Role of multiplex ligation-dependent probe amplification in hemophilia A: analysis of large F8 variants

Hemophilia A is caused by pathogenic variants in the F8 gene, with large deletions and duplications accounting for approximately 6% of severe cases and 1,4% of mild/moderate forms. Standard molecular diagnostic approaches typically include screening for introns 22 and 1 inversions, followed by sequencing of coding exons and flanking splice sites of the F8 gene. However, these methods frequently fail to detect large structural rearrangements, such as deletions and duplications, as the underlying genetic cause. This study is the first in Belarus to assess the utility of multiplex ligation-dependent probe amplification (MLPA) for identifying large structural alterations in the F8 gene. The study included 6 male patients with hemophilia A from 5 unrelated families and 3 female blood relatives (mothers or sisters), assessed for carrier status and mode of inheritance. MLPA identified hemizygous large CNV in all patients: deletions of exon 6 and exons 2–6, and duplications of exon 13 and exons 2–7. The same structural alterations were detected in heterozygous state in the female relatives examined. These findings demonstrate the high sensitivity and clinical value of MLPA in the genetic diagnosis of hemophilia A. The method provides a robust foundation for precise medical genetic counseling in families with a positive history, accurate risk assessment for disease transmission, and prevention of therapy-related complications.

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