F8 Mutations Research Articles

Risk Factors for the Progression from Low to High Titres in 260 Children with Severe Haemophilia A and Newly Developed Inhibitors.

In children with severe haemophilia A, inhibitors to factor VIII (FVIII) usually develop during the first 50 treatment exposure days and are classified as low or high titre depending on the peak inhibitor titre being greater or less than 5 Bethesda units/mL (BU/mL). Classification of the inhibitor may change with time, as some low-titre inhibitors progress to high titre following re-exposure to FVIII concentrate. The aim of this study was to investigate potential risk factors for such a progression in children with severe haemophilia A and newly diagnosed inhibitors. This study was a follow-up study of the PedNet Registry and included 260 children with severe haemophilia A and inhibitors born between 1990 and 2009 and recruited consecutively from 31 haemophilia centres. Clinical and laboratory data were collected from the date of each child's first positive inhibitor test for at least 3 years. At the time of first positive inhibitor test, 49% (n = 127) had low-titre inhibitors, with 50% of them progressing to high titre and only 25% maintaining low titres. The FVIII gene (F8) mutation type was known in 247 patients (95%), and included 202 (82%) null mutations. The progression to high-titre inhibitors was associated with null F8 mutations (odds ratio [OR]: 2.6; 95% confidence interval [CI]: 1.0–6.5), family history of inhibitors (OR: 7.2; 95% CI: 1.8–28.4) and the use of high-dose immune tolerance induction, defined as ≥100 IU FVIII concentrate/kg/d (OR: 3.9; 95% CI: 1.5–10.0). These results suggest that high-dose immune tolerance induction should be avoided as the initial strategy in patients who develop low-titre FVIII inhibitors.

The importance of mRNA structure in determining the pathogenicity of synonymous and non-synonymous mutations in haemophilia.

Mutational analysis is commonly used to support the diagnosis and management of haemophilia. This has allowed for the generation of large mutation databases which provide unparalleled insight into genotype-phenotype relationships. Haemophilia is associated with inversions, deletions, insertions, nonsense and missense mutations. Both synonymous and non-synonymous mutations influence the base pairing of messenger RNA (mRNA), which can alter mRNA structure, cellular half-life and ribosome processivity/elongation. However, the role of mRNA structure in determining the pathogenicity of point mutations in haemophilia has not been evaluated. To evaluate mRNA thermodynamic stability and associated RNA prediction software as a means to distinguish between neutral and disease-associated mutations in haemophilia. Five mRNA structure prediction software programs were used to assess the thermodynamic stability of mRNA fragments carrying neutral vs. disease-associated and synonymous vs. non-synonymous point mutations in F8, F9 and a third X-linked gene, DMD (dystrophin). In F8 and DMD, disease-associated mutations tend to occur in more structurally stable mRNA regions, represented by lower MFE (minimum free energy) levels. In comparing multiple software packages for mRNA structure prediction, a 101-151 nucleotide fragment length appears to be a feasible range for structuring future studies. mRNA thermodynamic stability is one predictive characteristic, which when combined with other RNA and protein features, may offer significant insight when screening sequencing data for novel disease-associated mutations. Our results also suggest potential utility in evaluating the mRNA thermodynamic stability profile of a gene when determining the viability of interchanging codons for biological and therapeutic applications.

Study on the Efficacy and Safety of Non-Factor Drugs Applied the Etiological Classification for Treatment of Hemophilia:a Multi-Centre, Prospective Single-Arm Clinical Trial

Objective: Research on the efficacy and safety of non-factor drugs applied the etiological classification for treatment of hemophilia.Diagnostic Methods: Between from June 2013 and June 2014, 73 patients were treated because of hemophilia. There were 69 males and 4 females, ages ranging from 0.4 to 83 years, with a mean of 16.13. We used MC-1000 Coagulometer (from MDC,Berlin, Germany) to detect the level of F¢ø/F¢ù:C, and detected the level of F¢ø/F¢ù inhibitor using Nigmegen assay. To detect mutations in the F8 and F9 gene, Intron 22 inversions were studied by inverse-shifting polymerase chain reaction (IS-PCR) and intron 1 inversions by long distance polymerase chain reaction. Point mutations were studied by sequencing the coding and promoter regions of the F8 and F9 genes.Therapeutic Methods: Patients with hemophilia were prospectively treated with Non-factor drugs without replacement therapy of F¢ø/F¢ù factor. Patients with mutations in f8 and f9 genes were treated with orally Compound Hemophilia Capsule (Shaanxi Yida Hemophilia Institute, Pat. No:ZL201410130047X, Shaanxi Drug Approval No.Z20150044). Each capsules contain the traditional Chinese medicine raw materials as shown in the following: Ginseng Radix et Rhizoma 0.035g, Astragali Radix 0.117g, Angelica Sinensis Radix 0.035g, Paeoniae Radix Alba 0.07g, Ligustri Lucidi Fructus 0.058g, Ecliptae Herba 0.058g etc. specification: 0.3g/capsule*40 capsule/bollte. The effect of drugs was to enhance the activity and synthesis of coagulation factors in hepatocyte. The dose of 1.2g for adults administered three times daily from 3 months to 6 months. The LD50 of the drugs is 32g/Kg in the mice’s acute toxicity test, which is equivalently to 533 times than the therapeutic dosage. In this study, no acute toxicity were observed. Other patients with normal f8 and f9 genes were treated with three kinds of drugs as shown in the following: Cyclophosphamide Injection (Shanxi Pude Pharmaceutical Co., Ltd, Shanxi province, China, H14023686). The dose of 0.2g for adults administered four-intravenous-drip daily from 4 weeks to 6 weeks. Compound Glycyrthizin Injection (Minophagen Pharmaceutical Co.,Ltd, Eisai China Inc. H20190124). The dose of 0.04g for adults administered four-intravenous-drip daily for 4 weeks. The effect of drugs was to stimulating the proliferation and protecting hepatocyte. Mycophemolate Mofetil Capsule (Cisen Pharmaceutical Co., Ltd. Shandong province. China, H20080764). The dose of 1.0g for adults administered twice daily by oral. The effect of drugs was immunosuppressive.Results: Patients were classified by HA (52 cases, 71.23%, F¢ø:C in 1.94±1.59%) and HB (21 cases, 28.77%, F¢ù:C in 2.13±2.06%). There were 58 patients (79.45%) who were detected mutations with maternally inherited, 5 patients(6.85%) who detected mutations without maternally inherited, and 10 cases(13.70%) who had normal f8 and f9 genes. 20 days later after treatment, the level of F¢ø:C was raised from 1.94±1.59 to 11.85±41.33%(P£¼0.01), and the level of F¢ù:C was raised from 2.13±2.06% to 10.10±19.97%(P£¼0.01). However, the patients with normal f8 and f9 genes had the better curative effects (The results were shown in Table/ Figure). After one year, the level of F¢ø:C was 38.89±63.47% in 21 patients with HA, and the level of F¢ù:C was 17.60±32.16% in 10 patients with HB. None of patients reported adverse effects in clinical trials.Conclusion: Non-factor drugs for treatment of hemophilia could promoted the level of F¢ø/F¢ù:C in varying degrees and reduced symptoms. Therefore, the study could provide a new insight into treatment of hemophilia. Further research was needed to understand the mechanism and curative effect.The Institutional Ethics Committee approved the study (Approval No: 20120319).Registration ID: WHO ICTRP & ChiCTR-OPC-16008561 [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Risk Factors for the Development of High-Titer Inhibitors in 260 Children with Severe Hemophilia a Born Between 1990 and 2009: The Remain Study

