Gene Therapy with Etranacogene Dezaparvovec for Hemophilia B.

BACKGROUND: Moderate-to-severe hemophilia B is treated with lifelong, continuous coagulation factor IX replacement to prevent bleeding. Gene therapy for hemophilia B aims to establish sustained factor IX activity, thereby protecting against bleeding without burdensome factor IX replacement. METHODS: In this open-label, phase 3 study, after a lead-in period (≥6 months) of factor IX prophylaxis, we administered one infusion of adeno-associated virus 5 (AAV5) vector expressing the Padua factor IX variant (etranacogene dezaparvovec; 2×1013 genome copies per kilogram of body weight) to 54 men with hemophilia B (factor IX activity ≤2% of the normal value) regardless of preexisting AAV5 neutralizing antibodies. The primary end point was the annualized bleeding rate, evaluated in a noninferiority analysis comparing the rate during months 7 through 18 after etranacogene dezaparvovec treatment with the rate during the lead-in period. Noninferiority of etranacogene dezaparvovec was defined as an upper limit of the two-sided 95% Wald confidence interval of the annualized bleeding rate ratio that was less than the noninferiority margin of 1.8. Superiority, additional efficacy measures, and safety were also assessed. RESULTS: The annualized bleeding rate decreased from 4.19 (95% confidence interval [CI], 3.22 to 5.45) during the lead-in period to 1.51 (95% CI, 0.81 to 2.82) during months 7 through 18 after treatment, for a rate ratio of 0.36 (95% Wald CI, 0.20 to 0.64; P<0.001), demonstrating noninferiority and superiority of etranacogene dezaparvovec as compared with factor IX prophylaxis. Factor IX activity had increased from baseline by a least-squares mean of 36.2 percentage points (95% CI, 31.4 to 41.0) at 6 months and 34.3 percentage points (95% CI, 29.5 to 39.1) at 18 months after treatment, and usage of factor IX concentrate decreased by a mean of 248,825 IU per year per participant in the post-treatment period (P<0.001 for all three comparisons). Benefits and safety were observed in participants with predose AAV5 neutralizing antibody titers of less than 700. No treatment-related serious adverse events occurred. CONCLUSIONS: Etranacogene dezaparvovec gene therapy was superior to prophylactic factor IX with respect to the annualized bleeding rate, and it had a favorable safety profile. (Funded by uniQure and CSL Behring; HOPE-B ClinicalTrials.gov number, NCT03569891.).

Emicizumab prophylaxis in haemophilia A with inhibitors: Three years follow-up from the UK Haemophilia Centre Doctors' Organisation (UKHCDO).

INTRODUCTION: The UK National Haemophilia Database (NHD) collects data from all UK persons with haemophilia A with inhibitors (PwHA-I). It is well-placed to investigate patient selection, clinical outcomes, drug safety and other issues not addressed in clinical trials of emicizumab. AIMS: To determine safety, bleeding outcomes and early effects on joint health of emicizumab prophylaxis in a large, unselected cohort using national registry and patient reported Haemtrack (HT) data between 01 January 2018 and 30 September 2021. METHODS: Prospectively collected bleeding outcomes were analysed in people with ≥6 months emicizumab HT data and compared with previous treatment if available. Change in paired Haemophilia Joint Health Scores (HJHS) were analysed in a subgroup. Adverse events (AEs) reports were collected and adjudicated centrally. RESULTS: This analysis includes 117 PwHA-I. Mean annualised bleeding rate (ABR) was .32 (95% CI, .18; .39) over a median 42 months treatment with emicizumab. Within-person comparison (n = 74) demonstrated an 89% reduction in ABR after switching to emicizumab and an increase in zero treated bleed rate from 45 to 88% (p < .01). In a subgroup of 37 people, total HJHS improved in 36%, remained stable in 46% and deteriorated in 18%, with a median (IQR) within-person change of -2.0 (-9, 1.5) (p = .04). Three arterial thrombotic events were reported, two possibly drug related. Other AEs were generally non-severe and usually limited to early treatment, included cutaneous reactions (3.6%), headaches (1.4%), nausea (2.8%) and arthralgia (1.4%). CONCLUSIONS: Emicizumab prophylaxis is associated with sustained low bleeding rates and was generally well-tolerated in people with haemophilia A and inhibitors.

