Single-cell Transcriptome Analyses in Facioscapulohumeral Muscular Dystrophy


Single Cell Analysis Approaches

Poster Number: 199


Lawrence Hayward, MD, PhD, Dongsheng Guo, PhD, Kathryn Wagner, MD, Oliver King, PhD, Charles Emerson Jr., PhD


1. University of Massachusetts Medical School, 2. University of Massachusetts Medical School, 3. Kennedy Krieger Institute, Johns Hopkins School of Medicine, Baltimore, MD, USA, 4. University of Massachusetts Medical School, 5. University of Massachusetts Medical School

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) causes progressive muscle wasting triggered by aberrant de-repression of DUX4, a pro-apoptotic transcription factor, in <1:200 muscle cells. A single-cell approach may be informative to gain mechanistic insights regarding FSHD from these rare cells.

OBJECTIVES: We are using single-cell analyses to characterize i) infrequent cells expressing DUX4 during distinct stages of myogenic reprogramming, ii) cellular heterogeneity among FSHD models and acutely isolated cells from affected muscle, and iii) immune cell phenotypes in FSHD muscle. We aim to define gene co-expression networks in subpopulations that may suggest novel FSHD therapeutic targets or biomarkers.

APPROACH: 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.

RESULTS: 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.

CONCLUSIONS: These results may provide insight into mechanisms contributing to the wide variance in disease severity among early-onset, adult-onset, and non-manifesting FSHD muscles and among corresponding iPSC myogenic lineages.

Support: Muscular Dystrophy Association, University of Massachusetts Wellstone MDCRC