Abstract
Facioscapulohumeral muscular dystrophy (FSHD) is hallmarked by the sporadic expression of the germline and cleavage-stage transcription factor DUX4 in myonuclei of affected muscle. Despite the sporadic nature of DUX4 expression, its presence in muscle activates a cascade of muscle disrupting events eventually leading to muscle atrophy and apoptosis of affected cells. Yet, with an estimated ratio of 1:100 - 1:1000 nuclei expressing DUX4 in primary myotube cultures, transcriptome analyses have systematically been challenged by the majority of nuclei being negative for DUX4 expression, weakening the DUX4 transcriptome signatures. More elaborate analysis of the FSHD-transcriptome has thus far been facilitated by DUX4-overexpression or DUX4-reporter systems biasing the data towards DUX4-associated pathways and limiting the analysis of non-cell autonomous effects. Identifying the ``pure'' FSHD transcriptome, i.e. including the study of cell-autonomous and non-cell autonomous DUX4 effects, and unraveling the cascade of events leading to FSHD development, has so far been very difficult. We employed single-cell RNA-sequencing (scRNAseq), combined with pseudotime trajectory modeling, to study FSHD disease etiology and cellular progression in human patient-derived primary myocytes. We identified a small FSHD-specific cell population in all 4 tested FSHD patient-derived primary cultures and detected promising new genes that are likely related to direct and indirect effects of DUX4. Furthermore, capitalizing on the heterogeneity in cellular state in FSHD cell cultures, we employed pseudotime trajectory modeling to generate detailed insights into FSHD etiology and cellular progression. We expect that pseudotime trajectories like our FSHD pseudotime model hold invaluable information not only for studying disease etiology, development and progression, but also for biomarker identification and therapeutic target selection
Published Version
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