Haemophilia management: time to get personal?

The possibility of alloimmunization in patients receiving protein replacement therapy depends on (at least) three risk factors, which are necessary concomitantly but insufficient alone. The first is the degree of structural difference between the therapeutic protein and the patient's own endogenous protein, if expressed. Such differences depend on the nature of the disease mutation and the pre-mutation endogenous protein structure as well as on post-translational changes and sequence-engineered alterations in the therapeutic protein. Genetic variations in the recipients' immune systems comprise the second set of risk determinants for deleterious immune responses. For example, the limited repertoire of MHC class II isomers encoded by a given person's collection of HLA genes may or may not be able to present a 'foreign' peptide(s) produced from the therapeutic protein - following its internalization and proteolytic processing - on the surface of their antigen-presenting cells (APCs). The third (and least characterized) variable is the presence or absence of immunologic 'danger signals' during the display of foreign-peptide/MHC-complexes on APCs. A choice between existing therapeutic products or the manufacture of new proteins, which may be less immunogenic in some patients or patient populations, may require prior definition of the first two of these variables. This leads then to the possibility of developing personalized therapies for disorders due to genetic deficiencies in endogenous proteins, such as haemophilia A and B. [Correction made after online publication 11 July 2011: several critical corrections have been made to the abstract].

Mosaicism and haemophilia.

Mosaicism may affect the haemophilia phenotype. Well-known instances include chromosomal mosaicism due to aneuploidy and pseudo-mosaicism due to varying patterns of X-chromosome inactivation. Chromosomal mosaicism in a chimera is a potential source of phenotypic variation. Gene mosaicism is commonplace. Its pattern and effect depend on the stage of development at which a mutation occurs. Proven or possible genetic mosaicism is an important consideration when predicting the likelihood of transmission of haemophilia to a future generation.

Mosaics and haemophilia.

Some mosaic conditions may affect the haemophilia phenotype. Well-known instances include chromosomal mosaicism because of aneuploidy and pseudo-mosaicism because of varying patterns of X-chromosome inactivation. Chromosomal mosaicism in a chimera is a potential source of phenotypic variation. Gene mosaicism is commonplace. Its pattern and effect depend on the stage of development at which a mutation occurs. Proven or possible genetic mosaicism is an important consideration when predicting the likelihood of transmission of haemophilia to a future generation. A mosaic is an individual who has two or more cell lines, genetically different with regard to chromosomes or genes. As techniques improve and studies accumulate, mosaics are being found to be more common than hitherto believed. Some mosaic conditions may affect the phenotype of haemophilia in males and of the carrier state in females. Cells may be mosaic with regard to chromosomes, as in some instances of aneuploidy, and in chimeras, and in females owing to the pattern of X-chromosome inactivation. Cells may be mosaic with regard to new gene mutations. The pattern of mosaicism depends upon the stage in embryogenesis or in germ-cell formation in which the mutation arose.

Hemophilia A: polymorphism detectable by a factor 8 antibody.

Plasma from 54 patients with hemophilia A was tested for neutralizing activity with a human antibody to factor VIII. The plasma from 52 patients had no demonstrable neutralizing activity. Two plasma samples had neutralizing activity equivalent to that of normal plasma despite the lack of factor VIII clotting activity. Apparently, most patients with hemophilia A do not synthesize factor VIII, whereas a few synthesize an inactive molecule with a presumed genetic structural mutation of the active site but with antigenic determinants in common with normal factor VIII. Thus, hemophilia A is a disease caused by more than a single genetic mechanism.

Antibodies to human T-cell leukemia virus membrane antigens (HTLV-MA) in hemophiliacs.

Along with homosexual men, Haitians, and intravenous drug abusers, hemophiliacs are at high risk of contracting acquired immunodeficiency syndrome (AIDS). An earlier study revealed that 36 percent of a group of the AIDS patients had antibodies to cell membrane antigens associated with the human T-cell leukemia virus (HTLV-MA), whereas only 1.2 percent of matched asymptomatic homosexual controls had these antibodies. In the present experiments, serum samples from 172 asymptomatic hemophiliacs were examined for the presence of antibodies to HTLV-MA. Such antibodies were detected in 5 to 19 percent of the hemophiliacs examined from four geographical locations, but in only 1 percent or less of laboratory workers, normal blood donors, donors on hemodialysis, or donors with chronic active hepatitis.

