Von Willebrand Factor antigen and age explain variation in baseline FVIII:C among nonsevere hemophilia A patients with the same F8 genotype (Arg593Cys and Asn618Ser)
J.I. LoomansAlice S. van VelzenCorien L. EckhardtMarjolein PetersJan AstermarkPaul BronsGiancarlo CastamanMarjon H. CnossenNatasja DorsC. Escuriola‐EttingshausenKarly HamulyákDaniel P. HartC. R. M. HaySaturnino HayaWaander L. van HeerdeCédric HermansMargaretha HolmströmVictor J. Imenez-YusteRussell KeenanRobert KlamrothChristoph KönigsMarieke J.H.A. KruipBritta A. P. Laros‐van GorkomFrank W.G. LeebeekRi LiesnerAnne MäkipernaaChristoph MaleEvelien P. Mauser‐BunschotenMaria Gabriella MazzucconiSimon McRaeKarina MeijerMichael A. MitchellMassimo MorfiniMarten R. NijzielJohannes OldenburgKathelijne PeerlinckPia PetriniHelen PlatokoukiSavita RangarajanSylvia Reitter-PfoertnerElena SantagostinoPiercarla SchincoFrans J. SmiersBritta SiegmundAnnarita TagliaferriT. T. YeePieter W. KamphuisenJohanna G. van der BomKarin Fijnvandraat
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Introduction and Objectives: Non-severe hemophilia A (baseline FVIII:C, 2-40 IU/dL) is caused by a mutation in the F8 gene. There is limited knowledge on the factors determining the variation in baseline FVIII:C. The aim is to identify the determinants of baseline FVIII:C in non-severe hemophilia A patients. Materials and Methods: We analyzed clinical data for non-severe hemophilia A patients, treated between 1980-2013, in European Haemophilia Treatment Centers (HTCs) participating in the INSIGHT/RISE project. We performed analyses on mutations that were present in ≥10 patients. Age (at FVIII:C measurement), F8 gene mutation, VWF:Ag, VWF:Act and HTC were analyzed as potential determinants by multivariate regression analyses. Results: We identified nine missense mutations present in ≥10 patients in 321 individuals, median age 23 years (IQR 7-47). From these individuals we had data on 667 FVIII:C measurements in 5 HTCs. Median baseline FVIII:C, VWF:Ag and VWF: Act were 17 IU/dL (IQR 11-22), 98 IU/dL (IQR 78-128) and 91 IU/dL (70-115) respectively. Baseline FVIII:C, VWF:Ag and VWF:Act all increased with age, both in the total population and within the two largest mutation groups (Asn618Ser, 113 patients; Arg593Cys, 107 patients). VWF:Ag, age and F8 mutation were significant predictors of baseline FVIII:C (p <0.0001-0.024). In mutations that were present in ≥10 patients the determinants age, F8 mutation, VWF:Ag and HTC together explained 61% of the variation in baseline FVIII:C. Within the specific mutation group Asn618Ser only 21% of the variance in baseline FVIII:C was explained by the combined potential determinants, with VWF:Ag and HTC as significant predictors (p = 0.008 and 0.013 respectively). Among individuals with the Arg593Cys F8 genotype the determinants age, VWF:Ag and HTC were significant predictors (p <0.0001 for age and VWF:Ag and p = 0.04 for HTC), together explaining 34% of the variance in baseline FVIII:C. Conclusion: In non-severe hemophilia A patients carrying the same F8 mutation the determinants age, VWF:Ag and HTC contribute to baseline FVIII:C to variable extends. With the studied determinants we can only explain 61% of the variance in baseline FVIII:C. This suggests that yet unknown factors influence FVIII:C in nonsevere hemophilia A.Cite
von Willebrand Disease
Compound heterozygosity
Heterozygote advantage
Ristocetin
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Summary. Patients with mild haemophilia A may have a discrepancy in the factor VIII (FVIII) level when measured with a one‐stage assay (FVIII:C1) compared with a two‐stage assay (FVIII:C2). This discrepancy usually results in the one‐stage level being higher than the two‐stage level. A F8 mutation resulting in a Tyr346→Cys substitution within the a1 interdomain region has been described which results in the converse assay discrepancy. We report four individuals (three families) who have this mutation. Mean FVIII:C1 level was 25 IU dL −1 compared with a mean FVIII:C2 level of 63 IU dL −1 . These individuals had presented opportunistically and did not have a clinically significant bleeding disorder. The bleeding phenotype correlated with the two‐stage assay result rather than the one‐stage result. FVIII replacement therapy does not appear to be required for these individuals.