▪The development of anti-FVIII antibodies (i.e., inhibitors) is the major side effect of severe hemophilia A treatment. Inhibitors mainly develop in children during the first 50 exposure days and are classified in low-and high-titer (i.e., peak titer < or > 5 UB/ml). High-titer inhibitors have the major clinical impact. At diagnosis however, the real nature of the antibody is not clear in all patients, since some low-titer inhibitors may progress to high-titer. The determinants of the evolution from low- to high-titer inhibitors are still unclear and the aim of the present study was to investigate potential risk factors associated with the progression from low- to high-titer inhibitors. This study is a follow-up study of the PedNet Registry and includes 260 children with severe hemophilia A and clinically relevant inhibitors, born between 1990 and 2009 and consecutively recruited from 31 hemophilia centers in 16 countries. Clinical and laboratory data were collected from the date of first positive inhibitor test and covered a minimum of 3-years follow-up. Factors potentially associated with progression from low- to high-titer inhibitor development were analyzed using univariate and multivariate logistic regression. F8 mutation type was known in 247 patients (95%), including 202 (82%) null mutations (i.e., large deletions, nonsense mutations and inversions). Positive family history of inhibitors was present in 37 of 99 (37%) with positive family history of hemophilia. At diagnosis 49% (n=127) had low-titer inhibitors, however, upon FVIII re-exposure, 50% of low-titer inhibitors progressed to high-titer and only 25% of patients (n=69) had persistent low-titer inhibitors. Within the first 3 years of follow-up, immune tolerance induction (ITI) was equally implemented in around 80% of low-and high-titer patients but it was started later in children with high-titers (median time to ITI start 4.5 vs 0.3 months; p<0.001) in whom daily regimens and high-dose FVIII were more frequently adopted (89, 67% vs 41, 50% and 98, 74% vs 35, 43%; p=0.01 and <0.001, respectively). Overall high-titer inhibitor development was associated with null F8 mutations (OR 2.8, 95%CI 1.4-5.5) and family history of inhibitors (OR 3.9, 95%CI 1.2-12.6). The progression from low- to high-titer inhibitors during follow up, was associated with the use of high-dose ITI regimens (i.e., >100 IU/kg/day) with an OR of 3.9 (95%CI 1.5-10.0), independent from the effects of F8 mutation type (adjusted OR 3.6, 95%CI 1.4-9.8) and family history of inhibitors (adjusted OR 6.7, 95%CI 1.1-42.6). No difference was found by comparing the use of daily versus non-daily ITI. In conclusion, in a cohort of 260 children with severe hemophilia A and inhibitors, 49% presented with low-titers at diagnosis and 46% of them progressed to high-titers during follow-up. Progression to high-titer inhibitors was associated with the use of high-dose ITI. These results suggest that intensive ITI should be avoided as initial strategy in low-titer inhibitor patients. DisclosuresMancuso:Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sobi/Biogen Idec: Consultancy, Speakers Bureau; Novo Nordisk: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Baxalta: Consultancy, Speakers Bureau; Bayer Healthcare: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kedrion: Consultancy. Fischer:Wyeth/Pfizer: Research Funding; Biogen: Consultancy; NovoNordisk: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Biotest Octapharma: Speakers Bureau; CSL Behring: Speakers Bureau; Baxter: Consultancy, Research Funding, Speakers Bureau; Freeline: Consultancy; Bayer: Consultancy, Research Funding, Speakers Bureau. Santagostino:Octapharma: Consultancy; Novo Nordisk: Consultancy; Kedrion: Consultancy; Sobi: Consultancy; Biogen Idec: Consultancy; Roche: Consultancy; Grifols: Consultancy; Pfizer: Consultancy; Baxalta: Consultancy; CSL Behring: Consultancy; Bayer: Consultancy. Escuriola:Baxalta, now part of Shire: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Investigator Clinical Studies, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Biotest: Consultancy, Honoraria, Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding; Octapharma: Consultancy, Honoraria, Research Funding; NovoNordisk: Consultancy, Honoraria, Research Funding. Liesner:BPL: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Speakers Bureau; Cangene: Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Baxalta Innovations GmbH, now a part of Shire: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; SOBI: Consultancy, Honoraria, Research Funding, Speakers Bureau; Octapharma: Consultancy, Honoraria, Research Funding, Speakers Bureau; Biogen: Consultancy, Honoraria, Research Funding; Grifols: Consultancy, Honoraria. Nolan:Sobi: Research Funding; Biogen: Research Funding.