Characterization of three abnormal factor IX variants (Bm Lake Elsinore, Long Beach, and Los Angeles) of hemophilia-B. Evidence for defects affecting the latent catalytic site.

Abnormal factor IX variant proteins were isolated from the plasmas of three unrelated severe hemophilia-B families that had been previously shown to contain functionally impaired molecules immunologically similar to normal factor IX. The families studied were: (1) a patient with markedly prolonged ox brain prothrombin time, designated factor IX Bm Lake Elsinore (IXBmLE); (b) three patients (brothers) with moderately prolonged ox brain prothrombin time, designated factor IX Long Beach (IXLB); and (c) a patient with normal ox brain prothrombin time designated factor IX Los Angeles (IXLA). Each variant molecule comigrates with normal factor IX (IXN) both in the sodium dodecyl sulfate and in the nondenaturing alkaline gel electrophoresis. All three variant proteins are indistinguishable from IXN in their amino acid compositions, isoelectric points, carbohydrate distributions and number of gamma-carboxyglutamic acid residues. Each variant protein undergoes a similar pattern of cleavage by factor XIa/Ca2+ and by factor VIIa/Ca2+/tissue factor, and is activated at a rate similar to that observed for IXN. All of the three variant proteins also react with an anti-IXN monoclonal antibody that interferes with the binding of activated IXN(IXaN) to thrombin-treated factor VIIIC. However, in contrast to IXaN, the cleaved IXBmLE has negligible activity (approximately 0.2%), and cleaved forms of IXLA and IXLB have significantly reduced activity (approximately 5-6%) in binding to antithrombin-III/heparin, and in activating factor VII (plus Ca2+ and phospholipid) or factor X (plus Ca2+ and phospholipid) +/- factor VIII. These data, taken together, strongly indicate that the defect in these three variant proteins resides near or within the latent catalytic site. This results in virtually a complete loss of catalytic activity of the cleaved IXBmLE molecule and approximately 95% loss of catalytic activity of the cleaved IXLA and IXLB molecules.

The use of porcine factor VIII concentrate (Hyate:C) in the treatment of patients with inhibitor antibodies to factor VIII. A multicenter US experience.

The response to a highly purified concentrate of porcine factor VIII was evaluated in 45 bleeding episodes in 38 patients with high responding inhibitor antibodies to factor VIII. A total of 437 infusions were given. The patients came from 25 hemophilia centers in the United States. The majority had a life- or limb-threatening hemorrhage for which other modalities had not been successful. In 32 of 45 episodes, a good to excellent response was obtained. Adverse reactions were minimal, occurring in 17 treatment episodes, and were mostly treated with antihistamines and/or hydrocortisone. No clear predictor of clinical response to porcine factor VIII concentrate was identified, including pretreatment human and porcine inhibitor levels, percentage of cross-reactivity between the human and porcine antibodies, and the presence of measurable levels of factor VIII after the porcine factor concentrate was given. Anamnesis to porcine factor VIII did occur in some instances. Porcine factor VIII is a valuable modality in the treatment of serious hemorrhages in patients with inhibitors to factor VIII. Its use should be considered early in the course of severe hemorrhage in these patients.

Frequency of recent retrotransposition events in the human factor IX gene.

Two germline retrotransposition mutations of recent origin were observed in 727 independent mutations (0.28%) in the human factor IX gene (F9) of patients with hemophilia B: 1) a 279 bp insertion in exon H originating from an Alu family of short interspersed elements not previously known to be active and, 2) a 463 bp insertion in exon E of a LINE1 element originating in the maternal grandmother. If the rates of recent germline mutation in F9 are typical of the genome, a retrotransposition event is estimated to occur somewhere in the genome of about one in every 17 children born. Analysis of other estimates for retrotransposition frequency and overall mutation rates suggests that the actual rate of retrotransposition is likely to be in the range of one in every 2.4 to 28 live births.

