to define gene co-expression networks in subpopulations that may suggest novel FSHD therapeutic targets or biomarkers. We induced iPSC-derived myogenic lineages from 8 individuals (2 with early-onset FSHD, 3 with late-onset FSHD, and 3 controls) using a gene-free directed differentiation protocol. Single-cell RNA-seq reads were obtained using a 10X platform and aligned to the human reference genome GRCh38 using STAR. Cell clustering and downstream expression analyses were performed using Seurat v3 and Markov affinity-based imputation. Proliferating myogenic progenitors (S1 cells), primary myoblasts (S2 cells), and induced secondary myoblasts (iSM cells) showed distinct single-cell gene expression patterns. S1 cells with 2-4 D4Z4 repeats (74I and 85I, early-onset phenotype) showed a higher frequency of DUX4 biomarker expression compared to S1 cells harboring 5-8 D4Z4 repeats (15A and 17A, adult-onset phenotype). Proliferating iSM cells and biopsy-derived cells did not express detectable DUX4 biomarkers. Further studies are in progress to characterize phenotypes of immune cells isolated from FSHD tissues. These results may provide insight into mechanisms contributing to the wide variance in disease severity among early-onset, adultonset, and non-manifesting FSHD muscles and among corresponding iPSC myogenic lineages.
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