Search

Recombinant factor VIII (FVIII) products represent a life-saving intervention for patients with hemophilia A. However, patients can develop antibodies against FVIII that prevent its function and directly increase morbidity and mortality. The development of anti-FVIII antibodies varies depending on the type of recombinant product used, with previous studies suggesting that second-generation baby hamster kidney (BHK)-derived FVIII products display greater immunogenicity than do third-generation Chinese hamster ovary (CHO)-derived FVIII products. However, the underlying mechanisms responsible for these differences remain incompletely understood. Our results demonstrate that BHK cells express higher levels of the nonhuman carbohydrate α1-3 galactose (αGal) than do CHO cells, suggesting that αGal incorporation onto FVIII may result in anti-αGal antibody recognition that could positively influence the development of anti-FVIII antibodies. Consistent with this, BHK-derived FVIII exhibits increased levels of αGal, which corresponds to increased reactivity with anti-αGal antibodies. Infusion of BHK-derived, but not CHO-derived, FVIII into αGal-knockout mice, which spontaneously generate anti-αGal antibodies, results in significantly higher anti-FVIII antibody formation, suggesting that the increased levels of αGal on BHK-derived FVIII can influence immunogenicity. These results suggest that posttranslational modifications of recombinant FVIII products with nonhuman carbohydrates may influence the development of anti-FVIII antibodies., (© 2022 by The American Society of Hematology.)

Inhibitors of factor VIII (FVIII) remain the most challenging complication of FVIII protein replacement therapy in hemophilia A (HA). Understanding the mechanisms that guide FVIII-specific B cell development could help identify therapeutic targets. The B cell-activating factor (BAFF) cytokine family is a key regulator of B cell differentiation in normal homeostasis and immune disorders. Thus, we used patient samples and mouse models to investigate the potential role of BAFF in modulating FVIII inhibitors. BAFF levels were elevated in pediatric and adult HA inhibitor patients and decreased to levels similar to those of noninhibitor controls after successful immune tolerance induction (ITI). Moreover, elevations in BAFF levels were seen in patients who failed to achieve FVIII tolerance with anti-CD20 antibody-mediated B cell depletion. In naive HA mice, prophylactic anti-BAFF antibody therapy prior to FVIII immunization prevented inhibitor formation and this tolerance was maintained despite FVIII exposure after immune reconstitution. In preimmunized HA mice, combination therapy with anti-CD20 and anti-BAFF antibodies dramatically reduced FVIII inhibitors via inhibition of FVIII-specific plasma cells. Our data suggest that BAFF may regulate the generation and maintenance of FVIII inhibitors and/or anti-FVIII B cells. Finally, anti-CD20/anti-BAFF combination therapy may be clinically useful for ITI.

Anti-drug antibody formation following factor VIII (FVIII) replacement therapy is the most important treatment-related complication in patients with severe haemophilia A. A significant number of these antibodies show neutralising activity against FVIII and are referred to as FVIII inhibitors. Alloimmunity to FVIII, given the absence of endogenous circulating FVIII protein, may be predictable to some extent; however, only 30% of patients develop inhibitors. Genetic and environmental risk factors have been identified, contributing to the likelihood of inhibitor development. Multiple immunological theories have been proposed which in part explain the outcomes of many epidemiological studies. Significant differences exist among replacement therapies, including the source, FVIII sequence, glycosylation, formulation components, impurities and aggregation potential, which significantly complicate interpretation of the results from these studies. In this review, we present recent advances in the understanding of the cellular mechanisms of inhibitor formation and highlight some areas of uncertainty requiring further investigation., (© 2020 British Society for Haematology and John Wiley & Sons Ltd.)

Introduction : The purpose of this article is to review which data about hemophilia are currently provided by the Cochrane database of systematic reviews (CDBSR). Methodological consideration: All statements about hemophilia in the Cochrane Collaboration are based on evidence generated in randomized controlled clinical trials. Areas covered : There is a high degree of evidence that prophylaxis preserves joint function in children with hemophilia compared to on-demand treatment. Also, that recombinant factor VII activated (rFVIIa) and activated prothrombin complex concentrates (APPCs) have similar efficacy and safety. In patients with hemophilia or von Willebrand disease who undergo minor oral surgery or dental extractions, antifibrinolytic therapy has not been shown to be effective in the prevention of oral bleeding; the possible benefit of physical exercise in hemophilia remains unclear. The effectiveness and safety of the gene therapy have not been proven. Evidence suggests that prophylaxis with bypassing agents may be effective in reducing bleeding in hemophilic patients with inhibitor. Expert opinion : Hemophilia physicians should follow the recommendations of the CDBSR.

Background/objective: Hemophilia A is a genetic disorder through which patients suffer from recurrent bleeding. This can be caused by a defect in human plasma coagulation factor VIII. High incidence of FVIII inhibitors in some severe hemophilia A patients after FVIII therapy is a considerable complication. Determination of good predictive factors can improve the safety of this treatment. HLA-II have been shown as a predictive element for inhibitor development. The goal of this study is to determine the association between HLA-DRB1*15:03, HLA-DRB1*11 and HLA-DRB1*01:01 alleles and FVIII inhibitors in severe hemophilia A patients in Iran., Materials/methods: HLA-DRB1 genotyping was performed using Multiplex sequences Specific Primers (PCR-SSP) in two groups of severe hemophilia A patients comprising 51 and 50 individuals with and without FVIII inhibitors respectively. The levels of inhibitor were determined through Nijmegen-modified Bethesda assay. HLA-DRB1 allele frequencies were compared between groups by using multiple logistic regression models., Results: HLA-DRB1*01:01 allele frequency was significantly higher in patients without inhibitor OR adj : 2.7 (95%CI: 1.08, 6.97; P = 0.034). There wasn't any statistically significant difference in HLA-DRB1*11 allele frequency between groups OR adj : 0.7 (95%CI: 0.27, 1.82; P = 0.47). There was no connection between HLA-DRB1*15:03 and inhibitor development OR adj : 0.94 (95%CI: 0.38, 2.35; P = 0.94)., Conclusion: An association between HLA-DRB1*01:01 and paucity of FVIII inhibitor showed that this allele has probably a protective effect in severe hemophilia A patients in Iran. Determination of the predictive and protective alleles are beneficial in pre-treatment activities and decrease the risk of unsuccessful therapy with FVIII in each population., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A major problem of hemophilia A (HA) treatment is the development of factor VIII (FVIII) inhibitor, which usually occurs shortly after initiating replacement therapy. Several studies showed the correlation between inhibitor development and polymorphisms in inflammatory and immune response genes of HA patients; however, literature data are not available to prove this association in Iranian population. The aim of this study was to investigate a possible association between FVIII inhibitor formation and the polymorphisms of 16 inflammatory and immune response genes in Iranian severe HA patients (FVIII activity < 1%). This case-control study was performed on 55 patients with severe HA inhibitors and 45 samples without inhibitors from Iranian Comprehensive Hemophilia Care center. After extraction of whole genomic DNA from blood samples and design of primers for 16 genes, the genotyping was performed by Tetra primer ARMS PCR, and the validation of single nucleotide polymorphisms was determined by DNA sequencing. The data indicated that there was a significant association between inhibitor development, and F13A1 (TT), DOCK2 (CC& CT), and MAPK9 (TT) genotypes. Moreover, a considerably increased inhibitor risk carrying T, C, and T allele for F13A1, DOCK2, and MAPK9 genes was observed in patients with inhibitors, respectively. In contrast, there was no statistically significant difference between the genotypic and allelic frequencies for other genes in patients with inhibitors compared to patients without inhibitors. These results demonstrate that only polymorphisms in F13A1, DOCK2, and MAPK9 genes are associated with the risk of developing FVIII inhibitors in Iranian HA patients.

