Genetic analysis of F8 mutations in five hemophilia a carriers

Hemophilia A (HA) is an X-linked recessive bleeding disorder caused by mutations in the F8 gene, which exhibits complex molecular mechanisms and high genetic heterogeneity. This study aimed to perform carrier screening and genetic analysis in five phenotypically normal females to identify F8 variants and assess their implications for genetic counseling and prenatal diagnosis.

Genomic DNA was extracted from peripheral blood samples. High-throughput sequencing was used to screen for mutations in 455 genes associated with genetic diseases. Long-range PCR (LR-PCR) was employed to detect the inversion of introns 1 and 22 of the F8 gene. Sanger sequencing validated small deletions. To clarify the complex variations of Subject 4, qPCR, CNV-seq and SV-seq analyses were conducted on her affected uncle.

Subject 1 carried an intron 22 inversion (Inv22) of the F8 gene. Subjects 2 and 4 carried intron 1 inversions (Inv1). Subject 3 had a small deletion (c.3168_3187del), and Subject 5 had a frameshift deletion (c.4379delA). Notably, Subject 4 was found to carry a rare complex structural variant involving Inv1. QPCR suggested potential duplications in the corresponding genomic region of Subject 4's uncle. SV-seq identified two duplications on the X chromosome in her uncle, which may disrupt F8 gene function.

All five subjects carried pathogenic F8 gene variants, and one affected individual had a pathogenic variant of the gene. Although carriers are asymptomatic, they can transmit the mutant allele to their offspring. Molecular genetics in carriers is crucial for improving genetic screening, prenatal diagnosis, and the development of targeted therapies for HA. This study further enriches the mutation spectrum of the F8 gene and underscores the importance of comprehensively detecting inversions, copy number variations, and structural rearrangements in the molecular diagnosis of hemophilia A.