Identification of a homozygous recessive variant in PTGS1 resulting in a congenital aspirin-like defect in platelet function.

We have identified a rare missense variant on chromosome 9, position 125145990 (GRCh37), in exon 8 in PTGS1 (the gene encoding cyclo-oxygenase 1, COX-1, the target of anti-thrombotic aspirin therapy). We report that in the homozygous state within a large consanguineous family this variant is associated with a bleeding phenotype and alterations in platelet reactivity and eicosanoid production. Western blotting and confocal imaging demonstrated that COX-1 was absent in the platelets of three family members homozygous for the PTGS1 variant but present in their leukocytes. Platelet reactivity, as assessed by aggregometry, lumi-aggregometry and flow cytometry, was impaired in homozygous family members, as were platelet adhesion and spreading. The productions of COX-derived eicosanoids by stimulated platelets were greatly reduced but there were no changes in the levels of urinary metabolites of COX-derived eicosanoids. The proband exhibited additional defects in platelet aggregation and spreading which may explain why her bleeding phenotype was slightly more severe than those of other homozygous affected relatives. This is the first demonstration in humans of the specific loss of platelet COX-1 activity and provides insight into its consequences for platelet function and eicosanoid metabolism. Notably despite the absence of thromboxane A2 (TXA2) formation by platelets, urinary TXA2 metabolites were in the normal range indicating these cannot be assumed as markers of in vivo platelet function. Results from this study are important benchmarks for the effects of aspirin upon platelet COX-1, platelet function and eicosanoid production as they define selective platelet COX-1 ablation within humans.

Delivery of AAV-based gene therapy through haemophilia centres-A need for re-evaluation of infrastructure and comprehensive care: A Joint publication of EAHAD and EHC.

INTRODUCTION: Adeno-associated virus (AAV)-based gene therapy for haemophilia presents a challenge to the existing structure of haemophilia centres and requires a rethink of current collaboration and information exchange with the aim of ensuring a system that is fit-for-purpose for advanced therapies to maximise benefits and minimise risks. In Europe, a certification process based on the number of patients and facilities is offered to the haemophilia centres by European Haemophilia Network (EUHANET). AIM AND METHODS: This joint European Association for Haemophilia and Allied Disorders (EAHAD) and European Haemophilia Consortium (EHC) publication describes criteria for centres participating in gene therapy care that require a reassessment of the infrastructure of comprehensive care and provides an outlook on how these criteria can be implemented in the future work of haemophilia centres. RESULTS: The core definition of a haemophilia treatment centre remains, but additional roles could be implemented. A modifiable 'hub-and-spoke' model addresses all aspects associated with gene therapy, including preparation and administration of the gene therapy product, determination of coagulation and immunological parameters, joint score and function, and liver health. This will also include the strategy on how to follow-up patients for a long-term safety and efficacy surveillance. CONCLUSION: We propose a modifiable, networked 'hub and spoke' model with a long term safety and efficacy surveillance system. This approach will be progressively developed with the goal of making haemophilia centres better qualified to deliver gene therapy and to make gene therapy accessible to all persons with haemophilia, irrespective of their country or centre of origin.

Factor VIII/IX inhibitor testing practices in the United Kingdom: Results of a UKHCDO and UKNEQAS national survey.

