Open AccessIdentification of deep intronic individual variants in patients with hemophilia A by next‐generation sequencing of the whole factor VIII gene
Author(s) -
Inaba Hiroshi,
Shinozawa Keiko,
Amano Kagehiro,
Fukutake Katsuyuki
Publication year - 2017
Publication title -
research and practice in thrombosis and haemostasis
Language(s) - English
Resource type - Journals
ISSN - 2475-0379
DOI - 10.1002/rth2.12031
Subject(s) - intron , genetics , gene , biology , in silico , rna splicing , dna sequencing , single nucleotide polymorphism , computational biology , genotype , rna
AbstractEssentials Intronic variants of the factor VIII gene ( F8 ) causing hemophilia A have been reported. We established an analysis method for whole F8 and investigated the variants within its introns. Rare variants located within introns of F8 in patients with hemophilia A are not uncommon. The c.6429+14194T>C variant was characteristically detected in patients with inversion.Background No genetic defects are found in the coagulation factor VIII gene ( F8 ) of approximately 2% of patients with hemophilia A. Recently, genomic variants causative of hemophilia A that were located deep within introns have been reported. Objectives We aimed to establish a comprehensive method of analysis of F8 using next‐generation sequencing ( NGS ) and investigate the variants located deep within the introns of F8 . Patients/Methods Forty‐five male patients with hemophilia A, including 31 with previously identified causative mutations, were investigated. Results Our NGS analysis allowed for the identification of genetic variants in roughly 99% of F8 . We confirmed that our NGS analysis can detect the single nucleotide variants and small deletions with high accuracy. After filtering, a total of 27 rare and unique individual variants from 16 patients remained. Three of these variants, c.144‐10810T>C, c.1010‐365A>G, and c.5219+9065A>G, were predicted as deleterious with high expected accuracy by Predict SNP 2 analysis. We also predicted the impact on splicing by in silico analysis using three different algorithms. Two patients with unknown causative mutations carried unique individual variants, c.144‐10810T>C and c.6723+193G>A. We inferred that the c.144‐10810T>C variant likely causes hemophilia, while the effect of the c.6723+193G>A variant remains unclear. Our analysis showed that the c.6429+14194T>C variant was significantly detected in patients carrying the intron 22 inversion. Conclusions Rare and unique individual variants located deep within the F8 introns in patients with hemophilia A are not uncommon. Future studies are necessary to determine the function and effect of these variants on F8 expression.