Premium
The challenge of genetically unresolved haemophilia A patients: Interest of the combination of whole F8 gene sequencing and functional assays
Author(s) -
Lassalle Fanny,
Jourdy Yohann,
Jouan Loubna,
Swystun Laura,
Gauthier Julie,
Zawadzki Christophe,
Goudemand Jenny,
Susen Sophie,
Rivard GeorgesEtienne,
Lillicrap David
Publication year - 2020
Publication title -
haemophilia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.213
H-Index - 92
eISSN - 1365-2516
pISSN - 1351-8216
DOI - 10.1111/hae.14179
Subject(s) - minigene , genetics , rna splicing , gene , biology , untranslated region , coding region , deep sequencing , human genome , alternative splicing , intron , computational biology , genome , messenger rna , rna
Background The causative variant remains unidentified in 2%–5% of haemophilia A (HA) patients despite an exhaustive sequencing of the full F8 coding sequence, splice consensus sequences, 5’/3’ untranslated regions and copy number variant (CNV) analysis. Next‐generation sequencing (NGS) has provided significant improvements for a complete F8 analysis. Aim The aim of this study was to identify and characterize pathogenic non‐coding variants in F8 of 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 entire F8 gene using an NGS capture method. We analysed F8 mRNA 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.