Facioscapulohumeral dystrophy transcriptome signatures correlate with different stages of disease and are marked by different MRI biomarkers

Figure 1. Representative examples of the two MRI-based imaging biomarkers used in this study; fatty infiltration and TIRM hyperintensity. (a) Axial T1-weighted and (b) TIRM image of the left upper leg of a 50-year-old FSHD patient (FSHD-09) showing marked fatty infiltration of nearly all muscles and focal hyperintensity in the VL muscle. The MRI-guided biopsy sites are marked with the yellow circles. In T1: Normal muscle is dark grey, fat infiltrated muscle is white. Note the relative sparing of the sartorius muscle and the severe fatty infiltration of the posterior compartment and quadriceps. (c) HPhlox staining of the FatPOS/TIRMPOS biopsy from the same patient demonstrates severe dystrophic changes indicated by a marked increase in fiber size variability, increased internal nuclei, regenerating fibers and fatty infiltration. (d) HPhlox staining of the FatPOS/TIRMNEG biopsy from the same patient shows fiber size variability, increased internal nuclei and few regenerating fibers corresponding to mild dystrophic changes. (e) TIRM hyperintensity frequencies in muscle biopsies of each disease state. p-values depict a Fisher’s exact test result (excluding the replicate samples). (f) Fat…

Figure 2. DUX4 and PAX7 signature expression in FSHD and control biopsies. (a) DUX4 signature expression in FSHD and control muscle biopsies. The threshold criterium for DUX4POS biopsy selection (cumulative normalized read count > 20) is marked with a red dashed line and the boxplots on the right depict the FSHD biopsies separated based on this classification. The two replicate FSHD samples sequenced in the two major sequence batches are highlighted in color. Both replicates are selected as DUX4POS in both sequence batches, indicating that the absence of a DUX4 signature in the controls is not due to a sequencing bias. (b) PAX7 signature expression in FSHD and control muscle biopsies. As a classification threshold for PAX7-affected versus non-affected biopsies could not be clearly defined based on the scores in control samples, for further subgroup comparisons the ten FSHD samples with the highest PAX7 score were classified as PAX7HIGH (i.e. likely non-affected) and the ten FSHD samples with lowest PAX7 scores were classified as PAX7LOW (i.e. most-affected). The boxplots on the right depict the FSHD biopsies included based on this classification. The two replicate FSHD samples sequenced in…

Figure 3. Linear correlation analysis between al metadata in this study’s dataset. (a) Pearson correlation scores r (top right values) and Spearman’s rank correlation scores rs (bottom left) for all comparisons between the two molecular signatures (DUX4 and PAX7 signatures), the two imaging-based biomarkers (TIRM hyperintensity and fat fraction) and all metadata of our dataset (see also methods). (b) p-values for all correlations. Color-coding follows the correlation values and specific scores are indicated in each box. Grey boxes indicate that no linear correlation score could be calculated. Note that all results for non-muscle-specific metadata (i.e. age, age at onset, disease duration, group, CSS, D4Z4 repeat size, BMI, sex, 6-MWT and MFM) may be biased by duplicate samples for participants that donated a muscle biopsy from both the TA and VL muscle. The two duplicate VL muscle biopsies from participant FSHD-09 and FSHD-13 are also included in the data. This analysis indicates the strength of linear (for Pearson) and monotonic (for Spearman) correlations. For some metadata, other correlations may still apply. See also Supplementary Figs. S3 and S4 for detailed visualization of all…

Figure 4. Correlations between DUX4 and PAX7 signature expression and TIRM hyperintensity and fat fraction. (a–c) DUX4 signature expression levels in control biopsies and FSHD biopsies separated based on imaging biomarker classifications, with in (a) TIRM hyperintensity, in (b) Fat fraction (FatPOS: > 15% Fat), an in (c) the combined biomarker (TIRMPOS and/or FatPOS). The threshold criterium for DUX4POS biopsy selection (cumulative normalized read count > 20) is marked with a red dashed line. The stacked bar plots on the right depict the relative frequency of DUX4POS biopsy classification in each subgroup. The numbers indicate the true frequencies. (d–f) PAX7 scores in control biopsies and FSHD biopsies separated based on imaging biomarker classifications, with in (d) TIRM hyperintensity, in (e) Fat fraction (FatPOS: > 15% Fat), an in (f) the combined biomarker (TIRMPOS and/or FatPOS). All p-values for the quantitative comparison of signature expression (boxplots) depict the results of a Student’s t-test comparing imaging biomarker-positive versus imaging biomarker-negative FSHD muscle biopsies respectively. For the frequency plots, the result of a Fisher’s exact test is depicted. For…

Figure 5. Significant non-muscle cell type contributions in FSHD, and their correlation with DUX4 and PAX7 signature expression. (a) Estimated relative contributions for all muscle and non-muscle cell types that show significantly different contributions in FSHD versus control muscle biopsies. See Supplemental Fig. S8 for the results of all (significant and non-significant) identified cell types. Results are based on the RNA deconvolution analysis (see “Methods” for details on PLIER analysis), based on cell types previously identified by Rubenstein AB et al.45 (in healthy human muscle biopsies). LV: latent vector best representing the respective cell type signature noted behind the LV number. FBN1 + FAPs: Fibrilin-1 positive fibro-adipogenic progenitors. LUM + FAPs: lumican-positive fibro-adipogenic progenitors. PCV-Endothelial cells: post-capillary venules endothelial cells. (b, c) Estimated relative contribution of the Type IIa myofiber content in controls and FSHD muscle biopsies showing a reduction in Type IIa myofiber content in both DUX4POS versus DUX4NEG FSHD biopsies (b) and in PAX7LOW versus PAX7HIGH FSHD biopsies (c). (d, e) Estimated relative contribution of the LUM+ FAP subtype…