Identification of deep intronic individual variants in patients with hemophilia A by next‐generation sequencing of the whole factor VIII gene

Abstract

Essentials 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. BackgroundNo 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.ObjectivesWe 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/MethodsForty‐five male patients with hemophilia A, including 31 with previously identified causative mutations, were investigated.ResultsOur 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 PredictSNP2 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.ConclusionsRare 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.

Severe hemophilia A caused by an unbalanced chromosomal rearrangement identified using nanopore sequencing

“…Because of the large number of repetitive elements such as Alu elements and LINE present in its sequence and its Xq28 location, the F8 appears to be extremely susceptible to genetic rearrangements . Whole F8 sequencing approaches previously reported were not able to detect F8 structural variant . While short‐read whole genome sequencing can identify chromosomal structural variations, repeated regions remain a challenge.…”

Identification of new F8 deep intronic variations in patients with haemophilia A

“…As an alternative to mRNA approach, several groups have developed whole F8 sequencing using next‐generation sequencing in order to identify deep intronic variants in patients in whom conventional genetic approaches failed 9,21 . However, the deleterious impact of deep intronic variation remains challenging to evaluate and functional studies such as mRNA or minigene studies should be systematically performed before reporting such variant as disease‐causing.…”

Intronic regions of the human coagulation factor VIII gene harboring transcription factor binding sites with a strong bias towards the short-interspersed elements

“…It suggests that the surveying the deep intronic variations which are overlapped to these regions might confirm the regulatory functions of them in the future studies. In this regard, the mutations which have reported within the introns of over 75 disease-associated genes, including the c.6429 + 14194T > C variant detected in patients carrying the intron 22 inversion of the hFVIII gene, highlight the importance of studying variation in deep intronic sequence as a cause of monogenic disorders [ 18 , 19 ]. The structural variants (SVs) including hFVIII inversion variants in introns 1 and 22, have been explained in nearly one-half of patients with severe hemophilia A [ 37 ].…”

“…Therefore, it would be essential to mining potentially regulatory non-coding regions that might be involved in hFVIII gene regulation. Indeed, many contributing DNA variants have been reported to date in the hFVIII gene including its deep intronic variations, highlight the importance of variation studies in the nucleotide sequences of the hFVIII gene including non-coding regions as a cause of monogenic disorders [ 18 , 19 ]. Moreover, it has been revealed that the genotypes and even genetic variants on multiple ethnic backgrounds of hFVIII gene have impact on bleeding, thrombosis and haemophilia outcomes [ 20 , 21 ].…”