Haemophilia-A in Indonesia: Modified Method for Detecting Intron Inversion and in Silico Approach for Functional Analysis of Three Novel Variants in FVIII Gene

<ns5:p> Introduction Haemophilia-A is a genetic disorder caused by genetic variations in the factor VIII (FVIII) gene on the q-arm of the X chromosome. Its prevalence reaches 80–85% of all haemophilia cases in the world. The intron-22 inversion is the most common type of genetic variation (50–75%) followed by intron-1 inversion (1–5%). However, the genetic variations of haemophilia-A in Indonesia are not yet extensively studied. This study aims to develop and modify a molecular technique to detect genetic mutation among hemophilia patients in Indonesia, including to find and classify any novel genetic variations within the <ns5:italic>FVIII</ns5:italic> gene. Methods Whole blood from severe haemophilia A subjects was used for genomic DNA extraction with a commercial kit. The FVIII gene was amplified by PCR using primers designed via Benchling, NCBI Primer-BLAST, and NetPrimer. PCR products were sequenced by Sanger sequencing and analysed with FinchTV and BioEdit. Novel variants were classified according to ACMG 2015 guidelines with CanVIG-UK updates. Predicted 3D amino acid structures were obtained from AlphaFold and analysed using BIOVIA Discovery Studio 2024. Results This study identified pathogenic genetic alterations in the <ns5:italic>FVIII</ns5:italic> gene among patients with haemophilia A. Using inverse-shifting PCR (IS-PCR), we detected intron 22 and intron 1 inversions with distinct amplicon sizes that differentiated patients from controls. The method proved reliable and reproducible across samples and experimental conditions. Sanger sequencing also revealed three novel variants in exon 14: c.2871G > C (p.Leu957Phe), c.3637A > T (p.Ile1213Phe), and c.3640C > T (p.Gln1214Ter). Based on ACMG/AMP guidelines, both missense variants classified as likely pathogenic while one nonsense variant classified as pathogenic. All variants were absent from population databases and associated with clinical findings of low level FVIII activity. Conclusions These findings underscore the utility of combining structural, molecular, and clinical data for accurate diagnosis. Our improved IS-PCR protocol and variant characterization approach offer a practical genetic diagnostic strategy for haemophilia-A in Indonesia. </ns5:p>