Identification of an F8 complex recombination in Chinese hemophilia a patient using long-read sequencing and optical genome mapping

Hemophilia A (HA) is an X-linked recessive bleeding disorder caused by pathogenic variants in the F8 gene, resulting in deficient coagulation factor VIII activity. Although intron 22 and intron 1 inversions (Inv22 and Inv1) accounts for approximately 50% of severe HA cases, complex structural rearrangements mediated by intron 22 homologous region (int22h) repeats have rarely reported and poorly characterized. In this study, we investigated a Chinese severe HA pedigree with a complex rearrangement of F8 gene by integrating 750 K SNP arrays, long-read sequencing (Oxford Nanopore Technologies, ONT), and optical genome mapping (OGM). This multi-platform strategy enabled comprehensive characterization of the structural variations affecting the F8 gene. We identified a complex rearrangement in the proband’s F8 gene, characterized by sequential duplications inserted within intron 22. These include a 7.75 kb direct duplication involving a portion of int22h-1, a 78.78 kb inverted duplication containing partial sequences of int22h-3 and int22h-2, and an 86.56 kb direct duplication spanning a portion of int22h-3 and exons 2–8 of the TMLHE gene. The structural variation in the F8 gene of the proband was inherited from the mother. Our findings revealed that the complex rearrangement of F8 is the genetic cause of HA in this pedigree. The combined use of OGM and ONT provides an effective approach for deciphering the characterization of complex structural variants and determining the orientation of inserted segments, although base-precision breakpoint mapping remains challenging in highly homologous regions.