Integrated genetic and epigenetic diagnosis of facioscapulohumeral muscular dystrophy using Oxford Nanopore long-read sequencing.

Facioscapulohumeral muscular dystrophy (FSHD) is a genetically and epigenetically complex disorder in which the comprehensive characterization of the D4Z4 repeat array remains challenging. We applied Oxford Nanopore long-read sequencing (ONT-LRS) to three patients with FSHD, one unaffected control, and a trio family to simultaneously characterize D4Z4 repeat size, haplotype, DNA methylation, and genomic structure. Results were validated by Southern blotting, single-molecule optical mapping and bisulfite sequencing. ONT-LRS enabled integrated characterization of D4Z4 repeat size, haplotype, single-molecule methylation, and genomic structure. Single-molecule methylation profiling revealed reduced and heterogeneous methylation across individual D4Z4 repeats. Reference-guided haplotype-resolved assembly generated consensus sequences spanning the D4Z4 array and flanking regions. By applying the assembled consensus sequences, we identified a deletion outside the D4Z4 region and precisely localized its breakpoint in the trio family. These findings demonstrate that ONT-LRS enables comprehensive genetic and epigenetic characterization of FSHD in a single assay, providing a potentially valuable approach for molecular diagnosis and facilitating investigation of genotype-phenotype relationships. 1. ONT-LRS enables direct measurement of D4Z4 repeat and repeat-level methylation, with high concordance to SB, SMOM, and bisulfite sequencing. 2. ONT-LRS is applied to generate the first consensus D4Z4 sequence in Chinese FSHD patients and to achieve accurate breakpoint mapping.