Objectives Development of neutralizing antibodies against factor VIII is the major complication in hemophilia care which makes replacement therapies ineffective. The reports showed that inflammatory cytokines play an important role in inhibitor production. In the present study, the relationship between inhibitor development and the polymorphisms of two cytokine genes was studied in severe hemophiliac patients from Iran. Methods In this case-control study, three polymorphisms of immune regulatory genes [TGF-β (rs1982037) and IL-2 (rs2069762, rs4833248)] were analyzed in 100 Iranian hemophilia A patients divided into 55 inhibitor positive and 45 inhibitor negative patients using Tetra primer ARMS PCR, and DNA sequencing. Results The analysis of polymorphisms in the TGF-β and IL-2 genes showed no association between the genotypes and the production of inhibitors (p > 0.05). Also, comparison of allele frequencies for TGF-β and IL-2 genes between two groups indicated no significant differences associated with the development of FVIII inhibitors (p > 0.05). Discussion In contrast with some reports involving the correlation between polymorphisms of the TGF-β1 and IL-2 genes and inhibitor development in the world, no statistically significant differences in analysis of the alleles and genotypes for TGF-β and IL-2 genes were found between the inhibitor and non-inhibitor Iranian patients. Thus, other genetic markers influencing the immune response to replacement therapy in patients with hemophilia should be identified. Conclusions Regarding our results in molecular predisposition for inhibitor development, further studies of effective genetic markers are required as a prerequisite for the development of novel immunogenic therapeutic approaches in the future.

Inhibitor development is the most serious complication in patients with hemophilia. We investigated association of HLA genotypes with inhibitor development in Korean patients with severe hemophilia A (HA). HLA genotyping was done in 100 patients with severe HA including 27 patients with inhibitors. The allele frequencies between inhibitor-positive and inhibitor-negative patients were compared. HLA class I alleles were not associated with the inhibitor status. In HLA class II, DRB1*15 [n = 100, odds ratio (OR) 0.217, P = 0.028] and DPB1*05:01 [OR 0.461, P = 0.026] were negatively associated with inhibitor development. In a subgroup of patients with intron 22 inversion, C*07:02 was positively associated with inhibitor development [n = 30, OR 5.500, P = 0.043]. In the subgroup of patients without intron 22 inversion, the negative association between DPB1*05:01 and inhibitor development was reinforced [n = 70, OR 0.327, P = 0.010], and positive association of DRB1*13:02 and DPB1*04:01 with inhibitor development was identified [OR 3.059, P = 0.037 for both]. Previously reported risk alleles were not consistently associated with inhibitor risk in our series. This study demonstrated the profile of HLA alleles associated with inhibitor risk in Korean patients with severe HA was different from that in patients of other ethnicities, which needs to be considered in risk assessment and management.

Factor VIII (FVIII)-neutralizing antibodies ("inhibitors") are a serious problem in hemophilia A (HA). The aim of this study was to characterize HLA-restricted T-cell responses from a severe HA subject with a persistent inhibitor and from 2 previously studied mild HA inhibitor subjects. Major histocompatibility complex II tetramers corresponding to both of the severe HA subject's HLA-DRA-DRB1 alleles were loaded with peptides spanning FVIII-A2, C1, and C2 domains. Interestingly, only 1 epitope was identified, in peptide FVIII 2194-2213 , and it was identical to the HLA-DRA*01-DRB1*01:01-restricted epitope recognized by the mild HA subjects. Multiple T-cell clones and polyclonal lines having different avidities for the peptide-loaded tetramer were isolated from all subjects. Only high- and medium-avidity T cells proliferated and secreted cytokines when stimulated with FVIII 2194-2213 T-cell receptor β (TCRB) gene sequencing of 15 T-cell clones from the severe HA subject revealed that all high-avidity clones expressed the same TCRB gene. High-throughput immunosequencing of high-, medium-, and low-avidity cells sorted from a severe HA polyclonal line revealed that 94% of the high-avidity cells expressed the same TCRB gene as the high-avidity clones. TCRB sequencing of clones and lines from the mild HA subjects also identified a limited TCRB gene repertoire. These results suggest a limited number of epitopes in FVIII drive inhibitor responses and that the T-cell repertoires of FVIII-responsive T cells can be quite narrow. The limited diversity of both epitopes and TCRB gene usage suggests that targeting of specific epitopes and/or T-cell clones may be a promising approach to achieve tolerance to FVIII.

Objectives: To investigate the genetic and acquired thrombophilic risk factors in pregnancy-associated complications and venous thromboembolism (VTE) and evaluate the association between particular thrombophilic risk factors and thromboembolic complications., Methods: In this study, pregnant women with pregnancy complications and VTE (N = 101) were the study group, and the control group were women with normal pregnancy (N = 102). All women underwent testing for factor V Leiden mutation (FVL), mutation of the coagulation factors II (FII20210), methylenetetrahydrofolate reductase (MTHFR), plasminogen activator inhibitor-1, antithrombin III (ATIII), protein C (PC) and protein S, lupus anticoagulant (LAC) antibodies, anticardiolipin antibodies and anti-beta-2-glycoprotein-1., Results: In this study group, mutations of the FVL was 15.8% (16/101), FII20210 5.9% (6/101) and the MTHFR at locus 677 was TT in 19.8% (20/101). Deficiency of ATIII and PC were rare: 3.0% and 1.0%, respectively. LAC were significantly higher in the study group than in the control group: 32.7% versus 3.9%; p < 0.0005. Pregnant women with VTE have been more frequent for FVL (41.7%; p < 0.005), PC deficiency (25.0%; p < 0.005) and LAC (33.3%; p < 0.005). Combination of FVL and MTHFR mutation was related to the risk of recurrent fetal death and habitual abortion., Conclusion: The inherited and the acquired thrombophilic risk factors were found to be up to 10 times more common in the study group than in the control group.