Novel hotspot detector software reveals a non-CpG hotspot of germline mutation in the factor IX gene (F9) in Latin Americans.

Two-base substitutions at each of two nucleotides in the factor IX gene (F9), but not part of CpG dinucleotides, were recently reported in a small population sample collected in Mexico, a significant observation of recurrent sites ("hotspots") of mutation (P=0.00005). When these new data were combined with previously collected mutation data into two progressively larger and inclusive Latin American samples, additional mutations were observed at one recurrent site, nucleotide 17747, and an additional recurrent nucleotide was observed such that the recurrent nucleotides in these larger samples were also significant (P=0.0003 and 0.0003). In contrast, in three non-Latin American control samples, there was at most only one nucleotide that recurred only once, most likely a chance recurrence (P>/=0.5). When the significance of substitutions was analyzed at each recurrent nucleotide individually, nucleotide 17747 was shown to be a significant recurrent nucleotide by itself in all the Latin American population samples (P

Use of porcine factor VIII for surgical procedures in hemophilia A patients with inhibitors.

We have described the use of polyelectrolyte fractionated porcine factor VIII (HYATE:C, Porton Speywood Ltd, Wrexham, UK) to provide hemostasis in 45 patients with hemophilia A complicated by inhibitor antibodies. The cases were collected from hemophilia care providers in seven nations and represent some of the experience with porcine factor VIII generated over the past 12 years. A wide variety of procedures was performed with varying degrees of associated hemostatic challenge in a difficult treatment group. The patients were representative of the hemophilia population in general and ranged in age from 1 to 67 years. The results of our survey should encourage clinicians to consider use of porcine factor VIII to provide hemostatic coverage in hemophilia patients with inhibitors who require surgical procedures. Hemostatic coverage was satisfactory in the vast majority of episodes; there have been only a small number of easily controlled, well-tolerated adverse reactions, which were usually self-limited in nature. No life-threatening reactions to porcine factor VIII were seen during coverage for surgical procedures in our survey, although serious reactions can occur as noted above. This is remarkable, since surgical patients usually are treated with higher doses of porcine factor VIII for greater periods of time than most other hemophilic patients treated for hemarthroses or soft-tissue bleeding, for example. Thrombocytopenia in association with porcine factor VIII therapy has been a major concern since the first crude preparations were used in the 1950s. Modern series, however, note the incidence of thrombocytopenia in only a minority of patients treated with the current polyelectrolyte fractionated preparations of porcine factor VIII. In our series, thrombocytopenia with platelet counts of less than 150 x 10(9)/L occurred in 11 of 54 treatment episodes (20%). The mechanism for thrombocytopenia demonstrated in a previous study involves porcine von Willebrand factor, which may copurify with porcine factor VIII and cause platelet aggregation and/or clearance from the circulation. The phenomenon of thrombocytopenia appears to be related to the administration of high doses of porcine factor VIII in some cases; however, in other patients, it may develop inconsistently at modest doses. It may be that there are idiosyncratic differences between patients that determine the dose of porcine factor VIII likely to cause thrombocytopenia. As yet, there is no way to predict which patient will develop thrombocytopenia while being treated with porcine factor VIII.(ABSTRACT TRUNCATED AT 400 WORDS)

Human T cell leukemia virus type III antibody, lymphadenopathy, and acquired immune deficiency syndrome in hemophiliac subjects. Results of a prospective study.