Genetic Risk Stratification to Minimize Inhibitor Risk with the Use of Recombinant Factor VIII Concentrates: A Sippet Analysis

▪BackgroundThe development of neutralizing antibodies against factor VIII is a common and serious complication of replacement therapy, occurring mainly in the early stages of treatment. Meta-analyses of observational studies have suggested a higher risk of inhibitor development with concentrates produced by recombinant technologies (rFVIII) than with those derived from human plasma (pdFVIII) containing von Willebrand factor, which was recently confirmed in a randomized trial. In this trial cumulative incidences of inhibitor development were 44.5% for rFVIII and 26.8% for pdFVIII, for a hazard ratio (HR) of 1.87 (95% confidence interval (CI95) 1.17-2.96). Given the particularly high risk with rFVIII , it has been suggested to restrict the use of rFVIII to low risk patients, and treat high-risk patients with pdFVIII. We investigated such a strategy in a post-hoc analysis of the SIPPET study, in which we used the FVIII genotype (F8 gene mutation) to classify patients by prior risk.MethodsSIPPET is an open label international randomized trial on which 251 previously untreated (n=142) or minimally treated (less than five exposure to blood components other than concentrate or cryoprecipitate, n=109) in 42 centers to be treated exclusively with a concentrate from the class of rFVIII or pdFVIII. Patients were tested for inhibitors before entry and at regular intervals during 50 exposure days, 3 years or the development of an inhibitor of at least 0.4 Bethesda units (BU). The trial ran from 2010 to 2014 and was terminated when the prespecified number of patients was included. Patients who had not reached 50ED by that time were censored. Patients were classified at high risk when they carried a null mutation (inversion, large deletion, frameshift, nonsense mutation) in the F8 gene and as low risk when they carried another causative variant (missense, splice site, polymorphisms, no mutation). We estimated cumulative incidences, hazard ratios and numbers needed to harm (NNH) for rFVIII vs pdFVIII for high- and low risk patients.ResultsAmong 251 patients, 76 developed an inhibitor (all > 0.7 BU) of which 50 were high- titer (> 5 BU). Among 197 patients classified as high risk, 65 developed an inhibitor (cumulative incidence 38.2%, CI95 30.8-45.6), whereas among the 38 patients classified as low risk 7 developed an inhibitor (cumulative incidence 23.9%, CI95 8.2-39.6). High and low risk patients were equally distributed over the two arms of the trial, i.e., 96 out of 126 treated with rFVIII were high risk, and 101 out of 125 treated with pdFVIII. Among high risk patients, cumulative incidence was 30.7% when treated with pdFVIII , and 46.5% when treated with rFVIII (risk difference 15.8%). Among low risk patients, no inhibitors developed with pdFVIII, whereas the cumulative incidence was 43.2% with rFVIII (risk difference 43.2%). This implies that the Number Needed to Harm was 5.6 overall, 6.3 for high-risk patients, and 2.3 in low risk patients.ConclusionRisk stratification by the type of F8 mutation does not identify previously untreated patients with hemophilia A who have a low inhibitor risk when exposed to rFVIII. Other means need to be found to reduce the occurrence of inhibitors with rFVIII. DisclosuresPalla:Pfizer: Other: travel support . Mannucci:NovoNordisk: Speakers Bureau; Kedrion: Speakers Bureau; Grifols: Speakers Bureau; Bayer: Speakers Bureau. Peyvandi:Bayer: Speakers Bureau; SOBI: Speakers Bureau; Kedrion Biopharma: Consultancy, Other: research funding paid to Luigi Villa Foundation, Research Funding; Alexion: Other: research funding paid to Luigi Villa Foundation, Research Funding; Octapharma: Consultancy; Ablynx: Membership on an entity's Board of Directors or advisory committees, Other: research funding paid to Luigi Villa Foundation, Research Funding; Biotest: Other: research funding paid to Luigi Villa Foundation, Research Funding, Speakers Bureau; CSL Behring: Speakers Bureau; LFB: Consultancy; Grifols: Speakers Bureau; Novo Nordisk: Other: research funding paid to Luigi Villa Foundation, Research Funding, Speakers Bureau.

