Variant analysis of F8 in 123 patients with hemophilia A: Identification of 18 novel variants

Hemophilia A (HA) is a bleeding disorder caused by pathogenic variants in the F8, which can impair FVIII activity by either affecting its protein structure or reducing its expression. Expanding the spectrum of F8 variants is essential for elucidating the genotype-phenotype relationship and facilitating accurate diagnosis. Data from 123 HA patients across 115 families were collected. Intron inversions were detected using inverse sequence polymerase chain reaction (IS-PCR), while Sanger sequencing and whole exome sequencing (WES) were employed to identify single nucleotide variants and small insertions or deletions (indels). Multiplex ligation-dependent probe amplification (MLPA) was used to detect large variants. Additionally, reverse transcription PCR (RT-PCR) was utilized to validate splice pattern changes in two patients with splice site variants. Among the 123 patients, 55 (44.7 %) had intron 22 inversions, 2 (1.6 %) had intron 1 inversions, 28 (22.8 %) had missense variants, 7 (5.7 %) had nonsense variants, 7 (5.7 %) had splice site variants, 18 (14.6 %) had small indels, and 6 (4.9 %) had large variants. A total of 18 novel variants were identified. Experimental validation showed that the c.144-26 A > C variant in a mild HA patient produced two additional truncated transcripts, leading to insufficient FVIII. The c.6116-1G > C variant in a severe HA patient caused a frameshift mutation by creating a new acceptor site. Genetic testing revealed mosaicism in the mothers of two patients. This study expands the variant spectrum of the F8 and experimentally validates the pathogenicity of the c.144-26 A > C and c.6116-1G > C variants, providing valuable insights for the molecular diagnosis of related variants.