Comparison of long-read sequencing strategies for resolving complex genotypes at Facioscapulohumeral dystrophy-associated loci

Abstract Background Facioscapulohumeral dystrophy (FSHD) is typically caused by contraction of the D4Z4 repeat array at chromosome 4q35 (FSHD1) or pathogenic variants in the SMCHD1 gene (FSHD2). While these account for the majority of cases, 1–2% of patients present with clinical features of FSHD but lack a known genetic cause, revealing a diagnostic gap. In Previous studies, we identified over 70 patients with structural rearrangements involving the 4q35 or 10q26 loci, some of which may be pathogenic in the absence of other FSHD-associated features. Methods Given their diagnostic relevance, we performed detailed structural analyses of these rearrangements using high-resolution long-read sequencing technologies (Oxford Nanopore and PacBio) for seven patients carrying different structural variants of the 4q35 or 10q26 loci. Results These approaches enabled us to resolve nucleotide-level architecture and methylation patterns across the 4q35 and 10q26 loci. We show that duplicated alleles arise from intrachromosomal recombination between LSau and β-satellite elements, producing variable deletions within D4Z4, with breakpoints differing among patients. These complex structural variants are not detectable using standard technologies like Bionano Optical Genome Mapping and require manual curation for identification. Importantly, determining the pathogenic relevance of these rearrangements necessitates integrating structural and epigenetic features typically associated with FSHD. Conclusion The results highlight the importance of thorough molecular analysis for FSHD patients who test negative for FSHD1 and FSHD2, advocating for expanded diagnostic strategies. Comprehensive evaluation of 4q35 structural variants is essential for improving diagnosis accuracy, guiding genetic counseling, and optimizing care for patients with atypical FSHD presentations. Graphical Abstract Hypothetical model for the formation of 4q35 intrachromosomal duplications. We speculate that the breakpoint occurs in the proximal region of a D4Z4 units that corresponds to LSau repeats and involves the distal βsatellite array. The duplicated array is then inserted in the distal part of the locus mapped by the most telomeric red probe used for MC.