Abstract Number: OC 56.3
Meeting: ISTH 2021 Congress
Background: With current molecular diagnosis, about 1% of haemophilia B patients remains genetically unresolved. In these cases, deep intronic variation could be causal.
Aims: To identify the causal variation in 6 unrelated mild-to-moderate HB patients in whom no genetic variation was found using conventional genetic exon-focused approaches.
Methods: The whole F9 was sequenced using Next Generation Sequencing capture method. All candidate variations were confirmed using Sanger sequencing. The putative splicing impact of these deep intronic variations was studied using both in silico analysis (Splicing Sequences Finder and MaxEntScan) and minigene assay.
Results: Next generation sequencing data revealed 3 candidate variants in F9 introns. The c.278-1806A>C was found in three patients with a mild phenotype, while the c.724-2385G>T and the c.723+4297T>A were found in a mild and a moderate haemophilia B patient, respectively. Additionally, a de novo 6-kb LINE retrotransposition in F9 intron 4 was found in the remaining patient with mild phenotype. In silico analysis strongly predicted that both c.724-2385G>T and c.723+4297T>A impacted the splicing. Conversely, no impact was predicted for the c.278-1806A>C. For all substitutions, an abnormal mRNA pattern was found using the minigene assay. The c.724-2385G>T led to the insertion of a 84 bp pseudo-exon due to the creation of a de novo donor splice site. The c.723+4297T>A led to the exonisation insertion of a 180 bp sequence by enhancing the strength of a pre-existing cryptic donor splice site. The c.278-1806A>C led to the retention of a 199 bp pseudo-exon.
Conclusions: With this comprehensive work combining next generation sequencing and functional assays, we report for the first time deep intronic variants that caused haemophilia B through splicing alteration. This study highlights the usefulness of whole F9 sequencing for the progressive reduction of genetically unexplained haemophilia B.
To cite this abstract in AMA style:Dericquebourg A, Fretigny M, Negrier C, Vinciguerra C, Jourdy Y. Identification and Characterization of F9 Deep Intronic Variations in Haemophilia B Patients [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 1). https://abstracts.isth.org/abstract/identification-and-characterization-of-f9-deep-intronic-variations-in-haemophilia-b-patients/. Accessed September 24, 2021.
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