Keiko Shinozawa

Novel FV mutation (W1920R, FVNara) associated with serious deep vein thrombosis and more potent APC resistance relative to FVLeiden.

Factor V (FV) appears to be pivotal in both procoagulant and anticoagulant mechanisms. A novel homozygote (FVNara), a novel mechanism of thrombosis associated with Trp1920→Arg (W1920R), was found in a Japanese boy and was associated with serious deep vein thrombosis despite a low level of plasma FV activity (10 IU/dL). Activated partial thromboplastin time-based clotting assays and thrombin generation assays showed that FVNara was resistant to activated protein C (APC). Reduced susceptibility of FVaNara to APC-catalyzed inactivation and impaired APC cofactor activity of FVNara on APC-catalyzed FVIIIa inactivation contributed to the APC resistance (APCR). Mixtures of FV-deficient plasma and recombinant FV-W1920R confirmed that the mutation governed the APCR of FVNara. APC-catalyzed inactivation of FVa-W1920R was significantly weakened, by ~11- and ~4.5-fold, compared with that of FV-wild-type (WT) and FVLeiden (R506Q), respectively, through markedly delayed cleavage at Arg506 and little cleavage at Arg306, consistent with the significantly impaired APC-catalyzed inactivation. The rate of APC-catalyzed FVIIIa inactivation with FV-W1920R was similar to that without FV, suggesting a loss of APC cofactor activity. FV-W1920R bound to phospholipids, similar to FV-WT. In conclusion, relative to FVLeiden, the more potent APCR of FVNara resulted from significant loss of FVa susceptibility to APC and APC cofactor activity, mediated by possible failure of interaction with APC and/or protein S.

Identification and characterization of an adenine to guanine transition within intron 10 of the factor VIII gene as a causative mutation in a patient with mild haemophilia A

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 patients 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 patients 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.

Molecular characterization of 3 factor V mutations, R2174L, V1813M, and a 5-bp deletion, that cause factor V deficiency

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.

Mutations to the probe of Cobas TaqMan HIV-1 ver. 1.0 assay causing undetectable viral load in a patient with acute HIV-1 infection

We encountered a human immunodeficiency virus (HIV)-1 in which the viral load was undetectable with the Cobas TaqMan HIV-1 ver. 1.0 (CTM v.1.0) in a patient with acute HIV-1 infection. The CTM v.1.0 assay showed more than 1,000-fold underestimation compared with the subsequent Cobas Amplicor Monitor v.1.5 assay. Because five mismatches to the CTM v.1.0 assay probe in the HIV-1 virus in the patient were disclosed by the manufacturer, partial gag regions of the HIV genome were directly sequenced from the patient’s plasma viral RNA. The detected single nucleotide point mutations were located near the 5′-end of the Cobas Amplicor Monitor probe. Clinicians should be very careful in making interpretations when indeterminate Western blot analysis results and a low or even undetectable HIV-1 viral load are encountered with the CTM HIV-1 ver. 1.0 assay in patients with suspected acute HIV infection. Repeating Western blot analysis is essential before considering a low HIV-1 viral load to be a false-positive result.

Emicizumab-mediated haemostatic function in patients with haemophilia A is down-regulated by activated protein C through inactivation of activated factor V

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 FVLeiden plasma was decreased by APC more than that observed with native FVLeiden 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.

Factor VIII haplotypes of Japanese population show similarity to those of Caucasian populations

