Comprehensive Analysis of Hemophilia A (CAHEA): Towards Full Characterization of the F8 Gene Variants by Long-Read Sequencing

Background Hemophilia A (HA) is the most frequently occurring X-linked bleeding disorder caused by heterogeneous variants in the F8 gene, one of the largest genes known. Conventional molecular analysis of F8 requires a combination of assays, usually including long-range polymerase chain reaction (LR-PCR) or inverse-PCR for inversions, Sanger sequencing or next-generation sequencing for single-nucleotide variants (SNVs) and indels, and multiplex ligation-dependent probe amplification for large deletions or duplications. Materials and Methods This study aimed to develop a LR-PCR and long-read sequencing-based assay termed comprehensive analysis of hemophilia A (CAHEA) for full characterization of F8 variants. The performance of CAHEA was evaluated in 272 samples from 131 HA pedigrees with a wide spectrum of F8 variants by comparing to conventional molecular assays. Results CAHEA identified F8 variants in all the 131 pedigrees, including 35 intron 22-related gene rearrangements, 3 intron 1 inversion (Inv1), 85 SNVs and indels, 1 large insertion, and 7 large deletions. The accuracy of CAHEA was also confirmed in another set of 14 HA pedigrees. Compared with the conventional methods combined altogether, CAHEA assay demonstrated 100% sensitivity and specificity for identifying various types of F8 variants and had the advantages of directly determining the break regions/points of large inversions, insertions, and deletions, which enabled analyzing the mechanisms of recombination at the junction sites and pathogenicity of the variants. Conclusion CAHEA represents a comprehensive assay toward full characterization of F8 variants including intron 22 and intron 1 inversions, SNVs/indels, and large insertions and deletions, greatly improving the genetic screening and diagnosis for HA.