Characterization of Missense Mutations in Factor VIII That Lead to Abnormal N-Linked Glycosylation

Most secreted proteins are glycosylated on the asparagine (N) residue with the consensus sequence N-X-S/T(X≠Proline).Coagulation factor VIII (FVIII) is heavily N-linked glycosylated with 5 consensus sites outside the B domain. However, the roles of these glycans are not well understood. Meanwhile, missense mutations which could create additional N-linked glycosylation sites have largely not been characterized in hemophilia A patients.In this study we first expressed individual domains of FVIII and determined that the A2, Cand C2 domains are efficiently secreted. The A1(N42,N239), A3 (N1810)and C1 (N2118)domains are glycosylated, whereas N582 in the A2 domain is not glycosylated. Only one hemophilia A missense mutation, S241C in the A1 domain, was found to abolish the consensus sequence for N-linked glycosylation at N239. We confirmed that the S241C mutant lost one glycan and became unstable inside cells. We also tested the other three glycosylation sites and found that elimination of the N-linked glycan at N2118 (N2118Q mutation) impaired the secretion of the C domain. This defect could not be rescued by adding another N-linked glycan (at N2062) in the C1 domain, indicating that the N2118 glycan is specifically required for the secretion of the C domain.We next searched the CHAMP F8 Mutation Database and the FVIII Variant Database and identified 19 missense mutations that potentially create an ectopic glycosylation site.These mutations are located in A1, A2, A3 and C1 domains, but none in the C2 domain. Only two mutations (I566T and M1772T) have previously been characterized.We found that all but one (I2071T) of these mutations gained an additional N-linked glycan. We further studied missense mutations in the A2 (A469T, A469S, I566T, M614T and G701S) and the C domain (W2062S, I2071T and D2131N) because these domains are secreted in cell culture. Whereas secretion of I566T, W2062S and D2131N mutants was comparable to their wild-type counterparts, secretion of other mutants decreased to 5%-30% of WT (P<0.05). Mutants that secreted into culture media nevertheless have low FVIII activity (<2%), indicating that these mutations cause cross reactive material positive hemophilia A.The consequences of additional N-linked glycan were further investigated using the A2 domain mutants, since this domain is normally unglycosylated. After treating with tunicamycin to block the N-linked glycosylation process in the endoplasmic reticulum (ER),the secretion of A2 domain with I566T andG701Smutants, which had relatively high secretion levels, decreased significantly. On the other hand, removing the additional glycan boosted the secretion of A469S and A469T, two low-secretion mutants.Tunicamycin treatment had no effect on another low secretion mutant,M614T.These results suggest that amino acid substitution in I566T andG701Smutationsis detrimental to the proper folding of the protein and the additional N-glycan plays a stabilization role. On the other hand, additional N-glycan plays a destabilization role in A469S and A469T mutations, contributing to disruption of folding in these mutants. For theM614Tmutation,the amino acid substitution alone is likely sufficient todestroy the protein folding.We also studied interactions of abnormally glycosylated mutants with ER chaperones.All the mutants with low secretion levels significantly induced expression of GRP78 to 1.5-2.0 folds(P<0.05), while mutants that maintain higher secretion levels did not affect GRP78 expression. The low secretion mutants also had increased binding to GRP78 and calreticulin, but not to calnexin.Therefore ER chaperones play a key role in the ER quality control of FVIII mutants. In conclusion, our results indicate that the effects of abnormal N-linked glycosylation on FVIII folding and secretionvary widely, from detrimental to beneficial. The impact of a particular glycan is likely determined by the location and the underlying amino acid change caused by the mutation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A New Classification of Mild/Moderate Hemophilia Α Based on a Multi-Dimensional FVIII(a) Functions Evaluated By a Plasma-Based Factor Xa Generation

In mild/moderate hemophilia A (MHA), the real hemostatic potential of their innate factor (F) VIII(a) may not be evaluated by FVIII activity (FVIII:C) obtained in a one-stage clotting assay. We often experience the discrepancies between clinical phenotype and FVIII:C in MHA patients. In order to elucidate the hemostatic potentials of the innate FVIII in MHA patients with a simple method, we here have established a plasma-based FXa generation assay (PB-XaG) to clarify the multi-dimensional functions of FVIII(a) in MHA determined by the interaction with the components of tenase complex consisting of FIXa, FX, and phospholipid (PL). XaG was evaluated in the patients’ plasmas and in the control plasma dilutions consisting of FVIII-deficient plasma and serial dilutions of recombinant FVIII (1-100 IU/dL) by a chromogenic substrate S-2765 together with human plasma-derived (pd)FIXa (0-20 nM), pdFX (0-200 nM), and PL vesicles (0-114 µM) initiated by thrombin (0.01 nM). The interaction between FVIIIa and each component of tenase complex was evaluated by Michaelis-Menten’s kinetic parameters, Vmax and apparent Km (Kmapp). We investigated the PB-XaG in the plasmas obtained from 17 MHA patients, carrying F8 mutations and FVIII:C as shown in the table. The PB-XaG showed that the group A, consisting of the case 1, 2, 3 and 4 with mutations at the residue(s) essential to formation of the inter-domain surface in the FVIII molecule, possessed ~1.8-7.1-fold increases in Kmapp for FIXa (10±0.9, 49±17, 31±6 and 9±3 nM, respectively) compared to FVIII:C-matched control (6.5±1.3 nM), whilst Kmapp for FX and PL in this group were of little difference from that in control, indicative of the reduced affinity for FIXa. The group B, including the case 5, 6, 7, 8 with mutations at the A2 or A3 domain remote from the PL-binding region on the C2 domain (residue 2303-2332) and the case 9 with the mutation at the PL-binding region, demonstrated ~1.7-4.8-fold increases in Kmapp for PL (4.3±2.4, 5.0±1.0, 6.5±1.3, 12±4.0 and 3.0±0.6 µM, respectively) compared to control (2.0±0.4 µM), whilst Kmapp for FIXa and FX in this group were similar to that in control, indicative of the reduced affinity to PL. Noteworthy, the apparent catalytic efficiencies defined as Vmax/Kmapp were 0.033 min-1 for the case 6 and 0.019 min-1 for the case 8 with mutations at the A3 domain, 0.3 or 0.1-fold lower than that for the case 9 (0.19 min-1) with mutation at the PL-binding region. The PB-XaG showed that the group C consisting of the case 10, 11, 12, 13, 14 and 15 demonstrated ~1.6-7.5-fold increases in Kmapp for FX (22±9, 23±9, 24±9, 10±4, 4.6±1.6 and 5.8±3.0 nM, respectively) compared to control (4.5±0.8 nM), whilst Kmapp for FIXa and PL in this group were similar to that in control, indicative of the reduced affinity to FX. Interestingly, the case 16 classified in the group C’ showed enhancing affinity with FX represented by a half decrease of Kmapp for FX compared to FVIII:C-matched control (2.0±0.9 and 4.8±1.5 nM, respectively). By contrast, the case 17 with mutation at the thrombin cleavage site categorized in the group D demonstrated any little XaG initiated by thrombin for all axes of FIXa, FX and PL. Taken together, the function of FVIII(a) in MHA could be evaluated multi-dimensionally and clearly classified into the some types of interaction with FIXa, FX, PL and thrombin by the PB-XaG. Our plasma-based XaG assay would be a useful tool for functional analyses of FVIII(a) to clarify the mechanism(s) of hemostatic heterogeneity in mild/moderate HA. [Display omitted] DisclosuresNogami:Chugai Pharmaceutical Co., Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding; F. Hoffmann-La Roche Ltd.: Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sysmex Corporation: Patents & Royalties, Research Funding. Shima:F. Hoffmann-La Roche Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees; Sysmex Corporation: Patents & Royalties, Research Funding; Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Blood Group O Protects against Inhibitor Development in Severe Hemophilia A Patients.