The impact of treatment-related factors on inhibitor development in previously untreated patients (PUPs) with haemophilia A is still debated. We present the results of a collaborative, individual patient data meta-analytic project. Eligible data sources were published cohorts of PUPs for which patient-level data were available. The exposures of interest were factor (F)VIII type (recombinant [rFVIII] vs plasma-derived [pdFVIII]) and treatment intensity (≥ vs < 150 IU/kg/week) at first treatment. Family history of inhibitors, F8 mutations, age, treatment regimen (on-demand vs prophylaxis), secular trend and surgery were analysed as putative confounders using different statistical approaches (multivariable Cox regression, propensity score analyses, CART). Analyses accounted for the multi-centre origin of the data. We included 761 consecutive, unselected PUPs with moderate to severe haemophilia A from 10 centres in Egypt, Germany, Israel and Italy. A total of 27 % of patients developed inhibitors; 40 % and 22 % of patients treated with rFVIII and pdFVIII (unadjusted HR 2.2, 95 % CI 1.6-2.9), respectively; 51 % and 24 % of patients receiving high- and low-intensity treatment (unadjusted HR 2.9, 95 % CI 2.0-4.2), respectively. In adjusted analyses, only treatment intensity remained an independent predictor; the effect of FVIII type was largely due to confounding, but with a significant interaction between FVIII type and treatment intensity. This patient-level meta-analysis confirms, across different statistical approaches, that high-intensity treatment is a strong risk factor for inhibitor development. The possible role of FVIII type in subgroups is suggested by the test for interactions but could not be proven because of the limited subgroups sample sizes.

Missense mutations are the most common F8 gene defects among the patients with non-severe haemophilia A. This type of mutation is typically associated with low (5%) inhibitor risk. In the present retrospective study we analysed the clinical data of 16 haemophiliacs with the T295A missense mutation treated at Bonn Haemophilia Centre. In total, three patients developed inhibitors: two patients experienced low-titer and one high-titer inhibitors. Both patients with low titer inhibitors underwent successful ITI. The third patient, at the age of 81, developed initially low-titer inhibitors (3 BU/ml) after rFVIII therapy because of knee surgery. He experienced spontaneous multiple large skin haematomas and haemarthrosis. Immunosuppressive therapy was not applicable because of the infectious origin of discitis (Th3-Th4). Immunoadsorption was performed, but the inhibitor titer increased up to 42 BU/ml nine weeks after termination. A successful treatment of discitis with antibiotics finally allowed a weekly therapy (four times) with rituximab (375 mg/m(2)). This resulted in a decrease of inhibitor titre to 0.7 BU/ml eight weeks after the fourth rituximab application. Patient had endogenous FVIII levels of 3-5%. Twelve months after rituximab therapy (after B cells recovery) he relapsed with low-titer inhibitors and therefore was treated with single rituximab dose (375 mg/m(2)) again. This resulted in his depletion of B cells, measurable endogenous FVIII levels and non measurable inhibitors. This study demonstrated T295A variant to be associated with significantly increased (3/16 patients, 17%) inhibitor development.

The two plasma inhibitors, protein Z-dependent protease inhibitor (ZPI) and tissue factor pathway inhibitor (TFPI), effectively inhibit the activity of activated factor X (FXa); however, neither inhibitor exhibits any reactivity with the homologous protease activated factor IX (FIXa). In this study, we investigated the molecular basis for the lack of reactivity of FIXa with these plasma inhibitors and discovered that unique structural features within residues of the 39-loop are responsible for restricting the inhibitor specificity of FIXa. This loop in FXa is highly acidic and contains three Glu residues at positions 36, 37, and 39. On the other hand, the loop is shorter by one residue in FIXa (residue 37 is missing), and it contains a Lys and an Asp at positions 36 and 39, respectively. We discovered that replacing residues of the 39-loop (residues 31-41) of FIXa with corresponding residues of FXa renders the FIXa chimera susceptible to inactivation by both ZPI and TFPI. Thus, the inactivation rate of the FIXa chimera by ZPI in the presence of protein Z (PZ), negatively charged membrane vesicles, and calcium ions approached the same diffusion-limited rate (>10(7) m(-1) s(-1)) that has been observed for the PZ-dependent inhibition of FXa by ZPI. Interestingly, sequence alignments indicated that, similar to FXa, residue 36 is a Glu in both mouse and bovine FIXa and that both proteases are also susceptible to inhibition by the PZ-ZPI complex. These results suggest that structural features within residues of the 39-loop contribute to the resistance of FIXa to inhibition by plasma inhibitors ZPI and TFPI.

We have identified 1,135 haemophilia A patients with missense mutations associated with mild (46%), moderate (22%), severe (16%), and mixed haemophilia phenotypes (11%). Altogether, we detected 374 different missense mutations of which 195 are not listed in the HAMSTeRS database. While missense mutations are strongly underrepresented within the factor VIII (FVIII) B-domain, they are evenly distributed throughout the entire F8 cDNA sequence. Only 36 (5%) of 720 patients with missense mutations and known inhibitor status showed an association with inhibitor formation. Inhibitor prevalence was four-fold higher for severe haemophilia compared to mild/moderate phenotypes. Mutations associated with inhibitor formation were especially clustered within the C1/C2 domain compared to the other domains (8.7% C1/C2 domain vs. 3.6% non-C1/C2-domain; p-value: 0.01). Three different missense mutations (T314A [T295A], S2010P [S1991P], R2169H [R2150H]) were associated twice with inhibitor formation. Importantly, we found that the risk of inhibitor formation in association with FVIII missense mutations is significant higher if the amino acid substitution belongs to another physicochemical class than the original residue (p-value 0.039). For this purpose distinct classes of substitutions were grouped in association with side chains properties: class I, small/hydrophobic; class II, neutral; class III, acidic; class IV, basic. Thus, although missense mutations were associated with an overall lower risk of inhibitor formation compared to other F8 gene mutation types, different missense mutations correlate with specific risks for inhibitor formation. These differences have to be identified in assigning risk profiles to aid in choice of preventative treatments designed to prevent inhibitor formation.

Hemostasis encompasses the tightly regulated processes of blood clotting, platelet activation, and vascular repair. After wounding, the hemostatic system engages a plethora of vascular and extravascular receptors that act in concert with blood components to seal off the damage inflicted to the vasculature and the surrounding tissue. The first important component that contributes to hemostasis is the coagulation system, while the second important component starts with platelet activation, which not only contributes to the hemostatic plug, but also accelerates the coagulation system. Eventually, coagulation and platelet activation are switched off by blood-borne inhibitors and proteolytic feedback loops. This review summarizes new concepts of activation of proteases that regulate coagulation and anticoagulation, to give rise to transient thrombin generation and fibrin clot formation. It further speculates on the (patho)physiological roles of intra- and extravascular receptors that operate in response to these proteases. Furthermore, this review provides a new framework for understanding how signaling and adhesive interactions between endothelial cells, leukocytes, and platelets can regulate thrombus formation and modulate the coagulation process. Now that the key molecular players of coagulation and platelet activation have become clear, and their complex interactions with the vessel wall have been mapped out, we can also better speculate on the causes of thrombosis-related angiopathies.

