Identification and structural characterization of F8 gene variants in Indonesian patients with nonsevere hemophilia A: Evidence from targeted next generation sequencing and in silico analyses

Abstract

Hemophilia A is a rare genetic disorder caused by deficiency of coagulation factor VIII resulting from pathogenic variants in the F8 gene. The aim of this study was to identify potential pathogenic variants using targeted next generation sequencing (NGS), followed by in silico structural and pathogenicity analyses. Genomic DNA extracted from saliva samples of 11 patients with nonsevere hemophilia A was analyzed using targeted sequencing of F8 exons 23–26, while targeted NGS was performed on two selected patients for broader variant detection. Identified variants were further evaluated using computational pathogenicity prediction and three-dimensional protein structural modeling. No variants were detected within exons 23–26. Nevertheless, targeted NGS identified a missense variant, c.2095A>G (p.Met699Val), in exon 13 in patient HM1 and a splice-site variant, c.1271+1G>A, in intron 8 in patient HM7. Variant assessment predicted that the variants were classified as likely pathogenic and pathogenic, respectively. Structural comparison between wild-type and mutant factor VIII proteins showed no major differences in overall conformation, secondary structure, or ProMotif features. Nonetheless, physicochemical and atomic interaction analyses indicated local structural perturbations that may affect protein stability and function. These findings suggest that the identified F8 variants, particularly p.Met699Val, may contribute to factor VIII dysfunction despite preservation of the global protein structure. However, further validation using molecular dynamics simulations and functional experimental studies is required to clarify its structural impact and confirm pathogenicity.