC-containing regenerating muscle fibre in an area of muscle exhibiting less overt signs of pathology. (F) Tibialis anterior section…

Figure 5. Regenerating muscle fibres in DM2 muscle biopsies. (A) Histopathological section from a quadriceps biopsy of a DM2 patient immunolabelled for Novocastra Clone RNMy2/9D2. A regenerating muscle fibre containing Dev MyHC is highlighted by an arrow. (B) Adjacent section from the same muscle as in (A), stained with H&E with the regenerating muscle fibre indicated by an arrow. (C) and (D) Dev MyHC immunolabelled muscle biopsy sections from tibialis anterior muscles from two individuals with DM2, with Dev MyHC-containing regenerating myofibres highlighted by arrows. Scale bars represent approximately 30 μm.