Increasing evidence supports the link between ABO(H) blood group determinants and hemostasis. In particular, the ABO-related different glycosylation patterns of von Willebrand factor strongly influence its clearance and functional levels, and this may contribute to the inter-individual variations in the half-life of infused Factor VIII (FVIII) in hemophilia A (HA) patients. We investigated the role of ABO blood groups in regulating FVIII immunogenicity by evaluating their distribution in patients with severe (FVIII < 1 IU/dL) HA according to inhibitor development and other known relevant factors. In a cohort of Italian severe HA patients (n = 209), the ABO blood group distribution was similar to that in the healthy general population. However, the distribution of inhibitors, developed in 56 patients overall (26.8%), was significantly different in the four ABO phenotypes (O, 18.2%; A, 31.9%; B, 39.1%, AB, 25%; p = 0.033); this difference seemed more pronounced when only high-titer inhibitors (overall, 21.1%) were considered (O, 11.4%; A, 27.7%; B, 34.8%; p = 0.011). Relative risks in O versus non-O blood group were 0.55 (95% CI: 0.33-0.92) and 0.40 (95% CI: 0.21-0.77) for any and high-titer inhibitors, respectively. In a multivariate logistic regression, O blood group was shown to lower (approximately twofold) inhibitor risk, similarly with plasma-derived FVIII, whereas high-risk F8 mutations were associated with increased risk. However, the estimated effect of O blood type on inhibitor development was free from any significant correlation to other covariates, including presence of high-risk F8 mutations and type of replacement FVIII used. In this retrospective cohort of severe hemophiliacs, blood group O appears to protect against inhibitor development, with independent effects from other covariates. Larger prospective studies are needed to confirm this finding and to delve deeper into its pathophysiologic mechanisms.

Hereditary angioedema with F12 mutation: Clinical features and enzyme polymorphisms in 9 Southwestern Spanish families

BackgroundInformation on F12 mutation hereditary angioedema (HAE) is still limited, but Spain is now recognized as having one of the highest concentrations of cases in Western Europe. ObjectiveTo describe unique features of HAE in Spanish carriers of the F12 mutation and investigate a potential role for angiotensin-converting enzyme (ACE) and aminopeptidase-P polymorphisms in disease expression. MethodsThis was a prospective observational cohort study of 35 individuals (80% females) from 9 unrelated families carrying the p.Thr309Lys mutation. We analyzed detailed medical records and complement activity (C4, C1q, C1 inhibitor) and screened for mutations in exon 9 of the F12 gene and 2 polymorphisms: XPNPEP2 c-2399A and the ACE insertion/deletion polymorphism. ResultsThe p.Thr309Lys mutation was found in all individuals. Three of the 9 index patients had a clinically negative family history, and 72% of males and 29% of females were asymptomatic. Sixteen females (44% estrogen dependent, 56% estrogen sensitive) were clearly symptomatic. The most common locations of attacks were the abdomen (63%), face (25%), and peripheral structures (6%). Triggers other than hyperestrogenic states included stress and minor trauma or pressure. Short-term treatment with C1-inhibitor concentrate and icatibant and long-term prophylaxis with tranexamic acid were useful. The combination of the I allele and A allele was detected in 17% of patients. ConclusionThe polymorphisms analyzed were not a major determinant of disease expression in our population. We recommend searching for F12 mutations in women with edema attacks without associated wheals and with normal C1-inhibitor levels, particularly when they develop symptoms during hyperestrogenic states or are of Western European or African origin.

