Key Points FVNara (W1920R), associated with serious deep vein thrombosis, is more resistant to APC relative to FVLeiden (R506Q). This mechanism results from significant decreases in FVa susceptibility to APC and FV cofactor activity for APC.
Summary. Haemophilia A is caused by various genetic mutations in the factor VIII gene (F8). However, after conventional analysis, no candidate mutation could be identified in the F8 of about 2% of haemophilia A patients. The F8 of a patient with mild congenital haemophilia A, in whom no candidate mutation was found in the exons or their flanking regions, was analysed in detail to identify the patient's aetiological genetic abnormality. We also characterized anti-FVIII antibody (inhibitor) development in this patient. Genomic DNA analysis revealed an adenine to guanine transition deep inside intron 10 (c.1478 + 325A>G) of F8 as a causative mutation. Analysis of the transcripts demonstrated that the majority of the patient's transcript was abnormal, with 226 bp of the intronic sequence inserted between exon 10 and 11. However, the analysis also indicated the existence of a small amount of normal transcript. Semi-quantification of ectopic F8 mRNA showed that about one-tenth of the normal mRNA level was present in the patient. After the use of a recombinant FVIII concentrate, the presence of an inhibitor was confirmed. The inhibitor was characterized as oligoclonal immunoglobulin IgG4 directed against both the A2 domain and light chain of the FVIII molecule with type I reaction kinetics of inhibition of FVIII activity. When no mutations are found by conventional analysis, deep intronic nucleotide substitutions may be responsible for mild haemophilia. The inhibitor development mechanism of the patient producing some normal FVIII was thought to be of interest.
We identified 3 mutations in the factor V (FV) gene (F5) associated with FV deficiency in 3 unrelated Japanese patients. Patient 1 had severe bleeding symptoms (plasma FV activity, <1%; FV antigen, 9%) and was a compound heterozygote for a novel 5-bp deletion in exon 22 and the V1813M mutation. Patient 2 had moderate bleeding symptoms (plasma FV activity, <1%; FV antigen, 4%) and was homozygous for the V1813M mutation. Patient 3 had very mild symptoms (plasma FV activity, 1%; FV antigen, 5%) and was homozygous for the novel R2174L mutation. A study of recombinant protein expression revealed that the FV coagulant-specific activities in conditioned media for the FV-R2174L and FV-V1813M mutants were reduced to approximately 40% and 28% of wild-type FV, respectively. The amounts of FV-R2174L protein and messenger RNA in the platelets of patient 3 were similar to those of healthy subjects; however, the amount of FV-V1813M protein in patient 2 was decreased. Our data suggest that the severity of the bleeding tendency in patients with FV deficiency is correlated not only with plasma FV activity but also with the amount of FV protein in the platelets.
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.
Activated protein C (APC) inactivates activated factor V (FVa) and moderates FVIIIa by restricting FV cofactor function. Emicizumab is a humanized anti-FIXa/FX bispecific monoclonal antibody that mimicks FVIIIa cofactor function. In recent clinical trials in haemophilia A patients, once-weekly subcutaneous administration of emicizumab was remarkably effective in preventing bleeding events, but the mechanisms controlling the regulation of emicizumab-mediated haemostasis remain to be explored. We investigated the role of APC-mediated reactions in these circumstances. APC dose-dependently depressed thrombin generation (TG) initiated by emicizumab in FVIII-deficient plasmas, and in normal plasmas preincubated with an anti-FVIII antibody (FVIII-depleted). FVIIIa-independent FXa generation with emicizumab was not affected by the presence of APC, protein S and FV. The results suggested that APC-induced down-regulation of emicizumab-dependent TG was accomplished by direct inactivation of FVa. The addition of APC to emicizumab mixed with FVIII-depleted FV-deficient plasma in the presence of various concentrations of exogenous FV demonstrated similar attenuation of TG, irrespective of specific FV concentrations. Emicizumab-related TG in FVIII-depleted FV plasma was decreased by APC more than that observed with native FV plasma. The findings indicated that emicizumab-driven haemostasis was down regulated by APC-mediated FVa inactivation in plasma from haemophilia A patients without or with FV defects.
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