Development of inhibitors to factor VIII, a serious complication of replacement therapy in haemophilia A patients, leads to increased bleeding, morbidity and mortality. There is no data on the risk factors for inhibitor development in Indian patients with severe haemophilia A. Our aim was to study the role of immune regulatory gene polymorphisms in the development of inhibitors. Fourteen immune regulatory gene polymorphisms (IL1β, IL4, IL10, TNFA and CTLA4) were analysed in 120 patients with severe haemophilia A, i.e. 50 inhibitor positive patients, and 70 inhibitor negative control patients, by PCR-RFLP, DNA sequencing and allele-specific PCRs. The IL10 promoter 'GCC' haplotypes overall (P: 0.002, OR: 3.452, 95% CI: 1.607-7.416), and 'GCC/ATA' (P: 0.011, OR: 3.492, 95% CI: 1.402-8.696) haplotype, associated with high and intermediate IL10 production, respectively, were significantly higher in inhibitor positive patients, whereas the 'non-GCC' haplotypes overall (P: 0.002,OR: 0.290, 95% CI 0.135-0.622) and 'ATA/ATA' haplotype (P: 0.025, OR: 0.278, 95% CI: 0.096-0.802), associated with low IL10 synthesis, were significantly higher among inhibitor negative patients. The TNFA rs1799724 C/T heterozygote prevalence was significantly higher in the inhibitor positive group (P: 0.021, OR: 3.190, 95% CI: 1.273-7.990), whereas the other polymorphisms showed no statistically significant association with the presence of inhibitors. Different immune regulatory gene polymorphisms play a significant role as possible risk factors for the development of inhibitors in severe haemophilia A patients., (© 2012 Blackwell Publishing Ltd.)

Unlabelled: The development of inhibitors in haemophilia B is one of the most important complications of replacement therapy, affecting mortality and morbidity. Inhibitor development is based on complex immunological factors, and to date, only little is known about its underlying mechanisms. Here, we present first results of the haemophilia B group of our Inhibitor-Immunology study., Patients, Methods: So far we have analysed 15 patients with haemophilia B. Four of them developed a high titre inhibitor; the remaining 11 had no inhibitor. We evaluated 9 SNPs in 8 genes (CD40, CTLA-4 , IL-1β, IL-10, TLR2 , TLR4, TLR9, TNF-α). We compared the distribution of these alleles between inhibitor and non-inhibitor haemophilia B patients and between haemophilia B patients and a normal male control population. HLA typing was performed in all patients. Results, discussion: There appears to be a trend towards a skewed distribution of TLR 9, IL-10 and CTLA4 alleles in haemophilia B patients. Due to the limited number these differences are, however, not statistically significant. The t-test of all patients with inhibitor versus without inhibitor was significant for HLA-A*03 and DPB1*0401 and borderline for DRB1*0201.

The mechanisms of Ebola (EBOV) pathogenesis are only partially understood, but the dysregulation of normal host immune responses (including destruction of lymphocytes, increases in circulating cytokine levels, and development of coagulation abnormalities) is thought to play a major role. Accumulating evidence suggests that much of the observed pathology is not the direct result of virus-induced structural damage but rather is due to the release of soluble immune mediators from EBOV-infected cells. It is therefore essential to understand how the candidate therapeutic may be interrupting the disease process and/or targeting the infectious agent. To identify genetic signatures that are correlates of protection, we used a DNA microarray-based approach to compare the host genome-wide responses of EBOV-infected nonhuman primates (NHPs) responding to candidate therapeutics. We observed that, although the overall circulating immune response was similar in the presence and absence of coagulation inhibitors, surviving NHPs clustered together. Noticeable differences in coagulation-associated genes appeared to correlate with survival, which revealed a subset of distinctly differentially expressed genes, including chemokine ligand 8 (CCL8/MCP-2), that may provide possible targets for early-stage diagnostics or future therapeutics. These analyses will assist us in understanding the pathogenic mechanisms of EBOV infection and in identifying improved therapeutic strategies.

Several genes that modify risk of factor VIII (FVIII) inhibitors in haemophilia A patients have been identified. Aside from the underlying mutations that cause haemophilia A, inhibitor risk appears to be modified by polymorphisms in various cytokines and immunomodulators including IL10, TNFα and CTLA4. HLA haplotypes have not been strong determinants of inhibitor risk. We sought to confirm previous observations on FVIII inhibitor risk-modifying genes and to test new candidate genes encoding various otherTH1/TH2 cytokines. We also sought to determine whether normal FVIII gene polymorphisms affect inhibitor risk in caucasians. We studied 915 caucasian, severe haemophilia A patients (282 inhibitor cases and 633 non-inhibitor controls). Genes were analysed using 368 tagging single nucleotide polymorphisms starting 20 kb 5' and ending 10 kb 3' of each gene's coding sequence; four other polymorphisms (factor V Leiden & prothrombin 20210 polymorphisms and two in HFE) were also evaluated. Haplotypes that increased inhibitor risk were found in IL10 (OR = 1.33, P = 0.04), IL12 (OR = 1.31, P = 0.04) and IL1α (OR = 2.16, P = 0.034). Protective haplotypes were seen in IL2 (OR =  .69, P = 0.008) and IL1β (OR = 0.75, P = 0.02). One rare haplotype in the FVIII gene increased the risk of inhibitor development by nearly fourfold (OR = 3.8, P = 0.004). We replicate previous findings for IL10; identify new associations with IL1, IL2 and IL12; and identify a rare FVIII haplotype in caucasians that is associated with increased inhibitor risk., (© 2011 Blackwell Publishing Ltd.)

The prevalence of inhibitors in Chinese haemophiliacs has not yet been reported. The aim of this study was to identify the prevalence of factor VIII (FVIII) inhibitors among haemophiliacs who are treated only with plasma-derived FVIII (pdFVIII), cryoprecipitate or fresh frozen plasma (FFP), and tried to explore the relationship between the generation of inhibitors and particular FVIII deficiency genotypes. Clinical information and blood samples of 1435 patients with haemophilia A (HA) were collected by six haemophilia centres in China. The Nijmegen modification of the Bethesda assay was used to detect inhibitors. Multiplex PCR, long-range PCR and direct sequencing were performed for genotyping. The overall prevalence of inhibitors in Chinese HA patients was 3.9% and the prevalence of severe haemophiliacs was 4.3%; 18 of the 56 patients with inhibitors had high titres. A total of 38 different mutations were identified in the 55 patients with inhibitors, including 15 intron 22 and 3 intron 1 inversions, seven large deletions, 14 small deletion/insertions, seven nonsense mutations, one splice site mutations and eight missense mutations. Of 38 mutations, 28 were novel. Patients with large deletions and nonsense mutations were prone to have high titre inhibitors, with incidence rates of 57.1% (4/7) and 42.9% (3/7), respectively. In conclusion, the prevalence of inhibitors in Chinese HA patients is much lower than that reported for other ethnic groups and the large deletion and nonsense mutations are high risk factors for high titre inhibitor development.

