Advanced Molecular Analysis in Hemophilia A in a Single Step: Next Generation Sequencing (NGS) and Copy Number...

Hemophilia A, an X-linked bleeding disorder caused by pathogenic variants in the F8 gene, requires precise genetic diagnosis for optimal management. Conventional stepwise sequence and copy number variation (CNV) analyses are time-consuming and may leave some cases unresolved.

To evaluate the effectiveness of combining next-generation sequencing (NGS)-based sequence and CNV analysis in a single step for resolving previously undiagnosed Hemophilia A cases and identifying differential diagnoses.

A cohort of 578 male patients with Factor VIII deficiency was assessed. Initial analyses included intron 22/intron 1 inversion testing and/or F8 sequencing. Twenty-seven unresolved cases underwent a targeted 57-gene coagulopathy panel using NGS with CNV analysis; CNVs were confirmed by multiplex ligation-dependent probe amplification (MLPA).

The integrated NGS approach resolved 19 of 27 unresolved cases, increasing the overall diagnostic yield from 95.3% to 98.6%. Ten CNVs in F8 (8 deletions, 2 duplications) were identified and confirmed by MLPA. Variants in VWF and LMAN1 genes led to diagnoses of von Willebrand disease (n = 6) and combined Factor V/VIII deficiency (n = 1). One F8 intronic variant was detected, while 8 cases remained unresolved by the NGS-based approach.

Integrating NGS-based CNV analysis into genetic diagnostic workflows enables simultaneous detection of sequence variants and CNVs, improves diagnostic yield, and facilitates identification of related bleeding disorders. This streamlined approach supports adoption as a first-line test for mild/moderate Hemophilia A and a secondary test for severe cases.