Haemophilia B
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von Willebrand Disease
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Factor VIII (FVIII) levels show a considerable variability in female carriers of haemophilia A. Presently, the reasons for this are poorly understood. The aim of the study was to elucidate the influence of genetic and non-genetic parameters on FVIII plasma levels in carriers (n = 42). Results were compared with age-matched healthy women without carriership of haemophilia A (n = 42). Each carrier was tested for the family-specific mutation, ABO blood group, FVIII level, von Willebrand factor (VWF) antigen and activity and C-reactive protein (CRP). FVIII levels were lower in carriers compared to non-carriers [74% (51-103) vs. 142% (109-169), P < 0.001]. No statistically significant differences were observed between the two groups with respect to VWF activity, prothrombin-time, hs-CRP, fibrinogen, body mass index (BMI), age and smoking status as well as the distribution of ABO blood groups. In non-carriers, FVIII was statistically significantly correlated with BMI, activated partial thromboplastin time (APTT), VWF antigen, hs-CRP and fibrinogen. In carriers, significant correlations between FVIII and APTT, VWF antigen and activity were found, whereas BMI, hs-CRP or fibrinogen did not correlate with FVIII. In non-carriers, the association of FVIII with ABO blood groups was statistically significant (P = 0.006), but not in carriers of haemophilia A (P = 0.234). The type of FVIII gene mutation did not influence FVIII levels. Carrier status is the major determinant of a carrier;s FVIII plasma level. Factors known to influence FVIII levels in the general population do not significantly affect FVIII activity in carriers, neither does the type of mutation influence FVIII levels.
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In order to detect coagulation factor VIII (FVIII) inhibitor in patients with severe hemophilia A (HA) and preliminarily study the genetic mutation in patients with inhibitor positive. Totally 58 patients with HA (FVIII: C < 1%) were enrolled. FVIII: C activity was measured by one-stage coagulation assay. FVIII inhibitor was screened by using APTT method and FVIII inhibitor in screened positive patients with HA was quantitatively analyzed by using Bethesda method. Using genomic DNA as template, 12, 14, 16 exons of FVIII in screened positive patients were amplified, and the mutations of amplified products were detected by direct sequencing. The results indicated that the FVIII inhibitor could be detected in 4 patients (6.9%) from 58 HA patients, no gene mutations in 12, 14, 16 exons of FVIII were found. It is concluded that the positive rate of FVIII inhibitor in HA patients is lower than that reported in literature. The causes of inhibitor production needs to further investigate.
genomic DNA
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To investigate the molecular mechanism of a Chinese hemophilia A patient in whom there was a discrepancy between the clinical bleeding symptoms and laboratory assay of FVIII activity (FVIII: C).FVIII: C was detected by chromogenic and one-stage methods, and FVIII: Ag by ELISA. The APTT corrected test was used to screen the FVIII inhibitor and PCR amplification to analyze all the exons and flanking sequences of F8 gene of the proband, PCR products were purified and sequenced directly. The corresponding gene sites of family members were detected according to the gene mutation sites. Two B domain deleted human FVIII mutant expression plasmids His99Arg and His99Ala (pRC/RS V - BDhFVIIIcDNA) were constructed and transfected into HEK293T transiently. FVIII: Ag and FVIII: C of the expression products were assayed.The proband APTT was prolonged, FVIII: Ag was 120% but FVIII: C <1% and no FVIII inhibitor in plasma. The results of anticoagulation and fibrinolytic functions were normal. The cross reacting material positive (CRM+) hemophilia A was diagnosed. Gene analysis revealed a A28828G substitution in exon 3 resulted in a H (His) to R (Arg) missense mutation and the same heterozygous was identified in his mother. In vitro expression of FVIII: Ag and FVIII: C of His99Arg were 180.0% and 5.8% , respectively, while FVIII: Ag and FVIII: C of His99Ala were 45.0% and 20.0% of that of wild type, respectively. His99Arg and His99Ala were diagnosed as CRM+ and CRM- mutations, respectively.Both the two F VIII mutations could express FVIII protein. However, CRM His99Arg mutant protein has little FVIII procoagulant activity and His99Ala has reduced FVIII function by routine methods.