One of the most severe and important complication in the treatment of patients with haemophilia A is the formation of neutralizing antibodies (FVIII inhibitors) that inhibit the clotting activity of substituted FVIII. Both genetic and environmental factors influence the susceptibility of patients to develop inhibitors. The objective of this study was to evaluate whether polymorphisms in different genes involved in the regulation of the immune system may confer susceptibility to inhibitor development in patients with HA. We analysed the distribution of polymorphisms in the CTLA4, PTPN22, IL10, TNFalpha, FOXP3 and IRF5 genes that have been reported to be associated with a number of autoimmune disease. In addition, we evaluated the distribution of IL10 haplotypes in haemophilic patients and healthy controls to assess whether specific polymorphisms in IL10 gene were associated to the risk of inhibitor development. We focused on a cohort of Italian unrelated haemophilic patients with and without a history of inhibitors. Genotyping was carried out with standard methods including RFLP, real time PCR and direct DNA sequencing. Our data show that, considering single nucleotide variations, genotype frequencies in patients with inhibitors were not significantly different from those observed in patients without inhibitors, suggesting a lack of association between these polymorphisms and the development of inhibitors. Moreover, no relationship was found between specific combinations of IL10 alleles and the antibody production. Previous contradictory association studies may depend on the different genetic background of the population examined. Further studies may contribute to a clearer understanding of this process.

Purpose of Review: Appropriate expression of immunomodulatory and anticoagulant proteins on endothelial cells is essential to prevent rejection of vascularized porcine organs after transplantation into primates. Here, we review the promoter sequences used for the establishment of transgenic pigs, as organ donors for xenotransplantation., Recent Findings: Transgenic pigs were produced using viral, chicken, mouse, human, and porcine promoter sequences with ubiquitous or cell type-specific activity. In addition to the expression of human complement regulatory proteins, which were efficient to prevent hyperacute rejection of pig-to-primate xenografts, novel transgenes, targeting cellular rejection mechanisms, abnormal-blood coagulation, or the risk of viral transmission, have been published or announced in preliminary reports., Summary: Accurate spatiotemporal expression of immunomodulatory and anticoagulant proteins on the endothelial cells of transgenic pigs is required for the successful xenotransplantation of vascularized organs into primates. Targeting transgene expression specifically to the cells critical for xenograft rejection may eliminate potential side effects of ubiquitous expression. Comparison of regulatory sequences from various species indicates that carefully selected porcine promoter sequences may be beneficial to achieve this aim.

The formation of antibodies against factor VIII or factor IX that inhibit replacement therapy is currently the most serious treatment-related complication faced by patients with haemophilia. This review highlights non-modifiable and modifiable risk factors that determine the development of these antibodies. The non-modifiable risk factors include patient genotype for haemophilia, immunogenotype, ethnicity and positive family history. Age, intensity of treatment and the type of clotting factor administered are identified as modifiable risk factors. These risk factors are likely to be identified more accurately in forthcoming prospective randomized controlled trials and current patient registries. Through a more complete picture of a patient's overall risk profile, individually tailored treatment schedules might be developed that could minimize the incidence of inhibitor formation and thus maximize therapeutic benefit.

In vitro thrombin generation, which reflects an individual's plasma coagulation potential and has been shown to correlate with the risk of venous thromboembolism (VTE), might represent a useful intermediate phenotype for the genetic dissection of VTE. As a proof of principle, we have investigated whether the thrombin generation assay can detect changes in the haemostatic balance associated with common genetic variation affecting the level or function of coagulation factors and inhibitors. The study population consisted of 140 healthy individuals. Plasma levels of coagulation factors and inhibitors and thrombin generation parameters determined at low tissue factor (TF) + or - thrombomodulin (TM) and at high TF + or - activated protein C (APC) were available from a previous study. All individuals were genotyped for F5 Leiden, F2 G20210A and 19 additional single nucleotide polymorphisms (SNPs) in haemostasis-related genes. The association of each SNP with plasma levels of the corresponding proteins and with thrombin generation parameters (lag time, peak height and endogenous thrombin potential [ETP]) was evaluated by statistical analysis. Not only F5 Leiden and F2 G20210A, but also several other common SNPs, significantly affected thrombin generation parameters. In particular, FGA A1069G (Thr312Ala) decreased the ETP(-APC), F2 A19911G increased the ETP(-APC), F10 IVS2 C+517G decreased the ETP(+APC), F12 C-46T decreased peak height at low TF, and TFPI T-287C and TFPI IVS7 T-33C decreased the ETP(+APC). These results indicate that the thrombin generation assay is sensitive to genetic variation in haemostasis-related genes, which makes it a promising tool to identify novel genetic risk factors of VTE.

Inhibitor development, because of its impact on patients' morbidity and quality of life, is presently the most serious complication of haemophilia A treatment. The identification of several genetic and non-genetic risk factors may be used for the stratification of inhibitor risk and the definition of prevention strategies, particularly for patients with a high-risk genetic profile. The most extensively studied genetic factor is the type of F8 mutation, i.e. large deletions, nonsense mutations and inversions, which are associated with a higher risk of inhibitor development. This is the basis for the increased risk in patients with inhibitor family history; however, concordance family studies showed that factors other than F8 mutations are involved. An emerging role is investigated for polymorphisms of immune-regulatory genes that may increase (IL-10 and TNF-alpha) or reduce (CTLA-4) inhibitor risk and whose heterogeneous ethnic distribution may correlate to the higher inhibitor risk in non-caucasian patients. A role for FVIII haplotypes, particularly in black haemophiliacs, has been recently proposed. Recent studies report an increased inhibitor risk for initial intensive treatments (surgery or severe bleeds requiring high-dose and/or prolonged treatment, presence of danger signals), whereas regular prophylaxis (absence of danger signals) exerts a protective effect. A clinical score including the type of F8 mutation, family history of inhibitors and intensive treatment has been recently validated for predicting inhibitor risk. Because of the lack of useful data regarding the role of different types of FVIII concentrates, the stratification of risk in patients starting replacement treatment together with the careful evaluation of indications, doses and duration of treatment at first exposures and further efforts for overcoming barriers to early implementation of prophylaxis are encouraged, particularly for patients with a predictable high inhibitor risk.

Development of inhibitory antibodies (inhibitors) to factor VIII (FVIII) is the most serious adverse event in replacement therapy of hemophilia A patients. The etiology and management of this condition remain major challenges for both researchers and clinicians. In the present review, we discuss recent advances in understanding the molecular mechanisms by which inhibitors inactivate FVIII and experimental approaches used for the mapping of inhibitor epitopes. We also present a comparative analysis of treatment of hemophilia A patients with inhibitors with currently available bypassing agents-activated prothrombin complex concentrate (FEIBA VH; Baxter Healthcare Corp., Westlake Village, CA) and recombinant activated factor VII (NovoSeven; Novo Nordisk, Princeton, NJ)-and describe some ongoing research programs aimed at developing new treatment options for these patients. Availability of sensitive and standardized laboratory assays that would assist in monitoring the effectiveness of bypass therapies is essential for designing customized treatment regimens and improvement in the management of health conditions of hemophilia patients with inhibitors.

