Haplotype-based non-invasive prenatal testing of F8 complex rearrangement via targeted capture sequencing of maternal plasma

Hemophilia A (HA) is an X-linked recessive bleeding disorder primarily caused by the intron 22 inversion (Inv22) in F8 gene, which accounts for approximately 45% of all cases. A minor proportion of HA results from complex structural rearrangements (CSRs) in F8 gene. However, the ability of non-invasive prenatal testing (NIPT) to detect F8-related CSRs has not been evaluated.

In this study, we applied NIPT to evaluate the risk of HA in a male fetus, whose mother had a history of delivering a boy affected by severe HA. Optical genome mapping (OGM) had previously identified the complex structural rearrangement (CSR) in F8 gene as the definitive genetic etiology of the first child's severe HA, and was confirmed to be maternally inherited. High-throughput sequencing was performed on the trios (the first child and both parents). Parental haplotypes were deduced through haplotype linkage analysis based on single nucleotide polymorphisms (SNPs). Subsequently, cell-free DNA in maternal plasma was subjected to sequencing, and fetal haplotypes were successfully inferred using the Hidden Markov Model (HMM) with parental haplotype information.

NIPT identified a male fetus who had inherited the CSR in the F8 gene from his mother, suggesting a diagnosis of HA. The NIPT findings were subsequently verified by OGM analysis of chorionic villi.

Our study demonstrates that haplotype-based NIPT is an effective approach for detecting CSRs in the F8 gene during prenatal screening.