Whole F8 gene sequencing identified pathogenic structural variant in the remaining unsolved patients with severe Haemophilia A.

Background No F8 genetic abnormality is detected in about 1-2% of patients with severe haemophilia A using conventional genetic approaches. In these patients, deep intronic variation or F8 disrupting genomic rearrangement could be causal. Objective The study aimed to identify the causal variation in families with history of severe haemophilia A for whom genetic investigations failed. Methods We performed whole F8 gene sequencing in 8 propositi. Genomic rearrangements were confirmed by Sanger sequencing of breakpoint junctions and/or qPCR. Results A structural variant disrupting the F8 was found in each propositus, so that all the 815 families with history of severe haemophilia A registered in our laboratory received a conclusive genetic diagnosis. These structural variants consisted of 3 balanced inversions, 3 large insertions of gained regions, and 1 retrotransposition of a mobile element. The 3 inversions were 105 Mb, 1.97 Mb, and 0.362 Mb in size. Among the insertions of gained regions, one corresponded to the insertion of a 34 kb gained region from chromosome 6q27 in F8 intron 6, the other was the insertion of a 447 kb duplicated region from chromosome 9p22.1 in F8 intron 14, and the last one was the insertion of a Xq28 349 kb gained in F8 intron 5. Conclusion All the genetically unsolved severe haemophilia A of this cohort were due to structural variant disrupting the F8. This study highlighted the effectiveness of whole F8 sequencing to improve the molecular diagnosis of HA when the conventional approach has failed.