Seven novel and four recurrent point mutations in the factor VIII (F8C) gene

Haemophilia A is a X‐linked bleeding disorder, caused by deficiency in the activity of coagulation factor VIII due to mutations in the corresponding gene. The most common defect in patients is an inversion of the factor VIII gene that accounts for nearly 45% of individuals with severe hemophilia A. Point mutations and small deletions/insertions are responsible for the majority of cases with moderate to mild clinical course and for half of the severe hemophilia A occurrences. The majority of these mutations are “private”, because of the high mutation rate for this particular gene. We report on eleven pathological changes in the factor VIII sequence detected in male patients with haemophilia A or in female obligate carriers. Seven of these mutations are novel [E204N, E265X, M320T, F436C, S535C, N2129M and R2307P] and four have been previously identified [V162M, R527W, R1966X, and R2159C]. Genotype‐phenotype correlations and computer prediction analysis on the effect of missense mutations on the secondary structure of the factor VIII protein are performed and the relationships evaluated. © 2001 Wiley‐Liss, Inc.     

Seven novel and four recurrent point mutations in the factor VIII (F8C) gene

Haemophilia A is a X‐linked bleeding disorder, caused by deficiency in the activity of coagulation factor VIII due to mutations in the corresponding gene. The most common defect in patients is an inversion of the factor VIII gene that accounts for nearly 45% of individuals with severe hemophilia A. Point mutations and small deletions/insertions are responsible for the majority of cases with moderate to mild clinical course and for half of the severe hemophilia A occurrences. The majority of these mutations are “private”, because of the high mutation rate for this particular gene. We report on eleven pathological changes in the factor VIII sequence detected in male patients with haemophilia A or in female obligate carriers. Seven of these mutations are novel [E204N, E265X, M320T, F436C, S535C, N2129M and R2307P] and four have been previously identified [V162M, R527W, R1966X, and R2159C]. Genotype‐phenotype correlations and computer prediction analysis on the effect of missense mutations on the secondary structure of the factor VIII protein are performed and the relationships evaluated. © 2001 Wiley‐Liss, Inc.     

Molecular etiology of factor VIII deficiency in hemophilia A

Hemophilia is a common X-linked coagulation disorder due to deficiency of factor VIII. The factor VIII gene has been cloned in 1984 and a large number of mutations that cause hemophilia A have been identified in the last decade. The most common of the mutations is an inversion of factor VIII that accounts for nearly 45% of patients vvith severe hemophilia A. This review lists all the factor VIII mutations identified to date and briefly discusses their functional significance. © 1995 Wiley-Liss, Inc.     

Hemophilia and von Willebrand's disease: genetic considerations

Recent progress in the biochemical characterization of coagulation factors VIII and IX has greatly contributed to our understanding of the inheritance of hemophilia and von Willebrand's disease and facilitated the recognition of carriers of these disorders. Factor VIII is a molecular complex which may be quantitated immunologically as factor VIII-related antigen. Within this complex reside the von Willebrand factor, absent in von Willebrand's disease, and factor VIII procoagulant activity and antigen. Hemophilia is an x-linked disorder; female carriers may be recognized by a disproportionate increase in factor VIII-related antigen or procoagulant antigens in relation to procoagulant activity. Prenatal diagnosis of hemophilia has been accomplished by measurements of clotting activity and antigens in fetal blood. Von Willebrand's disease has been classified on the basis of laboratory abnormalities, the biochemical characteristics of the von Willebrand factor, and its patterns of inheritance. In the most commonly observed form, there is autosomal dominant inheritance, and most patients are heterozygotes. These individuals manifest variably prolonged bleeding times and concordantly reduced activities associated with factor VIII. Rarely, there is an autosomal recessive pattern in which the homozygotes have much more severe clinical disease, including hemathroses. However, the biochemical defects in the von Willebrand factor appear to be quite diverse, defying any simple classification of this disorder.     