Development and application of molecular diagnostic method of hemophilia A

Objective To develop and validate a method for detecting factor 8 gene (F8) mutations in hemophilia A patients by Ion Torrent semiconductor sequencing. Methods Intron 22 and intron 1 inversions of F8 gene were identified by long distance PCR (LD-PCR), other mutations in the F8 gene were identified by Ion Torrent sequencing.Candidate variants were validated by Sanger sequencing. Sanger sequencing was applied to screen HA carriers from 11 female family members in the 8 pedigrees. One pregnant woman was offered prenatal diagnosis via analyzing the fetal DNA obtained through amniocentesis. Results Four missense mutations (c.1331A>C, 1648C>T, c. 6506G>A, c. 6544C>T), two frameshift mutations (c.2393_2394insT, c. 6320delG), one splicing mutation (IVS5+ 5G>A), one nonsense mutation (c.43C>T) and one Inv22 mutation were identified in all nine probands respectively. Among 11 female family members, 10 females were identified to be HA carriers, and one didn't carry the maternal pathogenic mutation. Prenatal diagnosis result showed that the fetus inherited the wild-type maternal allele and was predicted to be unaffected by HA. Conclusion The targeted Ion Torrent sequencing is a reliable and efficient method to detect F8 mutations in patients with Hemophilia A disease.(Chin J Lab Med, 2016, 39: 496-500) Key words: Hemophilia A; Factor Ⅷ; Mutation

Identification of mutations in the F8 and F9 gene in families with haemophilia using targeted high-throughput sequencing.

At present, many methods are available for the genetic diagnosis of haemophilia, including indirect linkage analysis, direct sequencing. However, these methods are time-consuming, labourious, and limited in their application. Therefore, the development of new, more effective techniques is necessary. To detect the F8 and F9 gene mutations in patients with haemophilia and their female relatives in 29 haemophilia A (HA) and 11 haemophilia B (HB) families. C and FIX:C were analyzed using one-stage method, and factor VIII and factor IX inhibitors were tested using the Bethesda method. Intron 22 and one inversions were identified using long-distance polymerase chain reaction (PCR) and standard PCR. Non-inversion mutations of the F8 and F9 gene were identified by targeted high-throughput sequencing. All mutations were verified by Sanger sequencing. Intron 22 inversion was detected in eight HA families and intron one inversion was detected in one HA family. Apart from the inversion mutations, 20 mutations were identified in HA families, including 17 previously reported and three novel mutations: c.5724G>A (p.Trp1908*), c.6116-1_6120delGAGTGTinsTCC (p.Lys2039Ilefs*13), and c.5220-2A>C. We found a complex rearrangement in HA: intron one inversion concomitant with exon one deletion. In HB, eight recurrent mutations were detected, including six missense mutations and two nonsense mutations. Targeted high-throughput sequencing is an effective technique to detect the F8 and F9 gene mutations, especially for the discovery of novel mutations. The method is highly accurate, time-saving and shows great advantage in uncovering large deletion mutations and also in distinguishing the wild-type genotype and heterozygous large deletions.

The thrombin generation assay distinguishes inhibitor from non-inhibitor patients with severe haemophilia A.

Patients with haemophilia A (HA) have impaired thrombin generation (TG) capacity and TG assay (TGA) values are linearly related to plasma factor VIII (FVIII) levels. This study carried out in patients with unmeasurable FVIII (<1 IU dL(-1) ) was aimed at unravelling any difference in TG capacity in patients with or without inhibitors. Blood samples were collected from patients in a non-bleeding state, after a 5-day wash-out period from last treatment. TGA was performed in 102 patients with severe HA (15% with high-responding inhibitors; 51% with null F8 mutations, that as expected were more prevalent in inhibitor than in non-inhibitor patients). TG capacity was significantly lower in inhibitor than non-inhibitor patients and in those with null mutations than in those with non-null mutations. When the TG capacity was evaluated only in patients with null mutations with and without inhibitors it was lower in the presence of inhibitors. This study shows a greater TG impairment in inhibitor patients irrespective of FVIII levels, inhibitor titre and F8 mutation type, suggesting a role for the TGA in unravelling functional interferences of anti-FVIII inhibitors on coagulation system activation.

Hereditary angioedema with F12 mutation: first report of three cases associated with immune disorders.

Please submit letters for the editor's consideration within three weeks of receipt of Clinical Medicine. Letters should ideally be limited to 350 words, and sent by email to: clinicalmedicine{at}rcplondon.ac.uk Hereditary angioedema (HAE) is a rare genetic disorder that is characterised by

F8 gene mutation profile in Indian hemophilia A patients: Identification of 23 novel mutations and factor VIII inhibitor risk association

‘FVIII inhibitors’, especially in severe hemophilia A (HA) patients, is a serious adverse effect that complicates their clinical management. Many genetic and non-genetic risk factors have been proposed for FVIII inhibitor development, diverse in different population groups. This is the first study in Indian hemophiliacs that analyzes inhibitor risk in relation to the complete F8 mutation profile, in a case-control study that included 145 Indian severe HA patients, i.e. 69 inhibitor positive (with 18 inhibitor concordant/discordant family members), and 58 inhibitor negative patients, after informed consent.While 53.54% (68/127) index cases were positive for intron 22 or intron 1 inversions, 55 causative F8 mutations were detected in the 59 inversion negative patients, of which 23 were novel mutations (in 24 patients) and 32 were reported earlier (in 35 patients). A higher incidence of mutations, in the C1 and C2 domains in inhibitor positive patients, and in the A1 domain in inhibitor negative patients was observed, though not significantly different. The study suggests that large F8 rearrangements (significantly higher in the inhibitor positive patients) pose the highest risk, while missense mutations (significantly higher in the inhibitor negative patients) pose the lowest risk of inhibitor development in Indian hemophilia A patients.