INTRODUCTION: Inhibitor formation is the greatest challenge facing persons with haemophilia treated with factor concentrates. The gold standard testing methodologies are the Nijmegen-Bethesda assay (NBA) for FVIII and Bethesda assay (BA) for FIX inhibitors, which are affected by pre-analytical and inter-laboratory variability. AIMS: To evaluate inhibitor testing methodology and assess correlation between self-reported and actual methodology. METHODS: Methodology was evaluated using a survey distributed alongside a UK National External Quality Assessment Service Blood Coagulation external quality assurance (EQA) exercise for FVIII and FIX inhibitor testing. RESULTS: Seventy four survey and EQA exercise responses were received (response rate 63.2%), with 50 paired survey/EQA results. 47.1% (33/70) reported using the NBA and 42.9% (30/70) the BA for FVIII inhibitor testing. Review of FVIII inhibitor assay methodology demonstrated discrepancy (self-reported to actual) in 64.3% (BA reporting) and 27.6% (NBA reporting). Pre-analytical heat treatment was used by 32.4%, most commonly 56°C for 30 minutes. Assay cut-offs of 0.1-1.0 BU/mL were reported. EQA samples (acquired FVIII and congenital FIX) demonstrated titres and coefficients of variation (CV) of 3.1 BU/mL (0.7-15.4 BU/mL; CV = 43%) and 18.0 BU/mL (0-117 BU/mL; CV = 33%), respectively. No significant assay or laboratory factors were found to explain this variance, which could have resulted in change in management for 6 patients (5 misclassified high-titre FVIII inhibitors and 1 false negative for a FIX inhibitor). CONCLUSIONS: Heterogeneity was seen at each stage of assay methodology. No assay-related factors were found to explain variation in inhibitor titres. Further standardization is required to improve inhibitor quantification to guide patient care.

Immune tolerance induction in severe haemophilia A: A UKHCDO inhibitor and paediatric working party consensus update.

INTRODUCTION: In good risk patients (historic inhibitor peak < 200BU), the International Immune Tolerance Study demonstrated equal efficacy to induce tolerance between high (200iu/kg/day) and low dose (50iu/kg ×3 times/week) immune tolerance induction (ITI) regimens. However, the trial stopped early on account of the excessive bleed rate in the low dose ITI arm. METHODS: United Kingdom Haemophilia Centre Doctors' Organization (UKHCDO) Paediatric and Inhibitor working parties considered available ITI data alongside the bi-phenotypic antibody emicizumab (Hemlibra®) efficacy and safety data to develop a consensus guideline for the future UK ITI guideline. RESULTS: This revision of UKHCDO ITI guidance incorporates the recommendation to use emicizumab as a prophylaxis haemostatic agent to reduce bleeding rates and to facilitate low dose and reduced frequency of FVIII CFC for ITI in the majority of children. CONCLUSION: This consensus protocol will facilitate future evaluation of ITI outcomes in the evolving landscape of haemophilia therapeutics and ITI strategies.

PERSEPT 3: A phase 3 clinical trial to evaluate the haemostatic efficacy of eptacog beta (recombinant human FVIIa) in perioperative care in subjects with haemophilia A or B with inhibitors.

INTRODUCTION: Surgical procedures in persons with haemophilia A or B with inhibitors (PwHABI) require the use of bypassing agents (BPA) and carry a high risk of complications. Historically, only two BPAs have been available; these are reported to have variable responses. AIM: To prospectively evaluate the efficacy and safety of a new bypassing agent, human recombinant factor VIIa (eptacog beta) in elective surgical procedures in PwHABI in a phase 3 clinical trial, PERSEPT 3. METHODS: Subjects were administered 200 µg/kg (major procedures) or 75 µg/kg eptacog beta (minor procedures) immediately prior to the initial surgical incision; subsequent 75 µg/kg doses were administered to achieve postoperative haemostasis and wound healing. Efficacy was assessed on a 4-point haemostatic scale during the intra- and postoperative periods. Anti-drug antibodies, thrombotic events and changes in clinical/laboratory parameters were monitored throughout the perioperative period. RESULTS: Twelve subjects underwent six major and six minor procedures. The primary efficacy endpoint success proportion was 100% (95% CI: 47.8%-100%) for minor procedures and 66.7% (95% CI: 22.3%-95.7%) for major procedures; 81.8% (95% CI: 48.2%-97.7%) of the procedures were considered successful using eptacog beta. There was one death due to bleeding from a nonsurgical site; this was assessed as unlikely related to eptacog beta. No thrombotic events or anti-eptacog beta antibodies were reported. CONCLUSION: Two eptacog beta dosing regimens in PwHABI undergoing major and minor surgical procedures were well-tolerated, and the majority of procedures were successful based on surgeon/investigator assessments. Eptacog beta offers clinicians a new potential therapeutic option for procedures in PwHABI.