The most important complication in the treatment of hemophilia A patients today is the development of inhibitory antibodies against infused factor VIII (FVIII). Inhibitor development is caused by a complex interplay between both genetic and environmental factors. The risk of developing inhibitors is greatest in previously untreated patients with severe hemophilia A. Several genetic factors, such as a positive family history of inhibitors, ethnicity, FVIII genotype, and certain polymorphisms in immune modulatory genes, are associated with the risk of inhibitor development. Treatment-related factors, such as intensive treatment with FVIII for bleeds or surgery, are associated with a higher inhibitor risk. However, regular prophylaxis seems to have a protective effect on inhibitor development. Knowledge about the risk factors of inhibitor development is a condition for predicting and in the future possibly even preventing the development of inhibitors in patients with severe hemophilia A. This review summarizes the current knowledge on the potential risk factors of inhibitor development. At present, many uncertainties still remain that will require collaborative investigation.

Inhibitor formation is a major complication of haemophilia treatment. In a prevalent case-control study, we evaluated blood product exposure, genotype and HLA type on haemophilia A inhibitor formation. Product exposure was extracted from medical records. Genotype was determined on stored DNA samples by detection of virtually all mutations-SSCP (DOVAM-S) and subcycling PCR. HLA typing was performed by PCR amplification and exonuclease-released fluorescence. Cases experienced higher intensity factor, 455 vs. 200 U per exposure, P < 0.005, more frequent central nervous system (CNS) bleeding, seven of 20 (35.0%) vs. one of 57 (1.7%), P = 0.001 and more commonly from inhibitor families, seven of 20 (35.0%) vs. zero of 57 (0%), P < 0.001, and African-American, 12 of 63 (19.0%) vs. six of 117 (5.1%), P = 0.015. Among the latter, CNS bleeding was more commonly the initial bleed, 60% vs. 0%, P < 0.001, and survival was shorter, 14 vs. 38 yr, P = 0.025. Inhibitor formation was uncommon in those with missense mutations, two of 65 (3.1%) vs. 31 of 119 (26.0%), P = 0.008, and unrelated to factor VIII immunogenic epitope, P = 0.388, or HLA type, P > 0.100. Genotype was not associated with race. Time to immune tolerance was shorter for titres <120 vs. > or = 120 BU/mL, six vs. 16 months, P < 0.01, but unaffected by tolerizing dose regimen, P > 0.50. Inhibitor formation is associated with high intensity product exposure, CNS bleeding, African-American race and low frequency of missense mutations. The ideal time to initiate prophylaxis to reduce CNS bleeding and inhibitor formation will require prospective studies.

BDDrFVIII is a B-domain deleted recombinant factor VIII (rFVIII) product for haemophilia A. Manufacture uniquely includes purification chromatography by synthetic-affinity ligand rather than murine-based monoclonal antibody, as well as an albumin-free cell culture process. BDDrFVIII was studied in 204 patients, including 62 subjects <16 years old, in two studies. A double-blind, randomized, pharmacokinetic (PK) crossover study, utilizing a central laboratory assay (one-stage (OS)) for both drug potency assignment and plasma FVIII-activity measurements, demonstrated that BDDrFVIII was PK-equivalent to a full-length rFVIII. Favourable efficacy and safety were observed: during defined routine prophylaxis in a patient population significant for preexisting target joints, nearly half (45.7%) of patients had no bleeding, and a low-annualized bleed rate (ABR) was achieved (median 1.9); 92.5% of haemorrhages (n = 187) required < or =2 infusions. Three subjects (1.5%, across both studies) developed de novo inhibitors (low-titre, transient), and the primary safety endpoint, based on a prospective Bayesian analysis, demonstrated the absence of neoantigenicity for BDDrFVIII. The PK-equivalence, based on central testing to align test and reference articles, and the novel Bayesian analysis of inhibitor safety in these investigations reflect robust experimental designs with relevance to future studies. This extensive dataset demonstrates the safety and efficacy of BDDrFVIII for haemophilia A.

Haemophilia A (HA) is a bleeding disorder caused by mutations within the X-linked F8 gene. A series of 42 unrelated Moldovan patients with HA had their disease-causative mutation determined to provide clinically valuable genotyping information for a historically underserved population and to utilize factor VIII (FVIII) structural information to analyse the effects of haemophilic missense substitutions. DNA samples were analysed to detect intron 22 and intron 1 inversions followed by heteroduplex analysis of PCR-amplified fragments containing all exonic sequences. Missense sites identified by DNA sequencing were visualized in the recently described crystal structures of human FVIII. Of the 26 different point mutations, 12 were novel. Gel electrophoresis identified samples with a second major DNA band that migrated abnormally; these amplified products were sequenced. Thirteen intron 22 inversions and one intron 1 inversion were found. Two patients had large, partial gene deletions and there were six frameshift, two non-sense, two splicing and 16 missense genotypes. Two subjects with an intron 22 inversion and one with a large, partial gene deletion developed an alloimmune inhibitor. Their localization suggests intra- and possibly inter-molecular interactions that are important for the structural integrity and/or procoagulant function of FVIII.

Thrombophilia screening is controversial. In a retrospective family cohort, where probands had thrombosis and a thrombophilic defect, 2479 relatives were tested for thrombophilia. In antithrombin-, protein C-, and protein S-deficient relatives, annual incidences of venous thrombosis were 1.77% (95% CI, 1.14-2.60), 1.52% (95% CI, 1.06-2.11), and 1.90% (95% CI, 1.32-2.64), respectively, at a median age of 29 years and a positive family history of more than 20% symptomatic relatives. In relatives with factor V (FV) Leiden, prothrombin 20210G>A, or high FVIII levels, these were 0.49% (95% CI, 0.39-0.60), 0.34% (95% CI, 0.22-0.49), and 0.49% (95% CI, 0.41-0.51), respectively. High FIX, FXI, and TAFI, and hyperhomocysteinemia were not independent risk factors. Annual incidence of major bleeding in antithrombin-, protein C-, or protein S-deficient relatives on anticoagulants was 0.29% (95% CI, 0.03-1.04). Cumulative recurrence rates in relatives with antithrombin, protein C, or protein S deficiency were 19% at 2 years, 40% at 5 years, and 55% at 10 years. In relatives with FV Leiden, prothrombin 20210G>A, or high levels FVIII, these were 7%, 11%, and 25%, respectively. Considering its clinical implications, thrombophilia testing should address hereditary deficiencies of antithrombin, protein C, and protein S in patients with first venous thrombosis at young age and/or a strong family history of venous thrombosis.

