Single-cell RNA sequencing in facioscapulohumeral muscular dystrophy disease etiology and development

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is characterized by sporadic de-repression of the transcription factor DUX4 in skeletal muscle. DUX4 activates a cascade of muscle disrupting events, eventually leading to muscle atrophy and apoptosis. Yet, how sporadic DUX4 expression leads to the generalized muscle wasting remains unclear. Transcriptome analyses have systematically been challenged by the majority of nuclei being DUX4 neg , weakening the DUX4 transcriptome signature. Moreover, DUX4 has been shown to be expressed in a highly dynamic burst-like manner, likely resulting in the detection of the downstream cascade of events long after DUX4 expression itself has faded. Identifying the FSHD transcriptome in individual cells and unraveling the cascade of events leading to FSHD development may therefore provide important insights in the disease process. We employed single-cell RNA sequencing, combined with pseudotime trajectory modeling, to study FSHD disease etiology and cellular progression in human primary myocytes. We identified a small FSHD-specific cell population in all tested patient-derived cultures and detected new genes associated with DUX4 de-repression. We furthermore generated an FSHD cellular progression model, ref lecting both the early burst-like DUX4 expression as well as the downstream activation of various FSHD-associated pathways, which allowed us to correlate DUX4 expression signature dynamics with that of regulatory complexes, thereby facilitating the prioritization of epigenetic targets for DUX4 silencing. Single-cell transcriptomics combined with pseudotime modeling thus holds valuable information on FSHD disease etiology and progression that can potentially guide biomarker and target selection for therapy.

Lymphocytes contribute to DUX4 target genes in FSHD muscle biopsies

“…1B). In a recent single cell RNA-seq of combined FSHD1 and FSHD2 unfused myocytes, we found DUX4 expression in 27/5133 (0.5%) FSHD cells 32 .…”

“…We previously evaluated the discriminatory power of the 3 DUX4 target gene scores on unfused FSHD myocytes profiled by single cell RNA-seq, and although significant discriminators, no score achieved an AUC>0.56 24 . However, 27/5133 myocytes expressed DUX4 32 , which offers greater power for assessment of DUX4 association with the target gene scores in differentiated muscle cells, compared to the 3 DUX4 positive myotube samples. Surprisingly, while the early and late DUX4 target gene expression scores correlated in this single cell data set (Choi et al, vs Geng et al, p< 2.2×10 −16 , Pearson’s r =0.54, Choi et al, vs Yao et al, p <2.2×10 −16 , Pearson’s r= 0.38, Geng et al, vs Yao et al, p <2.2×10 −16 , Pearson’s r= 0.86 Fig.…”

“…Myoblasts and in vitro differentiated myotube data are either new data (primary cell lines) or data previously published by ourselves 23,36 . Single cell RNA-seq of FSHD and control myocytes was previously published by van den Heuvel et al, 2019 32 . A sample was assessed as DUX4 positive if a single detectable DUX4 read was found in normalised RNA-seq data.…”

“…A muscle cell contribution to DUX4 in FSHD muscle biopsies is also likely, but via dynamic, stochastic DUX4 expression rather than the continuous expression seen in lymphocytes 32,40 . Time points for these transient bursts of DUX4 expression could include in satellite cells, when DUX4 expression may interfere with the normal function of the transcription factor PAX7 19,23,24 , and during myogenic differentiation, where DUX4 target gene expression may interfere with normal myogenesis 32,40,48 . Here we provide evidence for transient DUX4 expression at both these time points.…”

“…DUX4 can induce expression of immune system-related genes in myoblasts 20 , and inflammatory genes are dysregulated in FSHD muscle biopsies 21,32 . Recently a large library of 114 control and FSHD lymphoblastoid cell lines (LCLs) (EBV immortalised B-lymphocytes) from 12 FSHD1 affected families was published 33 .…”