Rare variants in GP1BB are responsible for autosomal dominant macrothrombocytopenia.

The von Willebrand receptor complex, which is composed of the glycoproteins Ibα, Ibß, GPV, and GPIX, plays an essential role in the earliest steps in hemostasis. During the last 4 decades, it has become apparent that loss of function of any 1 of 3 of the genes encoding these glycoproteins (namely, GP1BA, GP1BB, and GP9) leads to autosomal recessive macrothrombocytopenia complicated by bleeding. A small number of variants in GP1BA have been reported to cause a milder and dominant form of macrothrombocytopenia, but only 2 tentative reports exist of such a variant in GP1BB By analyzing data from a collection of more than 1000 genome-sequenced patients with a rare bleeding and/or platelet disorder, we have identified a significant association between rare monoallelic variants in GP1BB and macrothrombocytopenia. To strengthen our findings, we sought further cases in 2 additional collections in the United Kingdom and Japan. Across 18 families exhibiting phenotypes consistent with autosomal dominant inheritance of macrothrombocytopenia, we report on 27 affected cases carrying 1 of 9 rare variants in GP1BB.

Factor VIII cross-matches to the human proteome reduce the predicted inhibitor risk in missense mutation hemophilia A.

Single missense mutations in the F8 gene encoding the coagulation protein factor VIII give rise predominantly to non-severe hemophilia A. Despite only a single amino acid sequence difference between the replacement, therapeutic factor VIII and the patient's endogenous factor VIII, therapeutic factor VIII may still be perceived as foreign by the recipient's immune system and trigger an immune response (inhibitor). Inhibitor formation is a life-long risk for patients with non-severe hemophilia A treated with therapeutic factor VIII, but remains difficult to predict. The aim of this study was to understand whether fortuitous, primary sequence cross-matches between therapeutic factor VIII and proteins in the human proteome are the reason why certain F8 mutations are not associated with inhibitor formation. We predicted which therapeutic factor VIII differences are potentially perceived as foreign by helper T cells - a necessary precursor to inhibitor development - and then scanned potentially immunogenic peptides against more than 100,000 proteins in the proteome. As there are hundreds of disease-causing F8 missense mutations and the human leukocyte antigen gene complex governing peptide presentation to helper T cells is highly polymorphic, these calculations pose a huge combinatorial challenge that we addressed computationally. We found that cross-matches between therapeutic factor VIII and the human proteome are commonplace and have a profound impact on the predicted risk of inhibitor development. Our results emphasize the importance of knowing both the F8 missense mutation and the human leukocyte antigen alleles of a patient with missense mutation hemophilia A if his underlying risk of inhibitor development is to be estimated.

Treatment regimens and outcomes in severe and moderate haemophilia A in the UK: The THUNDER study.

INTRODUCTION: The THUNDER study provides an analysis of treatment patterns and outcomes in UK patients with severe or moderate haemophilia A (SHA/MHA) in 2015. METHODS: Patients with SHA or MHA registered with the UK National Haemophilia Database (NHD) were segregated by severity, inhibitor status and age. Haemophilia joint health score (HJHS) was derived from NHD records and treatment regimen and annualized bleed/joint-bleed rate (ABR/AJBR) from Haemtrack (HT) in HT-compliant patients. RESULTS: We report 1810 patients with SHA and 864 with MHA. Prophylaxis was used in 94.9% (n = 130/137) of HT-compliant children <12 years with SHA, falling to 74.1% (n = 123/166) aged ≥40 years. Median ABR increased with age (1.0, IQR 0.0-5.0, <12 years; 3.0 IQR, 1.0-8.0, ≥40 years). Inhibitors were present in 159 (8.8%) SHA and 34 (3.9%) MHA. Median ABR increased from 2.0 (<12 years) to 21.0 (≥40 years) in SHA inhibitor patients using prophylaxis. Prophylaxis was used by 68.8% of HT-compliant MHA patients (n = 106) (median FVIII baseline 0.01 IU/mL) associated with a median (IQR) ABR of 3.0 (1.0-7.0). Median HJHS (n = 453) increased with age in SHA and MHA. Median (IQR) HJHS was higher in SHA inhibitor (17.0, 0.0-64.5) than non- or past inhibitor patients (7.0, 0.0-23.0). CONCLUSIONS: Increasing ABR with age persists despite current prophylaxis regimens. SHA and MHA had similar ABR/AJBR and HJHS, leading to a suspicion that a subgroup of MHA may be relatively undertreated. More intensive prophylaxis may improve outcomes, but this requires further study.