Combined coagulation factor VIII and factor IX deficiency (CDF8F9) in a patient from Lithuania

Evaluation of clinical data, determination of FVIII and FIX levels and genetic analysis of F8 and F9 genes by direct sequencing. We report on a patient having severe haemophilia B (FIX:C <1 IU dl-1) and mild haemophilia A (FVIII:C 18 IU dl-1 ). FIX deficiency was known since childhood, whereas mild haemophilia A was confirmed at the age of 42 due to unexpected bleeding complications after dental extraction despite adequate substitution with plasma derived FIX concentrate. F9 gene analysis showed a point mutation in exon 2 (c.223C>T, p.R75X), whereas F8 gene analysis revealed a point mutation in exon 4 (c.545A>C, p.D182A). The mother of the patient was heterozygous for F8 mutation, but not for F9 mutation suggesting a de novo F9 mutation. Accidentally, further family from Germany with mild Haemophilia A was identified to have the same F8 mutation. F8 Haplotype analysis revealed that the p.D182A mutation most likely represents a founder mutation with common ancestors of the German and the Lithuanian family. Our results confirm the rare event of Haemophilia A and haemophilia B in the same patient originating from two distinct genetic defects in F8 and F9 genes.

High-Throughput Genotyping of Haemophilia A and B Using Next-Generation Sequencing Technology in Lille University Hospital

IntroductionThe identification of molecular defects in haemophilia is essential for the optimization of patient treatment and the formal characterization of female carriers. The Sanger method is the gold standard for sequencing F8 and F9 genes but is time-consuming and expensive. We aimed to develop a high-throughput method to genotype haemophilia A (HA) and B (HB) patients using the Next-Generation Sequencing (NGS) technology for an exhaustive and less expensive analysis of F8 and F9 genes.Material & MethodsWe developed a small panel containing F8 and F9 for exons and introns/exons junctions sequencing. We used two different methods for library preparation (AmpliSeq™, Life Technologies™ and HaloPlex™, Agilent™), performed in the same PCR emulsion system (Ion One Touch 2™, Life Technologies™) and sequenced with a Ion 316™ chip in a PGM™ Ion Torrent sequencer, or a Ion PI™ chip in a Proton™ sequencer (Life Technologies™) respectively. The promoter and 3' regions of F8 and F9 were always studied by Sanger. NGS analysis was first performed in 62 samples (HA: n= 42; HB: n=13; carriers: n=7) previously characterized for F8 or F9 mutations by Sanger method or Multiplex Ligation-Probe Amplification (MLPA). All types of mutations were studied (nonsense, missense, splice, small insertion/deletion and exons deletion/duplication) and were distributed in all exons of F8 and F9. NGS analysis was further performed in 42 haemophilia patients (HA: n=31, HB: n=11; of which 36 presented a mild phenotype), with unknown mutation status. All patients were included by the local Comprehensive Care Haemophilia Center of Lille University Hospital after written informed consent. Data were analyzed with SeqNext™ software (JCI Medical System™). A Normalized Reads Depth (NRD) ratio was used to detect exons deletion/duplication.ResultsAll exons were well covered by AmpliSeq™ (average number of reads, ANR = 300) and overall by HaloPlex™ (ANR = 15000, except small parts of exons 14 and 19 in F8). The average quality value for mutation detection was 60 (risk of false result <0.0001%).In previously-genotyped patients, 92% (57/62) of F8 and F9 mutations were detected by AmpliSeq™ and 85% (53/62) by HaloPlex™. The detection rate of small insertion/deletion in homopolymers of exon 14 in F8 was only 20% (1/5) with both methods and 71% (5/7) in other exons of F8. Four deletions and one duplication of exons accounting for a severe haemophilia phenotype were identified (3 HA, 1 HB and 1 HA-carrier). In 5 uncharacterized patients by Sanger method, neither AmpliSeq™ nor HaloPlex™ were able to find a mutation suggesting that the molecular defect is located in introns of F8 or F9.In never-genotyped patients, a mutation was detected in 90% (38/42) of cases (including 34 missense, 2 nonsense and 2 splice mutations in both F8 and F9). A duplication of exons 10 to 14 was also detected in a severe HA patient and was confirmed by MLPA. In NGS negative patients, no mutation was found in promoter or 3' regions. Of the ten candidate mutations identified in our cohort, seven were predicted to be deleterious by in silico analysis and/or co-segregation studies. No mutation was found in 10% (4/42) of never-genotyped patients with mild haemophilia A, in consistence with the available data for the mild phenotype. The technical development and laboratory protocol was easier and less expensive ($530 vs $602 including reagents and technical/medical staff) with AmpliSeq™ than HaloPlex™.ConclusionWe confirmed that NGS is able to detect the main types of mutations in F8 and F9 genes, albeit with a lower mutation detection rate with HaloPlex™ compared to AmpliSeq™. These detections were associated with an important depth of reads and high Quality Values, except for exons 14 and 19 in F8 with HaloPlex™. AmpliSeq™ seems also an interesting screening method for the detection of exons deletion/duplication using the NRD ratio. However, both strategies fail to detect small insertion/deletion located in homopolymers of exon 14 in F8, whom identification will still rely on Sanger sequencing. AmpliSeq™ protocol performed in the PGM™ sequencer appears as a new interesting tool in genotyping of HA and HB patients of the Lille University Hospital. DisclosuresZawadzki:Pfizer Pharmaceutical Company: Research Funding.

