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 Fat^POS/TIRM^POS 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 Fat^POS/TIRM^NEG 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 fractions of all individual muscle biopsies grouped by disease state. p-values depict a Mann–Whitney U test results (excluding the replicate samples). The red dashed line indicates the classification threshold for Fat^POS versus Fat^NEG FSHD muscle biopsies (15% fat fraction) and the boxplots on the right depict the FSHD biopsies separated based on this classification. p-values: ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. MRI scans were exported from Agfa IMPAX (https://global.agfahealthcare.com). Histology sections were digitized using a Philips UFS (www.usa.philips.com/healthcare/resources/landing/philips-intellisite-pathology-solution) and images were assessed using 3DHISTECH’s CaseViewer (v2.3, www.3dhistech.com). All data plots are generated in R (v4.0.3, www.R-project.org) using the packages gplots (v3.1.1) and ggplot2 (v3.3.3). Figure and panel layout was further adapted in Adobe Illustrator CC 2018 (www.adobe.com).