Current treatment of patients with inhibitors to factor VIII

The development of inhibitors against factor VIII or IX in hemophilic or nonhemophilic patients is a relatively rare condition, but basic and clinical studies of these inhibitors have been extremely fruitful and have led to the following: a better understanding of the structure-function relationships of the factor VIII and IX molecules; increased knowledge of the genetic defect in classic hemophilia and hemophilia B; newer insights into antibody formation against clotting factors; and an increased number of therapeutic options for the treatment of patients who have inhibitors to factor VIII or IX. The prevalence of inhibitors to factor VIII:C in the hemophiliac population of the U.S. was obtained in the NHLBI multi-center trial using 1500 patients and is estimated to be 14.5%. The vast majority of these inhibitors occurred in severely affected hemophiliacs less than twenty years of age with factor VIII:C levels of less than 0.03 u/ml. However, occasional older patients or those with F. VIII:C greater than 0.03 u/ml did develop inhibitors.     

Desensitization to factor VIII in a patient with classic hemophilia and C2 deficiency

Factor VIII therapy has been reported to cause anaphylactic reactions in patients with hemophilia. Desensitization attempts have been complicated by severe allergic reactions that have prevented the achievement of protective factor VIII levels. We report successful administration of factor VIII by a graded dose desensitization protocol in a 36-year-old man with hemophilia A who had previously experienced anaphylactic reactions to factor VIII infusions. The reactions were manifested by urticaria, choking, and bronchospasm and were not prevented by pretreatment with antihistamines and corticosteroids. Intradermal skin test with factor VIII was positive. Serum levels of circulating immune complexes were slightly elevated. Persistently low serum C2 levels were consistent with genetic C2 deficiency. These findings suggest the possibility of Type I (IgE mediated) and Type III (immune complex) immunopathogenic mechanisms. Our experience suggests that administration of factor VIII by graded dose desensitization protocol may offer a practical therapeutic approach for management of hemorrhage in patients with classic hemophilia who are allergic to factor VIII.     

Danazol increases factor VIII and factor IX in classic hemophilia and Christmas disease

We gave danazol (600 mg per day orally for 14 days), an attenuated androgen, to four adults with classic hemophilia and one adult with Christmas disease. The levels of factor VIII in the patients with classic hemophilia ranged from 1 to 3 per cent before treatment and rose to 3 to 8 per cent during the treatment period. The level of factor IX in the patient with Christmas disease rose from 5 to 14 per cent. The rise in clotting-factor activity was usually observed within five to six days after the initiation of therapy and peaked between 7 and 13 days. The drug had no untoward effects. During the 70 patient-days of therapy, only two patients required plasma products, each on one occasion. These data suggest that danazol therapy may decrease the hemorrhagic tendency and reduce the need for transfusions of plasma products in classic hemophilia and Christmas disease. Controlled clinical trials will be required to establish its value in these applications.     

Re-evaluation of plasmas from patients previously diagnosed as having von Willebrand's disease with the factor VIII-related antigen and ristocetin cofactor assay.

Diagnosis of deficiencies of coagulation factor VIII can be difficult to establish in some cases. The use of the factor VIII-related antigen and the use of the ristocetin cofactor assays have increased the reliability of diagnosis of factor VIII deficiency in patients with hemophilia A or von Willebrand's disease, and in carriers of hemophilia A. The authors re-evaluated samples, from frozen storage, of blood from patients previously diagnosed as having von Willebrand's disease. This diagnosis was based on clinical history, family history, bleeding time, factor VIII procoagulant activity, and response to ristocetin in platelet-aggregation studies. Eleven cases were studied by the review of previously obtained data and the addition of the factor VIII-related antigen and ristocetin-cofactor assays. In two of eleven cases, the diagnosis was changed to possible hemophilia A carrier state.     

Mutational‐screening in the factor VIII gene resulting in the identification of three novel mutations,one of which is a donor splice mutation

Hemophilia A is an X‐linked bleeding disease caused by mutations in the coagulation factor VIII gene. The identification and characterization of pathogenic mutations allows the recognition of new mechanisms of functional disturbances of factor VIII. To screen for mutations exons 1‐26 of the factor VIII gene have been amplified genomically and analyzed by SSCP followed by direct sequencing of respective exons showing abnormal electrophoretic mobility on SSCP analysis. In the present study we report the detection of four mutations in the factor VIII gene, of which three are novel. The mutational analysis of a patient with severe hemophilia A has revealed that the a ®c transversion at position 3 of the donor‐splice‐site of intron 23 results in the skipping of exon 23. A novel nonsense mutation Q1778X in exon 16 of factor VIII gene has been identified in a second hemophilia A case. Furthermore two missense mutations have been ascertained: a novel, S183R, causing a mild phenotype of hemophilia A and R282H, previously described in association with severe hemophilia A. Hum Mutat 13:504, 1999. © 1999 Wiley‐Liss, Inc.     