Proband
Mutation Testing
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von Willebrand Disease
Null allele
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Abstract Background Hemophilia A (HA) is an X-linked recessive bleeding disorder characterized by qualitative and quantitative deficiency of factor VIII (FVIII). The development of inhibitor antibodies against FVIII is the most challenging complication of treatment. Mutations in the FVIII gene is one of the genetic factors that leads to development of FVIII inhibitors especially intron 22 inversion (Inv22). Objectives This study was carried out to assess the frequency of Inv22 of FVIII gene in Egyptian patients with hemophilia A and its role as a risk factor for developing inhibitors. Patients and methods Seventy-two patients with severe HA and 48 patients with moderate HA were enrolled in the current study. All patients were treated on demand with either plasma-derived factor VIII or recombinant factor VIII concentrates. Genotyping of FVIII Inv22 was performed by LD-PCR while the presence and magnitude of inhibitor activity in blood was determined by the Bethesda assay. Results Around 23% of all hemophilia cases had positive Inv22. Intron 22 inversion mutation was detected in 6 and 33% of patients with moderate and severe HA respectively. Twenty-one cases (18%) of all hemophilic patients developed inhibitors. Thirty-7% of patients with Inv22 had inhibitor in their blood, almost all, but one, had severe HA. The risk of an inhibitor development during replacement therapy was four folds higher among Inv22 positive cases as compared with mutation negative peers (OR 4.3, 95% CI 1.6–11.9, P = 0.003). Conclusions The prevalence of Inv22 of F VIII in Egyptian hemophiliacs is nearly like that of other population. This mutation was more frequently detected among severe hemophilic patients as compared with moderately affected peers. The presence of Inv22 mutation significantly predispose to FVIII inhibitor development.
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Introduction : Von Willebrand disease (VWD) type 2N is characterized by a defective binding of factor VIII (FVIII) to von Willebrand factor (VWF) resulting in diminished plasma FVIII levels and a clinical phenotype mimicking mild haemophilia A. Several mutations in the FVIII binding site of VWF have been reported. Aim: This study aims to examine the effect of genotype on clinical phenotype in a cohort of VWD 2N patients. Methods: Patients with at least one genetically confirmed 2N mutation were selected retrospectively from a cohort of patients with suspected VWD. Clinical and laboratory phenotypes including bleeding scores (BS) were obtained and analysed. Results : Forty‐two VWD 2N patients with a mean age of 44 years were included. Eleven patients were homozygous or compound heterozygous (genetically confirmed group) and 31 patients were heterozygously affected (carriers group). Statistically significant differences between genetically confirmed VWD 2N patients and carriers were found in FVIII activity, VWF antigen levels, VWF‐FVIII binding capacity, FVIII/VWF antigen ratio (all P <0.001), VWF‐ristocetin activity (p=0.001) and VWF collagen binding ( P = 0.002). Median BS was 6 in genetically confirmed VWD 2N patients compared with 3 in carriers ( P = 0.047). Haemarthrosis, muscle haematomas and postpartum haemorrhage were only reported in genetically confirmed 2N patients. Conclusion : Phenotypic analysis showed that all laboratory parameters are lower in genetically confirmed VWD 2N patients compared with heterozygous 2N carriers. The clinical phenotype in genetically confirmed VWD 2N patients is comparable to mild haemophilia A patients and more severe than heterozygous 2N carriers.
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Heterozygote advantage
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