Background: Black patients with hemophilia A (factor VIII deficiency) are twice as likely as white patients to produce inhibitors against factor VIII proteins given as replacement therapy. There are six wild-type factor VIII proteins, designated H1 through H6, but only two (H1 and H2) match the recombinant factor VIII products used clinically. H1 and H2 are found in all racial groups and are the only factor VIII proteins found in the white population to date. H3, H4, and H5 have been found only in blacks. We hypothesized that mismatched factor VIII transfusions contribute to the high incidence of inhibitors among black patients., Methods: We sequenced the factor VIII gene (F8) in black patients with hemophilia A to identify causative mutations and the background haplotypes on which they reside. Results from previous Bethesda assays and information on the baseline severity of hemophilia, age at enrollment, and biologic relationships among study patients were obtained from review of the patients' medical charts. We used multivariable logistic regression to control for these potential confounders while testing for associations between F8 haplotype and the development of inhibitors., Results: Of the 78 black patients with hemophilia enrolled, 24% had an H3 or H4 background haplotype. The prevalence of inhibitors was higher among patients with either of these haplotypes than among patients with haplotype H1 or H2 (odds ratio, 3.6; 95% confidence interval, 1.1 to 12.3; P=0.04), despite a similar spectrum of hemophilic mutations and degree of severity of illness in these two subgroups., Conclusions: These preliminary results suggest that mismatched factor VIII replacement therapy may be a risk factor for the development of anti-factor VIII alloantibodies., (2009 Massachusetts Medical Society)

Continuous expression of activated factor VII (FVIIa) via gene transfer is a potential therapeutic approach for hemophilia patients with or without inhibitory antibodies to human factor VIII (FVIII) or IX (FIX). Here, we investigate whether gene transfer of an engineered canine FVIIa (cFVIIa) transgene can affect hemostasis in a canine model of hemophilia, a good predictor of efficacy of hemophilia treatments. Purified recombinant cFVIIa exhibited 12-fold higher tissue factor-dependent activity than purified recombinant zymogen cFVII. Subsequently, we generated a serotype 8 recombinant adeno-associated viral vector expressing cFVIIa from a liver-specific promoter. Vector delivery via the portal vein in hemophilia A and B dogs was well tolerated, and long-term expression of cFVIIa resulted in a shortening of the prothrombin time, partial correction of the whole blood clotting time and thromboelastography parameters, and a complete absence of spontaneous bleeding episodes. No evidence of hepatotoxicity, thrombotic complications, or inhibitory immune response was found. These data provide the first evidence for in vivo efficacy and safety of continuously expressed FVIIa as a FVIII/FIX-bypassing agent in a large animal model of hemophilia, avoiding the risk of inhibitor formation associated with bolus FVIII or FIX infusion.

Purpose of Review: Vascular injury is a complex process that is central to the rejection of solid-organ xenografts in the preclinical pig-to-primate model of xenotransplantation. This review summarizes recent work that provides insights into the mechanisms of vascular injury in GalT KO pig xenografts., Recent Findings: Several groups have reported further evidence for the importance of preexisting and elicited non-GalT antibodies, which are capable of fixing complement and activating graft endothelial cells. One important study showed that without complete suppression of these antibodies, there is a progressive activation and injury of xenograft endothelium, resulting in the development of thrombotic microangiopathy and graft loss. The mechanism of molecular incompatibilities affecting the control of coagulation across the species barrier has been examined in finer detail. The failure of pig thrombomodulin to promote activation of human protein C was found to be due to a deficiency in cofactor activity rather than to its capacity to bind human thrombin. On a positive note, pig tissue factor pathway inhibitor appears to be capable of efficiently regulating the human tissue factor pathway, contrary to earlier reports. We and others remain optimistic that overexpressing complement regulators anticoagulant proteins or both on the GalT knockout background will provide a significant protective effect, but definitive testing of this approach in the preclinical model is still forthcoming., Summary: Preventing the activation of xenograft endothelium and consequent intravascular coagulation is critical to the future success of solid-organ xenotransplantation.

Genetic factors involved in the interindividual variability of antithrombin have not been identified. We studied two polymorphisms of the gene coding for antithrombin (SER-PINC1) in 298 Spanish Caucasian blood donors: rs3138521, a DNA length polymorphism located on the promoter region and rs2227589, a SNP located on intron 1 that has been described as a mild thrombotic risk factor. We detected a complete linkage disequilibrium between these polymorphisms (D'=0.999). The rs3138521 polymorphism has no functional consequences. However, the rs2227589 SNP significantly associated with plasma anti-FXa activity and antithrombin levels: carriers of the A allele had slightly but significantly lower anticoagulant activity and levels than GG subjects (97.0+/-7.3% vs. 94.6+/-8.4%; p=0.032; 99.5+/-5.8% vs. 94.8+/-5.6%; p=0.001; respectively). Our results identified a functional effect of the rs2227589 polymorphism not explained by its linkage with the promoter polymorphism that support the moderate thrombotic risk associated with the A allele.

Background and Objectives: Genes encoding protein C anticoagulant pathways are candidates for atherothrombotic and other aging-related disorders., Methods: Using a tagSNP approach, and data from the Cardiovascular Health Study (CHS), we assessed associations of common polymorphisms of PROC, PROS1 and PROCR with: (i) plasma protein C, soluble protein C endothelial receptor (sEPCR) and protein S levels measured in a subsample of 336 participants at study entry; and (ii) risk of incident clinical outcomes [coronary heart disease (CHD), stroke, and mortality] in 4547 participants during follow-up. Secondarily, we explored associations between plasma protein C, protein S and sEPCR levels and other candidate genes involved in thrombosis, inflammation, and aging., Results: The PROCR Ser219Gly polymorphism (rs867186) was strongly associated with higher sEPCR levels, explaining 75% of the phenotypic variation. The PROCR Ser219Gly variant was also associated with higher levels of circulating protein C antigen. An IL10 polymorphism was associated with higher free protein S levels. The minor alleles of PROC rs2069901 and PROS1 rs4857343 were weakly associated with lower protein C and free protein S levels, respectively. There was no association between PROCR Ser219Gly and risk of CHD, stroke, or mortality. The minor allele of another common PROCR tagSNP, rs2069948, was associated with lymphoid PROCR mRNA expression and with increased risk of incident stroke and all-cause mortality, and decreased healthy survival during follow-up., Conclusions: A common PROCR variant may be associated with decreased healthy survival in older adults. Additional studies are warranted to establish the role of PROCR variants in ischemic and aging-related disorders.

This is a two-part unit on haemophilia and inhibitors. This article, part 1, examines the condition, the problem of inhibitors and treatment options for patients with inhibitors. It also discusses the practical challenges nurses may face in patients' surgical management.

