Increased METTL3 Expression and m6A Methylation in Myoblasts of Facioscapulohumeral Muscular Dystrophy.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by the aberrant expression of the double homeobox 4 (DUX4) gene. In this study, an analysis of human FSHD muscle biopsies revealed differential expressions of six m6A regulators, including writers, readers and eraser proteins. In immortalized human FSHD myoblasts, we found higher levels of mRNA and protein expression of a major m6A regulator, methyltransferase-like protein 3 (METTL3), in comparison with myoblasts from unaffected siblings (UASbs). Quantification of the overall RNA m6A levels in the FSHD myoblasts revealed significant elevation compared with their UASb, which was reversed to UASb levels following treatment with an antisense oligonucleotide targeting the DUX4 mRNA. Using Oxford Nanopore direct-RNA sequencing, we mapped m6A across the transcriptome and identified genes harboring differential methylated m6A sites, including several involved in iron homeostasis. Western blot protein quantification showed that FSHD myoblasts had higher levels of ferritin-heavy chain-207 isoform and mitoferrin-1. In addition, our data showed elevation in mitochondrial ferrous iron in FSHD myoblasts. Our findings suggest that m6A RNA modifications play a pivotal role in FSHD pathophysiology and may serve as biomarker for this disease.