Recombinant factor VIII products and inhibitor development in previously untreated patients with severe haemophilia A: Combined analysis of three studies.

INTRODUCTION: Standard treatment of congenital haemophilia A is based on replacement therapy with coagulation factor VIII (FVIII) products. A major complication of FVIII therapy is the occurrence of IgG alloantibodies (inhibitors) that neutralize FVIII activity. AIM: The aim of the analysis was estimating the risk of high-titre inhibitor associated with the second-generation full-length product compared to third-generation full-length product and other recombinant FVIII (rFVIII). METHODS: We conducted a combined analysis of individual patient data from three large studies in previously untreated patients (PUPs) with severe haemophilia A. RESULTS: A total of 1109 PUPs were treated from 1993 to 2013 including 787 PUPs treated from 2004 onwards (primary analysis cohort). A total of 322 patients (29.0%) developed an inhibitor, of which 192 (17.3%) a high-titre inhibitor. In the primary analysis set, 29.9% of patients developed an inhibitor and 17.2% a high-titre inhibitor. The combined analysis indicated a lower risk of high-titre inhibitor development for the third-generation rFVIII product compared to the second-generation rFVIII product (primary analysis: adjusted hazard ratio (HR) = 0.72, 95% CI: 0.49 to 1.06). Adjusted HR for all inhibitor development was significantly lower for the third-generation product compared to the second-generation product. CONCLUSION: The trend of an increased risk of inhibitor development in PUPs for one recombinant product illustrates that extrapolation from one recombinant factor VIII product to other products might not be justified.

A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders.

Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.

Treatment burden, haemostatic strategies and real world inhibitor screening practice in non-severe haemophilia A.

Inhibitor formation in non-severe haemophilia A is a life-long risk and associated with morbidity and mortality. There is a paucity of data to understand real-world inhibitor screening practice. We evaluated the treatment burden, haemostatic strategies, F8 genotyping and inhibitor screening practices in non-severe haemophilia A in seven London haemophilia centres. In the 2-year study period, 44% (377/853) patients received at least one haemostatic treatment. Seventy-nine percent of those treated (296/377) received factor VIII (FVIII) concentrate. F8 genotype was known in 88% (331/377) of individuals. Eighteen per cent (58/331) had 'high-risk' F8 genotypes. In patients with 'standard-risk' F8 genotypes treated on-demand with FVIII concentrate, 51·3% episodes (243/474) were screened within 1 year. However, poor screening compliance was observed after 'high-risk' treatment episodes. In patients with 'standard-risk' F8 genotypes, 12·3% (28/227) of treatment episodes were screened in the subsequent 6 weeks after surgery or a bleed requiring ≥5 exposure days. Similarly, in the context of 'high-risk' F8 genotypes after any FVIII exposure, only 13·6% (12/88) of episodes were screened within 6 weeks. Further study is required to assess optimal practice of inhibitor screening in non-severe haemophilia A to inform subsequent clinical decisions and provide more robust prevalence data to further understand the underlying immunological mechanism.

Factor VIII brand and the incidence of factor VIII inhibitors in previously untreated UK children with severe hemophilia A, 2000-2011.

