Nanopore Ultra-Long Sequencing for FSHD: From Molecular Diagnosis to Preimplantation Genetic Testing

Facioscapulohumeral muscular dystrophy (FSHD) is a genetically and epigenetically complex autosomal dominant myopathy that presents formidable challenges to molecular diagnosis and reproductive intervention. The disease is caused by aberrant derepression of the DUX4 retrogene within the D4Z4 macrosatellite repeat array at chromosome 4q35, triggered either by pathological contraction of the array on a permissive 4qA haplotype (FSHD1, ~95% of cases) or by mutations in epigenetic modifier genes SMCHD1, DNMT3B, and LRIF1 that lead to global D4Z4 hypomethylation (FSHD2, ~5% of cases). Traditional approaches (Southern blotting, optical genome mapping, bisulfite sequencing) are discontinuous and labor-intensive. Nanopore ultra-long read sequencing spans the entire D4Z4 array in single reads, simultaneously resolving repeat number, haplotype, and allele-specific CpG methylation without bisulfite conversion. With the telomere-to-telomere (T2T-CHM13) reference genome, long-range haplotype phasing enables preimplantation genetic testing for monogenic conditions (PGT-M) for families with de novo pathogenic variants and somatic mosaicism, groups previously excluded from reproductive genetic intervention. This review systematically examines FSHD molecular mechanisms, the Nanopore diagnostic workflow integrated with T2T-CHM13, Nanopore-based PGT-M clinical data, and future perspectives including R11 pore chemistry, AI-driven bioinformatics, CRISPR-targeted enrichment, and multi-omics integration.