Proximity ligation assay to detect DUX4 protein in FSHD1 muscle: a pilot study

OBJECTIVE: Aberrant expression in skeletal muscle of DUX4, a double homeobox transcription factor, underlies pathogenesis in facioscapulohumeral muscular dystrophy (FSHD). Although previous studies of FSHD muscle biopsies detected mRNAs encoding DUX4 and its target genes, no studies had reported detection of DUX4 protein. Our objective was to develop a proximity ligation assay (PLA) for DUX4 and to determine if this assay could detect DUX4 protein in FSHD muscle sections. RESULTS: We developed a PLA protocol using two DUX4 antibodies previously reported by Stephen Tapscott’s group: P2G4, a mouse mAb specific for an epitope in the N-terminal region, and E5-5, a rabbit mAb specific for an epitope in the C-terminal region, in combination with commercial PLA secondary reagents. We validated the DUX4 PLA using cultured human myogenic cells in which DUX4 was ectopically expressed in a small fraction of nuclei. Using this two primary mAb PLA on an FSHD1 biceps biopsy, we observed nuclei with apparent DUX4 PLA signals associated with a small subset of myofibers (~ 0.05–0.1%). Though a limited pilot study, these results suggest that the two primary mAb PLA protocol could be useful for detecting DUX4 protein in FSHD muscle biopsies. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13104-022-06054-8.