Haemophilia A is the most common inherited bleeding disorder which is X-linked recessive. This disease is caused by a quantitative or qualitative abnormality of plasma factor VIII (FVIII), which is affected by a genetic mutation located in the coagulation FVIII gene. Diagnosis and replacement therapy strategies in haemophilia A patients are well established; however, some issues remain to be addressed. The most important issue concerning replacement therapy in haemophilia A patients is the development of inhibitors (alloantibody) against FVIII. This development leads to marked attenuation in the effectiveness of replacement therapy, leading to a substantial deterioration in the quality of life of patients. In general, the incidence of inhibitor development in treatment-naive patients with haemophilia A is estimated to be 20–30% [1]. However, it has been shown that its incidence in the Black population is markedly higher, about twice as high as in other racial groups [2]. The question of why this inhibitor develops with such high incidence in the Black population remains undetermined. Recently, Viel et al. reported that six wild-type FVIII proteins, the H1–H6 haplotypes, had different prevalence rates among racial groups [3]. They speculated that a mismatch of the FVIII haplotype between the FVIII concentrate and its recipients, particularly in immunodominant epitopes, caused an increase in the frequency of inhibitor development. In particular, they focused on four different amino acid polymorphisms (R484H, R776G, D1241E and M2238V), based on a non-synonymous single nucleotide polymorphism. By using a combination of those amino acids, they classified the FVIII protein into six haplotypes, namely, H1: RRDM, H2: RREM, H3: RREV, H4: HREM, H5: RRDV and H6: RGEM. It is presumed that amino acid positions 484 and 2238, located in the A2 and C2 domains, respectively, are components of the immunodominant epitope of FVIII. In Caucasian participants, positions 484 and 2238 have been observed as R and M , respectively, and have only shown the haplotypes of H1 and H2. However, Viel et al. confirmed the presence of H in place of R at position 484, or V in place of M at position 2238, in approximately 25% of Black participants. Thus, the haplotype frequencies in Black participants (H1: 0.354; H2: 0.374; H3: 0.222; H4: 0.040 and H5: 0.010) are different from those in Caucasian participants. Furthermore, they also analysed haplotypes in ethnic Chinese participants, and observed the H6 haplotype, but not the H3, H4 or H5 haplotype. We set out to analyse the haplotypes of FVIII proteins among 106 unrelated Japanese subjects at our institution (63 with haemophilia A and 43 with haemophilia B as a control group), as shown in Table 1. The study was approved by the Ethics Committee of Tokyo Medical University and written informed consent was obtained from each patient. The studies were carried out in accordance with the principles of the Declaration of Helsinki. Among the Japanese participants, only the H1 and H2 haplotypes (H1: 0.896 and H2: 0.104) were found at frequencies not significantly different from the Caucasian frequencies (H1: 0.926 and H2: 0.074). Despite the Chinese being geographical neighbours of the Japanese, the H6 haplotype which was observed in approximately 8% of Chinese was not detected in any Japanese. Currently in Japan, approximately 80% of haemophilia A patients receive replacement therapy using the recombinant FVIII concentrates Kogenate (Bayer) or Advate (Baxter). Data regarding the incidence of inhibitor development in Japanese patients with severe haemophilia A were previously presented as poster presentations on post-authorisation safety studies for Kogenate or Advate at the 22nd Congress of the International Society on Thrombosis and Hemostasis (ISTH) (2009) and the Hemophilia World Congress (World Federation of Hemophilia) (2010) by Fukutake et al., who reported that the incidence was similar to that in Caucasians. Although the role of the haplotypes of FVIII proteins as a risk factor for inhibitor development is not yet determined, the similarities of haplotype incidence may partially explain the similarities in inhibitor incidence between Japanese patients and Caucasian patients. Further studies are needed to clarify the role and characteristics of FVIII haplotypes in Japanese population.

Genotyping analysis of the factor V Nara mutation, Hong Kong mutation, and 16 single-nucleotide polymorphisms, including the R2 haplotype, and the involvement of factor V activity in patients with recurrent miscarriage

&NA; Recurrent miscarriage can arise from a large diversity of causes and the factors responsible have not been fully clarified. The coagulation factor V R506Q (Leiden) mutation is a well known risk factor for recurrent miscarriage, although it has not been found in Japanese populations. We examined whether the factor V Nara and Hong Kong mutations, the factor V gene (F5) 16 single-nucleotide polymorphisms (SNPs), including the factor V R2 haplotype, and plasma factor V activity (FV:C) were risk factors for recurrent miscarriage. A cross-sectional study was conducted among 88 patients with a history of unexplained recurrent miscarriage and 95 fertile controls. None of the patients or controls was homozygous or heterozygous for the factor V Nara or Hong Kong mutation. In the 16 SNPs of F5, frequencies of the G/T and T/T genotypes at Ser156Ser were significantly lower in patients than in controls (OR 0.45, 95% CI 0.22–0.91, OR 0.32, 95% CI 0.14–0.72) and the allele frequency of C at Leu1288Leu was significantly higher in patients than that in controls (OR 1.66, 95% CI 1.02–2.71). The mean FV:C values were not significantly different between patients and controls. However, the prevalence of patients with a high or low FV:C (>95th or <fifth percentile) was significantly greater than the controls (OR 3.59, 95% CI 1.11–11.60: OR 3.94, 95% CI 1.23–12.60). These results suggest that some SNPs of F5 and a high or low FV:C level might be associated with recurrent miscarriage.

A novel synonymous variant in the F8 gene, p.(Leu40=)/c.120C>A, likely causes mild haemophilia A

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

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.