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March 30, 2026Human Mutation0 citationsOpen Access

Spectrum of F8 Gene Variants in Malaysian Patients With Severe Hemophilia A: Discovery of 15 Novel Variants

ANAbdullah Aziz NursaedahMinistry of Health LYLam Kah YuenMinistry of Health AZAli Ernie ZuraidaMinistry of Health

Key Points

To characterize the spectrum of F8 gene variants in Malaysian patients with severe Hemophilia A.
Screened patients for Intron 22 and Intron 1 inversions using PCR methods.
Applied direct DNA sequencing to analyze essential regions of the F8 gene.
Employed multiplex ligation-dependent probe amplification (MLPA) for detecting large deletions or duplications.
Detected variants in 69 out of 70 patients, yielding a detection rate of 98.6%.
Identified 15 novel variants, with 13 classified as pathogenic and 2 as variants of uncertain significance (VUS).
Observed inhibitor development in 12.9% of patients with high-risk mutations.

Abstract

Hemophilia A (HA) (MIM No. 306700) is an X‐linked bleeding disorder caused by variants in the F8 gene, resulting in blood coagulation Factor VIII (FVIII) deficiency. This study was aimed at characterizing the F8 gene variant spectrum in 70 patients with severe HA using comprehensive molecular approaches. Patients were first screened for Intron 22 inversion (Inv22) using inverse‐shifting PCR, followed by multiplex PCR for Intron 1 inversion (Inv1). Patients without Inv22 and Inv1 underwent direct DNA sequencing to analyze the essential regions of the F8 gene, including the promoter, all 26 exons, and exon–intron boundaries. For samples where no variants were identified, multiplex ligation‐dependent probe amplification (MLPA) was performed to detect large deletions or duplications within the F8 gene. Variants were successfully detected in 69 patients, yielding a variant detection rate of 98.6%. Inv22 and Inv1 were identified in 35.7% and 1.4% of patients with severe HA, respectively. Sequencing analysis revealed a broad spectrum of variants, including missense, nonsense, and splice‐site variants, as well as small deletions, duplications, and deletion–insertion (delins). MLPA identified large deletions and duplications in 8.6% of cases. In this study, 15 novel variants were identified, with 13 classified as pathogenic and 2 classified as variants of uncertain significance (VUS) according to ClinGen guidelines. Inhibitor development was observed in 12.9% of patients carrying high‐risk mutations, including Inv22, nonsense variants, frameshift variants, and a large deletion. The variant spectrum, distribution, and their associations with disease severity and inhibitor development aligned with previous studies in HA populations. A combination of molecular methods employed in this study has facilitated the identification of underlying variants in patients with severe HA. These findings are essential for genetic confirmation, counseling, prediction of inhibitor development, patient management, and carrier screening.

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