Deep intronic ‘mutations’ cause hemophilia A: application of next generation sequencing in patients without detectable mutation in F8 cDNA

Abstract

Summary. Background: In a small group of typical hemophilia A (HA) patients no mutations in the F8 coding sequence (cDNA) could be found. In the current study, we performed a systematic screening of genetic and nongenetic parameters associated with reduced FVIII:C levels in a group of mostly mild HA (only one moderate) patients with no detectable mutations in F8 cDNA. Methods: We determined FVIII and VWF activity and antigen levels and performed VWF-FVIII binding (VWF:FVIIIB) and VWF-collagen binding assays (VWF:CB) as well as VWF multimer analysis. VWF was completely sequenced to exclude mutations. The F8 locus, including the introns, was sequenced using overlapping long-range PCRs (LRPCRs) combined with a next generation sequencing (NGS) approach. Moreover, the F8 mRNA was analyzed quantitatively and qualitatively by real-time PCR (qRT) and overlapping reverse transcription (RT) PCRs, respectively. Results: All VWF tests were normal. The LRPCRs demonstrated the integrity of the F8 locus. Eight unique polymorphisms were found in the patients, with two being recurrent. Furthermore, RT-PCRs analysis confirmed that two of the unique variants create detectable new cryptic splice sites in the patients that result in the introduction of intronic DNA sequences into the mRNA and create premature stop codons. Conclusion: By systematically excluding all possible causes of HA, we could with great certainty conclude that deep intronic mutations in F8, although rare, cause abnormal mRNA splicing, leading to mild HA.

Novel approach to genetic analysis and results in 3000 hemophilia patients enrolled in the My Life, Our Future initiative

“…For example, multiple groups have reported rare F8 deep intronic variants as the cause of hemophilia A in patients for whom a variant had not been found. [37][38][39] It is also likely that some of the patients diagnosed with hemophilia A, particularly those with mild disease, instead have low FVIII because of undiagnosed von Willebrand disease. 40 Thus, in patients diagnosed with hemophilia A where a clinically reportable variant was not identified, we recommended that the provider evaluate the patient for von Willebrand disease.…”

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

“…Pezeshkpoor et al. reported an analysis of deep intronic mutations using NGS in patients without detectable mutations in F8 cDNA . Their methodology combined analysis by NGS and of mRNA.…”

Antisense-based RNA therapy of factor V deficiency: in vitro and ex vivo rescue of a F5 deep-intronic splicing mutation

“…38 In fact, deep-intronic splicing mutations potentially amenable to this corrective approach have been identified in the F8, [39][40][41][42] FGB, 30 and FGG 43 genes. COS-1 and HepG2 cells transfected with a F5 minigene construct containing the c.12961268A.G mutation provided a suitable in vitro model to test the antisense molecules, even if the relative proportion of correctly spliced mRNA was much higher in this model (10%-20%) than in the patient's platelets (;0.5%, as estimated by real-time qPCR).…”