Factor VIII procoagulant antigen levels in normal pregnancy and preeclampsia

It has been suggested that the basis for the marked discordance in factor VIII parameters in preeclampsia is the result of proteolysis of the factor VIII procoagulant component (VIII:C) owing to activation of the coagulation system. To investigate this further, levels were compared of factor VIII:C and VIII:C (Ag), the immunologic equivalent of the procoagulant activity, in a series of preeclamptic patients and patients with uncomplicated pregnancies. No significant difference in the mean (+/- SD) level of factor VIII:C or VIII:C (Ag) could be detected between these groups. Our results do not support proteolysis of factor VIII:C as a mechanism to account for the discordant levels of the factor VIII parameters in preeclampsia.     

Immune coagulation disorders (excluding antiphospholipid syndrome]

During the course of replacement therapy, 22-30% of patients with severe hemophilia A develop alloantibody to factor VIII. Autoantibodies to coagulation factors rarely occur in elder individuals with previously normal hemostatic mechanisms or in patients with various underlying disorders. Although the great majority of the acquired inhibitors are directed to factor VIII, the antibodies may arise to every coagulation factor. The inhibitor antibodies directly inactivate specific clotting factor, or occasionally, they bind to a nonfunctional site, resulting in increased plasma clearance. In the last decade, we experienced 12 hemophilia A and 3 hemophilia B patients who developed factor VIII and factor VIII and factor IX inhibitor, respectively, 9 patients with autoantibody to factor VIII (acquired hemophilia), and 4 patients with acquired von Willebrand syndrome. Among 12 factor VIII inhibitors, 4 patients were identified to have inversion in the factor VIII gene, 1 with 4 bases deletion, and 1 with missense mutation resulting in G479R. Four of 9 patients with acquired hemophilia had underlying disorders of autoimmune hemolytic anemia, macroglobulinemia, urticaria, and pharyngeal cancer at the development of factor VIII inhibitor. Antibody to von willebrand factor was detected in 3 of 4 patients with acquired von Willebrand syndrome.     

B-Cell and T-Cell Epitopes in Anti-factor VIII Immune Responses

Adequate hemostasis is achieved for many hemophilia A patients by infusion of plasma-derived or recombinant factor VIII (FVIII), but unfortunately, a significant subset of patients develop an immune response in which anti-FVIII antibodies, referred to clinically as “inhibitors,” interfere with its procoagulant activity. Inhibitors are the subset of anti-FVIII antibodies that bind to surfaces on FVIII (B-cell epitopes) that are important for its proper functioning in coagulation. Less antigenic FVIII molecules may be designed by identifying and then modifying the amino acid sequences of inhibitor B-cell epitopes. Conversely, characterization of these epitopes can yield important information regarding functionally important surfaces on FVIII. The production of inhibitor antibodies is driven by T cells. T cells recognize FVIII as foreign when FVIII-derived peptides bind to major histocompatibility complex (MHC) class II molecules on the surface of antigen-presenting cells. The class II–peptide complexes must then be recognized by T-cell receptors (TCRs). T-cell stimulation requires sustained association of antigen-presenting cells and T cells through formation of a class II–peptide–TCR complex, and peptide sequences that mediate this association are termed “T-cell epitopes.” MHC class II tetramers that bind FVIII-derived peptides and recognize antigen-specific TCRs are proving useful in the characterization of human leukocyte antigen-restricted T-cell responses to FVIII.     