A cohort of 63 hemophiliac subjects was followed for clinical and immunologic abnormalities related to the acquired immune deficiency syndrome (AIDS). When evaluated in early 1984, antibody to human T cell leukemia virus type III (HTLV-III) was detected in the serum of 59 percent (24 of 41) of factor VIII or IX concentrate recipients, but in none (0 of six) of the cryoprecipitate/fresh frozen plasma recipients. HTLV-III-seropositive hemophiliac subjects, on average, had been exposed to twice as much concentrate during the previous year as seronegative hemophiliac subjects. The seropositive group had a significantly lower mean helper/suppressor T cell ratio and absolute helper T cell level than the seronegative group. By early 1984, 13 hemophiliac subjects in the study population had lymphadenopathy and one had AIDS. Antibody to HTLV-III was detected in the serum of 13 of these 14 hemophiliac subjects with overt clinical disease. The prevalence of lymphadenopathy or AIDS among HTLV-III-seropositive hemophiliac subjects was 54 percent (13 of 24). It is concluded that HTLV-III antibody occurs with high frequency in hemophiliac subjects, and is related to the amount of factor VIII or IX concentrate infused. Over half of HTLV-III-seropositive hemophiliac subjects in this population had overt clinical disease with either lymphadenopathy or AIDS.

Purification and properties of an abnormal blood coagulation factor IX (factor IXBm)/kinetics of its inhibition of factor X activation by factor VII and bovine tissue factor.

An abnormal blood coagulation factor IX has been isolated from the blood of a hemophilia B patient with a variant of the disease (hemophilia Bm) characterized by a normal concentration of factor IX antigen, negligible factor IX coagulant activity, and a prolonged prothrombin time with bovine tissue factor. The isolated protein (factor IXBm) had the same apparent molecular weight as normal factor IX (55,000) and the same mobility on two dimensional immunoelectrophoresis as normal factor IX. Factor IXBm underwent limited proteolysis induced by activated factor XI, in the presence of Ca2+ ions, or induced by the reaction product of tissue factor, factor VII and Ca2+ ions. A timecourse study showed that activated factor XI cleaved factor IXBm and factor IX at similar rates. However, in contrast to normal factor IX, the limited protelysis of factor IXBm did not generate procoagulant activity. In kinetic experiments purified factor IXBm behaved like a competitive inhibitor (Ki of 0.017 muM) of the activation of factor X by bovine tissue factor and factor VII. Normal factor IX was also found to inhibit the reaction but required a four-fold higher concentration to activate the same inhibitory effects as factor IXBm.

In vivo recovery and survival of monoclonal-antibody-purified factor VIII concentrates.

In response to reports of discrepant in vitro assays of high-purity concentrates, a double-blind crossover study of in vivo recovery and half-life of two brands of monoclonal-antibody-purified factor VIII concentrates (Monoclate and Hemofil-M) was performed in 23 patients with hemophilia A. In vivo recoveries were close to values predicted from the labelled unitage when plasma samples were assayed by a one-stage method. When a two-stage assay was used, lower recoveries were calculated and the recovery with Hemofil-M was slightly but significantly lower than that with Monoclate. The concentrates were re-assayed in vitro by the two-stage method. Monoclate (which is assayed by the manufacturer using a two-stage method) contained 97% of the labelled potency and Hemofil-M (which is assayed by the manufacturer using a one-stage method) contained 81% of the labelled potency. Differences in in vitro and in vivo assay methods contribute to disparities between expected and observed factor VIII recovery. Clearance of Hemofil-M was significantly faster than that of Monoclate, but volume of distribution at the steady state, mean residence time, and plasma half-disappearance times of the two concentrates were not significantly different.

Molecular epidemiology of factor IX germline mutations in Mexican Hispanics: pattern of mutation and potential founder effects.

Germline mutations in patients with hemophilia B generally have arisen within the past 150 years. Evidence suggests that these germline mutations generally result from endogenous processes. However, a unique pattern would be expected if a population were exposed to a physiologically important germline mutagen since mutagens generally produce characteristic patterns, or "fingerprints", of mutation. To determine the pattern of mutation in Mexican Hispanics, the regions of likely functional significance in the factor IX gene were screened by dideoxy fingerprinting (ddF) in 31 families with hemophilia B. Mutations were found in 30 of these families. Haplotype analysis was performed on individuals with identical mutations to help distinguish independent, recurrent mutations from founder effects. Analysis of these 30 mutations, along with 7 mutations reported previously in Mexican Hispanic families, reveals a pattern of independent mutation that is similar to the pattern of mutation observed in 127 U.S. Caucasian families (p = 0.89). These results may reflect either an underlying pattern of germline mutation due to endogenous processes or the presence of an ubiquitous mutagen. Further analyses of the recurrent mutations revealed that two mutations, T296M and R248Q, accounted for 19% of the mutations found in the Mexicans. Haplotype data suggest that the multiple occurrences of T296M and R248Q are associated with founder effects and that screening for these mutations may allow rapid mutation detection and carrier diagnosis in a significant minority of Mexican families with hemophilia B, These two mutations also are associated with founder effects in the U.S, Caucasian population. However, the haplotypes are different in these two populations, indicating independent origins. The occurrence of identical founder mutations in distinct populations provides evidence for the previous hypothesis that the number of different mutations giving rise to mild or borderline mild/moderate hemophilia B is small compared to deleterious mutations causing more severe disease.