The effect of recombinant factor VIII (rFVIII) brand on inhibitor development was investigated in all 407 severe hemophilia A previously untreated patients born in the United Kingdom (UK) between 1 January 2000 and 31 December 2011. Eighty-eight (22%) had been in the RODIN study. Information was extracted from the National Haemophilia Database. Because exposure days (EDs) were not known for some patients, time from first treatment was used as a surrogate for rFVIII exposure. An inhibitor developed in 118 (29%) patients, 60 high and 58 low titer, after a median (interquartile range) of 7.8 (3.3-13.5) months from first exposure and 16 (9-30) EDs. Of 128 patients treated with Kogenate Bayer/Helixate NexGen, 45 (35.2%, 95% confidence interval [CI] 27.4-43.8) developed an inhibitor compared with 42/172 (24.4%, 95% CI 18.6% to 31.4%) with Advate (P = .04). The adjusted hazard ratio (HR) (95% CI) for Kogenate Bayer/Helixate NexGen compared with Advate was 2.14 (1.12-4.10) (P = .02) for high titer and 1.75 (1.11-2.76) (P = .02) for all inhibitors. When excluding UK-RODIN patients, the adjusted HR (95% CI) for high-titer inhibitors was 2.00 (0.93-4.34) (P = .08). ReFacto AF was associated with a higher incidence of all, but not high-titer, inhibitors than Advate. These results will help inform debate around the relative immunogenicity and use of rFVIII brands.

A large-scale computational study of inhibitor risk in non-severe haemophilia A.

Over 500 missense F8 mutations have been reported to cause non-severe haemophilia A. Some F8 genotypes appear to confer a higher risk of inhibitor formation than others and individuals with the same F8 genotype may have differing risks of inhibitor formation. We present an in silico strategy demonstrating the heterogeneity of factor VIII (FVIII)-derived antigen presentation whilst identifying patterns of human leucocyte antigen (HLA) peptide binding that might predict future inhibitor risk. A well-validated computational tool, NetMHCII, enabled large-scale comparison of predicted antigen presentation between endogenous, mutated FVIII-derived peptides and wild-type, therapeutic FVIII-derived peptides spanning all F8 missense mutation positions reported to The Haemophilia A Mutation, Structure and Resource Site (HADB). We identify 40 F8 genotypes to be 'low risk' at a 50% inhibitory concentration (IC50 )-binding threshold of 300 nmol/l (P = 0·00005), defined as absence of novel peptide-major histocompatibility complex (MHC) surfaces for all 14 common HLA-DR alleles assessed. Analysing each of the possible 7280 F8 genotype/HLA-DR permutations individually at an IC50 threshold of 300 nmol/l, 65% are predicted to not generate a novel peptide-MHC surface that would be necessary to engage T cell help for subsequent anti-FVIII antibody generation. This study demonstrates the future importance of interpreting F8 genotype in the context of an individual's HLA profile to personalize inhibitor risk prediction.

Factor VIII gene (F8) mutation and risk of inhibitor development in nonsevere hemophilia A.

Neutralizing antibodies (inhibitors) toward factor VIII form a severe complication in nonsevere hemophilia A, profoundly aggravating the bleeding pattern. Identification of high-risk patients is hampered by lack of data that take exposure days to therapeutic factor VIII concentrates into account. In the INSIGHT study, we analyzed the association between F8 mutation and inhibitor development in patients with nonsevere hemophilia A (factor VIII 2-40 IU/dL). This analysis included 1112 nonsevere hemophilia A patients from 14 centers in Europe and Australia that had genotyped at least 70% of their patients. Inhibitor risk was calculated as Kaplan-Meier incidence with cumulative number of exposure days as the time variable. During 44 800 exposure days (median, 24 exposure days per patient; interquartile range [IQR], 7-90), 59 of the 1112 patients developed an inhibitor; cumulative incidence of 5.3% (95% confidence interval [CI], 4.0-6.6) after a median of 28 exposure days (IQR, 12-71). The inhibitor risk at 50 exposure days was 6.7% (95% CI, 4.5-8.9) and at 100 exposure days the risk further increased to 13.3% (95% CI, 9.6-17.0). Among a total of 214 different F8 missense mutations 19 were associated with inhibitor development. These results emphasize the importance of F8 genotyping in nonsevere hemophilia A.