Comprehensive genetic analysis of facioscapulohumeral muscular dystrophy by Nanopore long-read whole-genome sequencing

Facioscapulohumeral muscular dystrophy (FSHD) is a high-prevalence autosomal dominant neuromuscular disease characterized by significant clinical and genetic heterogeneity. Genetic diagnosis of FSHD remains a challenge because it cannot be detected by standard sequencing methods and requires a complex diagnosis workflow. We developed a comprehensive genetic FSHD detection method based on Oxford Nanopore Technologies (ONT) whole-genome sequencing. Using a case-control design, we applied this procedure to 29 samples and compared the results with those from optical genome mapping (OGM), bisulfite sequencing (BSS), and whole-exome sequencing (WES). Using our ONT-based method, we identified 59 haplotypes (35 4qA and 24 4qB) among the 29 samples (including a mosaic sample), as well as the number of D4Z4 repeat units (RUs). The pathogenetic D4Z4 RU contraction identified by our ONT-based method showed 100% concordance with OGM results. The methylation levels of the most distal D4Z4 RU and the double homeobox 4 gene (DUX4) detected by ONT sequencing are highly consistent with the BSS results and showed excellent diagnostic efficiency. Additionally, our ONT-based method provided an independent methylation profile analysis of two permissive 4qA alleles, reflecting a more accurate scenario than traditional BSS. The ONT-based method detected 17 variations in three FSHD2-related genes from nine samples, showing 100% concordance with WES. Our ONT-based FSHD detection method is a comprehensive method for identifying pathogenetic D4Z4 RU contractions, methylation level alterations, allele-specific methylation of two 4qA haplotypes, and variations in FSHD2-related genes, which will all greatly improve genetic testing for FSHD.