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 DUX4^POS 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 DUX4^POS 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 PAX7^HIGH (i.e. likely non-affected) and the ten FSHD samples with lowest PAX7 scores were classified as PAX7^LOW (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 the two major sequence batches are labeled and highlighted in color. (c) Receiver Operator Characteristic curve for FSHD versus control biopsy classification with either DUX4 or PAX7 signature expression. AUC; area under curve. p-values depict the results of a Student’s t-test (excluding the replicate samples). p-values: ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All data plots are generated in R (v4.0.3, www.R-project.org) using the packages gplots (v3.1.1), ggplot2 (v3.3.3) and pROC (v1.17.0.1). Figure and panel layout was further adapted in Adobe Illustrator CC 2018 (www.adobe.com).