Resolving structural variants in hemophilia: insights from long-read sequencing and molecular and clinical...

Structural variants (SVs) in F8 and F9 are major causes of severe hemophilia, yet their mechanistic basis and clinical impact remain incompletely understood. We aimed to implement an integrated workflow to define hemophilia-associated SVs at nucleotide resolution and to explore genomic signatures and clinical correlates. We analyzed 25 unrelated families non-recurrent SVs with hemophilia A (HA;n=20) or hemophilia B (HB;n=5) using genome walking, short-read sequencing, and nanopore whole-genome long-read sequencing (WGS-LRS). In HA, we resolved 15 partial F8 deletions (from 249 bp to ~0.5 Mb), one tandem duplication, and four complex rearrangements. In HB, we characterized three large F9 deletions (including one partial and two whole-gene deletions, up to >4 Mb) and two pathogenic exonic Alu insertions. In four cases, MLPA calls were incomplete or misleading, whereas WGS-LRS uncovered breakpoints and structures missed by standard workflows. Notably, repetitive elements were present at least one breakpoint in nearly all SVs (22/25), implicating local genomic architecture in SV formation. Breakpoint-junction analysis revealed short microhomologies in most events (F8: 18/20; F9: 3/5), supporting microhomology-mediated end joining or replication-based mechanisms as predominant pathways. WGS-LRS enabled methylation-based assessment of skewed X-inactivation in a carrier with a Xq28 deletion. Inhibitors were reported in 4/16 and 3/4 evaluable HA and HB cases, respectively. Overall, this study provides the first systematic application of nanopore WGS-LRS as a transformative genomic diagnostic approach for comprehensive SV analysis in hemophilia, enabling definitive SV delineation, correcting misclassification by conventional testing, and supporting mechanism-informed interpretation to improve molecular diagnosis and genetic counseling.