Anti factor VIII (FVIII) antibodies represent the main complication of replacement therapy in severe cases of haemophilia and most patients with inhibitor have gross gene rearrangements or point mutations that hamper the production of normal circulating FVIII. In this study we have investigated 82 haemophilia A patients with inhibitors. Seventy six were severe, three were moderate and three were mild. We screened the patients for the causative mutations using long range PCR for the recurrent intron 22 inversion (invint22), multiplex PCR for intron 1 inversion (invint1) and conformation sensitive gel electrophoresis followed by DNA sequencing for all other mutation types in the F8 gene. Diverse genetic defects were detected in the severe cases (with a predominance of severe molecular defects): F8 gene inversions, large deletions and non-sense mutations account for 71% of the mutations. Only missense and splicing mutations were identified in the non-severe patients and we confirmed that the presence of inhibitors correlates well with the presence of severe mutations, but a proportion of severe patients develops inhibitors despite the presence of diverse less severe mutations. When we have analysed the subgroup of patients who underwent immunetolerance, we have found that F8 gene large deletions are likely to be a high risk factor also for immunetolerance therapy unresponsiveness, while no clear evidence has been demonstrated for other mutation types.

Context: Although the roles of clotting proteins and enzymes that activate or inhibit fibrin production and lysis are well characterized, the underlying contribution of genetic variation in these constituents to risk of venous thrombosis (VT) has not been fully investigated., Objective: To describe the association of common genetic variation in 24 coagulation, anticoagulation, fibrinolysis, and antifibrinolysis candidate genes with risk of incident nonfatal VT in postmenopausal women., Design, Setting, and Participants: Population-based case-control study conducted in a large integrated health care system in Washington State. Participants were perimenopausal and postmenopausal women aged 30 to 89 years who sustained a first VT event between January 1995 and December 2002 (n = 349) and 1680 controls matched on age, hypertension status, and calendar year (n = 1680)., Main Outcome Measure: Risk of venous thrombosis associated with global variation within a gene as measured by common haplotypes and with individual haplotypes and single nucleotide polymorphisms (SNPs). Significance of the associations was assessed by a .20 threshold of the false-discovery rate q value, which accounts for multiple testing., Results: Only the tissue factor pathway inhibitor gene demonstrated global association with risk (q = .13). Five significant SNP associations were identified across 3 of the candidate genes (factors V, XI, and protein C) in SNP analyses. Two associations have been previously reported. Another 22 variants across 15 genes had P values less than .05 but q values between .20 and .35. Five of these confirm previously reported associations (fibrinogen genes and protein C), 2 were inconsistent with earlier reports (thrombomodulin and plasminogen activator inhibitor 1), and 15 were new discoveries., Conclusions: After accounting for multiple testing, 5 SNPs associated with VT risk were identified, 3 of which have not been previously reported. Replication of these novel associations in other populations is necessary to corroborate these findings and identify which genetic factors may influence VT risk in postmenopausal women.

The HLA class I/II alleles and the tumor necrosis factor alpha (TNFA) locus are closely linked in the MHC complex. We have characterized the causative factor VIII mutation, HLA alleles as well as 4 polymorphisms (-827C>T, -308G>A, -238A>G, and 670A>G) in the TNFA gene in 164 patients (124 severe, 26 moderate, and 14 mild) in 78 families with hemophilia A enrolled in the Malmö International Brother Study (MIBS). Inhibitors were identified in 77.8% of patients with a single haplotype (Hap 2) and 72.7% of the patients with the TNFA -308 A/A genotype within this haplotype compared with 39.7% for TNFA -308 G/G patients and 46.9% for TNFA -308 G/A heterozygotes (OR 4.0; 95% CI, 1.4-11.5; P = .008). The association between the -308 A/A genotype and inhibitors was enhanced in subgroups of patients with severe hemophilia (OR 19.2; 95% CI 2.4-156.5; P < .001) and with inversions (n = 75; OR, 11.8; 95% CI, 1.3-105.1; P = .013). Associations were found for the HLA A26 and B44 alleles, but these were not consistent in the subgroup analysis. Our data imply that the TNFA -308G>A polymorphism within Hap 2 is a useful marker and potential modulator of the immune response to replacement therapy in patients with hemophilia.

The formation of alloantibodies against factor VIII (FVIII) or factor IX (FIX) is the most severe complication of replacement therapy in patients with haemophilia. In the last decade, genetic factors have been shown to constitute a decisive risk determinant for the development of inhibitors. In severe haemophilia A and B, mutations that result in an absent or truncated FVIII/FIX protein are associated with a 20-80% risk of inhibitor formation. In mild to moderate haemophilia, missense mutations represent the main mutation type, with an inhibitor prevalence of 5%. These patients synthesize some endogenous, although non-functional protein that is sufficient to induce immune tolerance. However, in patients with missense mutations clustered in the A2 and C2 domains (C1/C2 junction), the risk of inhibitor formation is fourfold greater than in patients with mutations outside this region, indicating that inhibitor prevalence in missense mutations is also dependent on localization of the mutation. Recently, a significant association between inhibitor formation and polymorphisms in genes coding for cytokines (IL-10) and other immunoregulatory factors (TNF-alpha) has been shown. These genetic factors constitute the individual genetic risk profile of a haemophilic patient. This risk is imprinted and fixed; however, environmental factors such as treatment schedule may increase or decrease the inhibitor risk in an individual patient. Improved understanding of these complex interactions may lead to the development of preventive measures to minimize inhibitor formation.

The presence of inhibitory antibodies to factor VIII (FVIII) remains one of the most serious complications of haemophilia therapy. Accordingly, understanding risk factors that may contribute to inhibitor developments in young patients with haemophilia A continues to be an area of great interest. Previously untreated patient (PUP) population studies have been instrumental in understanding the aetiology of inhibitor development. These studies have revealed the importance of risk factors such as clotting factor exposure history, ethnicity, and FVIII genotype in the development of inhibitors, while also providing insights into potential risk factors that may be related to therapeutic practice. However, due to differences in study designs and patient populations among previous PUP studies, there are limitations to the value of these studies in deciphering the role of potential risk factors. Therefore, future PUP studies should be prospective, consistent in their study designs and consider all established parameters and also those that possibly may influence inhibitor formation, thereby facilitating a better understanding of the aetiology of inhibitor formation in haemophilia A patients.

The aim of the Malmö International Brother Study (MIBS) is to evaluate host genetic factors associated with the development of inhibitory antibodies in patients with hemophilia. Factor VIII gene mutations and genetic polymorphisms of the IL1beta, IL4, and IL10 genes, known to influence antibody production in autoimmune diseases, were analyzed in 164 patients (124 with severe, 26 with moderate, and 14 with mild disease) in 78 unrelated families with hemophilia A. Seventy-seven (47%) patients in 54 families had a history of inhibitors (57 high responding, 20 low responding). Inversions were found in 36 families (75 patients). There was no association between the development of inhibitor and the IL1beta Taq I RFLP alleles in exon 5 or the -590 C/T single nucleotide polymorphism (SNP) in the promoter region of IL4. There was, however, a strong association between an allele with 134 bp in one of the CA repeat microsatellites, IL10G, located in the promoter region of the IL10 gene, and the development of inhibitor (odds ratio [OR], 4.4; 95% confidence interval [95% CI], 2.1-9.5; P < .001). The association was consistent in the subgroup of families with severe hemophilia and inversions. IL10 is the first gene located outside the causative factor VIII gene mutation to be associated with inhibitor development.