Modification of Predicted Inhibitor Risk in Non-Severe Hemophilia-a By in silico Analysis of Human Proteome Homology with Wild-Type, FVIII-Derived Peptides

Neutralising antibodies (inhibitors) are increasingly recognized to be a life-time risk in non-severe hemophilia A patients exposed to factor VIII concentrates. It is currently not possible to reliably identify the variable inhibitor risk between individuals. Risk appears to differ between F8 genotypes but also for individuals living with the same F8 genotype. As a T cell dependent process, the wild-type, factor concentrate-derived peptide sequences spanning the F8 mutation position are presented by class II MHC and responsible for driving the T helper response and subsequent B cell response. We hypothesize that the primary sequences of the 20,469 proteins in the human proteome will, coincidentally, contain short primary sequences homologous to key immunogenic peptides derived from the therapeutic factor VIII. We present in silico data and correlation with published registry inhibitor data (Fisher's exact test) to demonstrate the potential impact of such "proteome protection" and future potential to more reliably stratify individuals between low/negligible risk and more significant risk of inhibitor formation.We utilize a well-validated, computational tool, NetMHC-II, to enable large scale, computational comparison of predicted antigen presentation between endogenous, mutated FVIII derived peptides and factor-concentrate derived, wild-type FVIII peptides spanning all 520 F8 missense mutations listed on www.hadb.org. NetMHC-II analyses peptide presentation by 14 class II MHC HLA-DR alleles, resulting in analysis of 7,280 (520 x 14) permutations of F8 -MHC-II. We identify 56% (n=4,077) of these permutations to be at low/negligible risk of inhibitor formation, at a binding threshold of 500nM, defined as absence of a novel peptide-MHC surface capable of driving a helper T cell response (p=0.005). When cross referenced with potential homologous sequences buried anywhere in the human proteome (http://www.ebi.ac.uk/reference_proteomes), a further 1,237 F8 -MHC-II combinations are afforded "proteome protection" due to direct sequence homology between FVIII-derived peptide and peptide(s) derived from other proteins. This increases the total number of F8 -MHC-II combinations predicted to be unable to drive a T cell response to 73% (n=5,314). The residual 1,966 (27%) F8 -MHC-II combinations are predicted to retain the ability to present novel-interface FVIII-derived peptides to T cells with an IC50<500nM (higher affinity).Previous work exploring in silico prediction of FVIII-derived peptide presentation may have overestimated the number of F8 -MHC II combinations deemed to be at risk of contributing to inhibitor formation. Our data suggests an additional mechanism "protecting" a larger proportion of those living with non-severe HA from inhibitor formation. The contribution of "proteome protection" further reduces the "at risk" F8 -MHC II permutations to be more representative of clinically observed inhibitor rates. We identify a potential novel tolerance mechanism and provide key data for future in vitro validation strategies. DisclosuresNo relevant conflicts of interest to declare.

Complexity and diversity of F8 genetic variations in the 1000 genomes

Hemophilia A (HA) is an X-linked bleeding disorder caused by deleterious mutations in the coagulation factor VIII gene (F8). To date, F8 mutations have been documented predominantly in European subjects and in American subjects of European descent. Information on F8 variants in individuals of more diverse ethnic backgrounds is limited. To discover novel and rare F8 variants, and to characterize F8 variants in diverse population backgrounds. We analyzed 2535 subjects, including 26 different ethnicities, whose data were available from the 1000 Genomes Project (1000G) phase 3 dataset, for F8 variants and their potential functional impact. We identified 3030 single nucleotide variants, 31 short deletions and insertions (Indels) and a large, 497kb, deletion. Among all variants, 86.4% were rare variants and 55.6% were novel. Eighteen variants previously associated with HA were found in our study. Most of these 'HA variants' were ethnic-specific with low allele frequency; however, one variant (p.M2257V) was present in 27% of African subjects. The p.E132D, p.T281A, p.A303V and p.D422H 'HA variants' were identified only in males. Twelve novel missense variants were predicted to be deleterious. The large deletion was discovered in eight female subjects without affecting F8 transcription and the transcription of genes on the X chromosome. Characterizing F8 in the 1000G project highlighted the complexity of F8 variants and the importance of interrogating genetic variants on multiple ethnic backgrounds for associations with bleeding and thrombosis. The haplotype analysis and the orientation of duplicons that flank the large deletion suggested that the deletion was recurrent and originated by homologous recombination.

Type and intensity of FVIII exposure on inhibitor development in PUPs with haemophilia A. A patient-level meta-analysis.

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.

The role of previously untreated patient studies in understanding the development of FVIII inhibitors.

Development of inhibitors against factor VIII (FVIII), the major complication of haemophilia A treatment today, is influenced by multiple factors. Genetic (F8 mutation, family history, ethnicity, polymorphisms in immune modulating genes) and non-genetic (intensive exposure to FVIII, presence of pro-inflammatory signals as might occur with large bleeds, infections, surgery, or other immune stimulants [e.g. vaccines]) risk factors as well as their complex inter-relationships contribute to the inhibitor risk profile of haemophilia patients, particularly in the previously untreated patient (PUP) population. Studies in PUPs have been fundamental to furthering the understanding of FVIII inhibitor development, as well as discovering previously unappreciated risk factors. The multi-factorial nature of inhibitor development makes it difficult to ascertain the contribution of FVIII products in inhibitor development through individual PUP studies. Sufficiently powered studies of large cohorts may overcome these limitations but interpretations should be conducted cautiously. Proper design and implementation of PUP safety studies will become even more important with the introduction of new molecules, such as extended half-life or human cell-line derived FVIII that propose reduced immunogenicity. Despite these difficulties, carefully performed clinical studies in PUPs may provide important insights into the natural history of the immune response to FVIII and may suggest targets for intervention to reduce immunogenicity.