Human recombinant DNA-derived antihemophilic factor (factor VIII) in the treatment of hemophilia A. recombinant Factor VIII Study Group

BACKGROUND. Current treatment of hemophilia A, a hereditary disorder affecting approximately 1 in 10,000 males, relies on plasma-derived factor VIII concentrates. We tested the safety and efficacy of a recombinant factor VIII preparation for the treatment of this disorder. METHODS. We conducted the investigation in three stages: comparing the pharmacokinetics of plasma-derived and recombinant factor VIII, assessing the efficacy of recombinant factor VIII for home therapy, and assessing its efficacy for major surgical procedures and hemorrhage. A total of 107 subjects with hemophilia, 20 of whom had not been treated previously, enrolled in the investigation. RESULTS. The in vivo recovery and elimination half-lives of recombinant factor VIII equaled or exceeded those of plasma-derived factor VIII. Seventy-six subjects participated in a home-treatment program, using recombinant factor VIII for 69 to 807 days (median, 618); home diaries of 56 subjects treated for 5 months were analyzed. Of 540 bleeding episodes, 399 (73.9 percent) required only one treatment with recombinant factor VIII. The projected annual consumption of recombinant factor VIII was similar to that of plasma-derived factor VIII concentrate. Twenty-six subjects received recombinant factor VIII for 22 surgical procedures and 10 serious hemorrhages; hemostasis was excellent in all cases. De novo formation of inhibitors occurred in only 1 of 85 previously treated subjects. Inhibitor antibodies also developed in 6 of 21 children, 20 of whom had not previously been treated; 5 had low levels (less than or equal to 7.5 Bethesda units) despite continued treatment with recombinant factor VIII. There was no evidence of new formation of antibody to foreign proteins, and recombinant factor VIII was well tolerated. CONCLUSIONS. Recombinant factor VIII has biologic activity comparable to that of plasma factor VIII and is safe and efficacious for the treatment of hemophilia A.     

Three cases of acquired factor VIII: C inhibitors in non-hemophilic patients

Summary During the last ten years we observed three non-hemophilic patients with factor(F) VIII: C inhibitors (2 women aged 68 and 80 and a man aged 51).In all three cases, a sudden bleeding tendency was observed shortly after an injury or surgery. Coagulation tests showed a prolonged aPTT and a decreased F VIII: C level. Other deficiencies of blood-clotting factors and acquired or hereditary von Willebrand's disease could be excluded. Therapy with F VIII: C concentrate, cryoprecipitate, or fresh-frozen plasma did not produce the expected increase in F VIII: C. Measurement of F VIII: C inhibitor levels (Bethesda Units, BU) revealed values in the range between 9 and 64 BU. The two patients subjected to long-term therapy with a combination of prednisone (initially 2–3 mg/kg BW) and azathioprine (2–3 mg/kg BW) responded positively; the F VIII: C concentration increased. The third patient, treated only with a low dose of prednisone (30 mg/day), did not show any reaction at all. Since hereditary hemophilia A could be excluded, the inhibitors apparently were acquired. Malignant tumors did not appear. In conclusion, long-term therapy of an acquired F VIII: C inhibitor with a combination of prednisone and azathioprine may lead to complete disappearance of the inhibitor, normalization of the coagulation tests, and complete remission of the bleeding tendency.Abbreviations aPCC activated prothrombin complex concentrate - aPTT activated partial thromboplastin time - BU Bethesda Units - BW body weight - CP cryoprecipitate - DIC disseminated intravascular coagulation - F III: C factor VIII procoagulant activity - FEIBA factor eight inhibitor bypassing activity - FFP fresh-frozen plasma     

Immunoaffinity purification of factor VIII

The development of factor VIII concentrates has greatly facilitated hemophilia care and has made the home care of hemophilia possible. However, factor VIII concentrate that has been produced using traditional methods contains large amounts of foreign proteins and viruses. This has resulted in the development of immunologic abnormalities in many hemophiliacs and has exposed many of these patients to blood-borne viruses such as the human immunodeficiency virus (HIV) and hepatitis viruses. Factor VIII circulates in plasma in complex with the von Willebrand factor (vWF). Both factor VIII and vWF have been purified and monoclonal antibodies (mAb) have been generated to both of these proteins. When bound to a solid support, these mAb's can be used to isolate selectively the proteins of interest. Recently, two separate procedures have been used in the immunoaffinity purification of factor VIII on a commercial scale. One product (Monoclate) has been prepared using a mAb to the vWF bound to a chromatography column. The other product (Hemophil M) uses immobilized mAb to the factor VIII molecule. Factor VIII concentrate purified using either of these approaches is far more pure than traditional factor VIII concentrates. In addition, the use of both viral purification and viral inactivation procedures has greatly reduced the risk of viral contamination. Early clinical studies have demonstrated that these products are effective in treating bleeding episodes and that the risk of viral infection with HIV or hepatitis viruses is low. Factor VIII concentrate produced using mAb technology appears to be the product of choice in previously untransfused hemophiliacs. Its role in the treatment of patients who have already been infected with HIV is less clear.     

