The challenge of genetically unresolved haemophilia A patients: Interest of the combination of wholeF8gene sequencing and functional assays

Background The causative variant remains unidentified in 2%-5% of haemophilia A (HA) patients despite an exhaustive sequencing of the fullF8coding sequence, splice consensus sequences, 5'/3' untranslated regions and copy number variant (CNV) analysis. Next-generation sequencing (NGS) has provided significant improvements for a completeF8analysis. Aim The aim of this study was to identify and characterize pathogenic non-coding variants inF8of 15 French and Canadian HA patients genetically unresolved, through the use of NGS, mRNA sequencing and functional confirmation of aberrant splicing. Methods We sequenced the entireF8gene using an NGS capture method. We analysedF8mRNA in order to detect aberrant transcripts. The pathogenic effect of candidate intronic variants was further confirmed using a minigene assay. Results After bioinformatic analysis, 11 deep intronic variants were identified in 13 patients (8 new variants and 3 previously reported). Three variants were confirmed to be likely pathogenic with the presence of an aberrant transcript during mRNA analysis and minigene assay. We also found a small intronic deletion in 6 patients, recently described as causing mild HA. Conclusion With this comprehensive work combining NGS and functional assays, we report new deep intronic variants that cause HA through splicing alteration mechanism. Functional analyses are critical to confirm the pathogenic effect of these variants and will be invaluable in the future to study the large number of variants of uncertain significance that may affect splicing that will be found in the human genome.