Production and therapeutic use of a factor XI concentrate from plasma.

Factor XI deficiency is an uncommon bleeding disorder usually manifested by excessive bleeding after surgery or trauma. Until recently the only effective therapy has been fresh-frozen plasma (FFP) infusion. We describe the efficacy and safety of a new factor XI concentrate produced from human donor plasma by a modification of the method used for antithrombin III concentrate. The mean recovery of factor XI in the circulation measured on 62 occasions was approximately 91% of the injected dose, and the mean half-disappearance-time was 52 h. The concentrate was used for 31 invasive procedures in 30 patients, including 16 patients who had a definite bleeding tendency on previous occasions, with normal haemostasis being achieved in all but 1. Only 1 patient (previously experiencing allergy to FFP) experienced adverse effects during infusion. Monitoring of liver function tests and viral antibody status in suitable patients has shown no evidence of transmission of hepatitis viruses, HIV-1 or parvovirus B19. We conclude that this concentrate provides effective treatment for patients with factor XI deficiency. Preliminary results suggest safety from virus transmission, but this needs to be established in further studies of previously untreated patients.

Comparison of six commercial plasma references for factor VIII, factor IX and von Willebrand factor. On behalf of the Subcommittee for Factor VIII and IX of the Scientific and Standardization Committee of the ISTH.

Six brands of normal reference plasma produced in the United States, with assigned assay values for factor VII and IX and, in four instances, ristocetin cofactor and van Willebrand antigen, were assayed in nine coagulation laboratories in academic institutions in the same country. Differences in mean assays of reference plasmas, as a percent of labelled potency, were significant and were greater than differences among laboratories. Standard methods of assigning potency to commercial reference plasmas are recommended.

Safety profile of porcine factor VIII and its use as hospital and home-therapy for patients with haemophilia-A and inhibitors: the results of an international survey.

A multicentre retrospective survey was conducted to re-assess the use of porcine factor VIII (HYATE:C), its side effects and the selection of patients for regular or home-therapy. 15,152,000 units of HYATE:C were used by 154 patients. The median inhibitor cross-reactivity to porcine VIIIC of 137 patients was 15%, 27% of patients lacking cross-reactivity. An absent, intermediate or brisk specific antiporcine anamnestic response was observed in 29, 40 and 31% of patients respectively. Seven patients were treated on-demand as home-therapy for a median 6.2, range 1.5-13 years, 23 further patients were treated regularly in hospital for a median of 3, range 2-7 years. This group used 8,319,000 U of porcine VIIIC for 2,000 bleeding episodes. The incidence of transfusion reactions was 0.001%, 0.64% and 2.3%, for domiciliary infusions, infusions in multiply treated in-patients, and unselected in-patient infusions, respectively. The risk of reactions was dose-related. A post-infusion fall in platelet count was common, but usually transient and clinically insignificant. This was also dose-related (r = -0.64, p = 0.002). Marked reductions in platelet count were occasionally seen, usually with intensive replacement therapy. The relative lack of side effects observed amongst patients treated at home is attributable to the low, median 33 U/kg, dose used by this group. A subgroup of inhibitor patients, identifiable by their absent or modest anamnestic response to porcine factor VIII may be treated regularly and safely with this product in small doses, over a period of years.