Nonviral transfer of the gene encoding coagulation factor VIII in patients with severe hemophilia A

BACKGROUND: We tested the safety of a nonviral somatic-cell gene-therapy system in patients with severe hemophilia A. METHODS: An open-label, phase 1 trial was conducted in six patients with severe hemophilia A. Dermal fibroblasts obtained from each patient by skin biopsy were grown in culture and transfected with a plasmid containing sequences of the gene that encodes factor VIII. Cells that produced factor VIII were selected, cloned, and propagated in vitro. The cloned cells were then harvested and administered to the patients by laparoscopic injection into the omentum. The patients were followed for 12 months after the implantation of the genetically altered cells. An interim analysis was performed. RESULTS: There were no serious adverse events related to the use of factor VIII-producing fibroblasts or the implantation procedure. No long-term complications developed, and no inhibitors of factor VIII were detected. In four of the six patients, plasma levels of factor VIII activity rose above the levels observed before the procedure. The increase in factor VIII activity coincided with a decrease in bleeding, a reduction in the use of exogenous factor VIII, or both. In the patient with the highest level of factor VIII activity, the clinical changes lasted approximately 10 months. CONCLUSIONS: Implantation of genetically altered fibroblasts that produce factor VIII is safe and well tolerated. This form of gene therapy is feasible in patients with severe hemophilia A.     

The mechanism of factor VIII inactivation by human antibodies. II. The effect of factor VIII R: antigen on the rate of interaction

Factor VIII R:Ag by binding to the procoagulant component (VIII:C) inhibits the extent and rate of interaction of human antifactor VIII antibodies with VIII:C. When this reaction is examined under ionic strength conditions (0.24M CaCl) which dissociate the two factor VIII components, the extent of the reaction is increased approximately two fold and the initial rate of interaction is increased three to four fold for both intact IgG antibody and its Fab' derivative. With isolated procoagulant component, increased ionic strength conditions only influence the rate of interaction. These studies further explain the peculiar time-dependence of this interaction.     

Recurrent inversion with concomitant deletion and insertion events in the coagulation factor VIII gene suggests a new mechanism for X-chromosomal rearrangements causing hemophilia A

Recurrent int22h-related inversions in the coagulation factor VIII gene (F8) are the most common cause of severe hemophilia A. Such inversions have repeatedly been hypothesized to be associated with concomitant deletions that are responsible for an increased risk of immune responses against therapeutic exogenous factor VIII. However, exact DNA breakpoints have not yet been reported. In a patient with persistent factor VIII-inactivating antibodies, molecular analysis of F8 including Southern Blot, long-range PCR and primer walking techniques revealed a combination of an int22h2-related inversion, deletion of exons 16-22 and insertion of a duplicated part of the X-chromosomal MPP1 gene. This novel genomic rearrangement was also detectable in the patient's mother, but absent in both maternal grandparents. The genetic defect most likely originated from a complex X-chromosomal recombination event during spermatogenesis due to the formation of a DNA loop stabilized by Alu and LINE repeat elements. Elucidation of such combined mutations may allow early identification of patients at high risk of developing factor VIII-neutralizing antibodies and will help to understand the mechanisms behind gross chromosomal rearrangements causing hemophilia A and other diseases.     

Mechanism of factor VIII inactivation by human antibodies. III. Proteolytic cleavage of factor VIII:C and C antigen by thrombin

Thrombin activation of factor VIII results in a marked, but transient, increase in factor VIII procoagulant activity. This proteolytic process has been examined by immobilizing factor VIII in a solid phase system using mouse monoclonal antibody specific for the factor VIII related antigen. These studies demonstrate that thrombin activation is the result of proteolytic cleavage from the factor VIII:C protein of a peptide fragment which contains both the factor VIII:C procoagulant and VIII:C antigen sites recognized by human antibodies.