Inhibitor development and mortality in non-severe hemophilia A.

Background: The life expectancy of non-severe hemophilia A (HA) patients equals the life expectancy of the non-hemophilic population. However, data on the effect of inhibitor development on mortality and on hemophilia-related causes of death are scarce. The development of neutralizing factor VIII antibodies in non-severe HA patients may dramatically change their clinical outcome due to severe bleeding complications.

Significance of F8 missense mutations with respect to inhibitor formation.

We have identified 1,135 haemophilia A patients with missense mutations associated with mild (46%), moderate (22%), severe (16%), and mixed haemophilia phenotypes (11%). Altogether, we detected 374 different missense mutations of which 195 are not listed in the HAMSTeRS database. While missense mutations are strongly underrepresented within the factor VIII (FVIII) B-domain, they are evenly distributed throughout the entire F8 cDNA sequence.

Measurements of DNA methylation at seven loci in various tissues of CD1 mice.

In humans, considerable variation in methylation at single loci and repetitive elements in various cells and tissues is observed. Recently, several inter- and intra-tissue correlations for DNA methylation have been reported. To investigate the extent and reproducibility of such correlations, we investigated inter- and intra-tissue methylation correlations among seven different loci in 9 different tissues in a population of 100 healthy seven-week-old CD1 outbred mice.

Methylation of L1Hs promoters is lower on the inactive X, has a tendency of being higher on autosomes in smaller genomes and shows inter-individual variability at some loci.

LINE-1 repeats account for ~17% of the human genome. Little is known about their individual methylation patterns, because their repetitive, almost identical sequences make them difficult to be individually targeted. Here, we used bisulfite conversion to study methylation at individual LINE-1 repeats.

Methylation at global LINE-1 repeats in human blood are affected by gender but not by age or natural hormone cycles.

Previously, we reported on inter-individual and gender specific variations of LINE-1 methylation in healthy individuals. In this study, we investigated whether this variability could be influenced by age or sex hormones in humans. To this end, we studied LINE-1 methylation in vivo in blood-derived DNA from individuals aged 18 to 64 years and from young healthy females at various hormone levels during the menstrual cycle.

Impact of polymorphisms of the major histocompatibility complex class II, interleukin-10, tumor necrosis factor-alpha and cytotoxic T-lymphocyte antigen-4 genes on inhibitor development in severe hemophilia A.

Background: Approximately 25% of severe hemophilia A (HA) patients develop antibodies to factor VIII protein.

Patients: In the present case-controlled cohort study, 260 severely affected, mutation-type-matched HA patients were studied for association of human leukocyte antigen (HLA) class II molecules and polymorphisms in the genes encoding interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-alpha) and cytotoxic T-lymphocyte antigen-4 (CTLA-4) and development of inhibitors.

Results: Our results demonstrate a higher frequency of DRB1*15 and DQB1*0602 alleles as well as of the haplotype DRB1*15/DQB1*0602 in inhibitor patients [odds ratio (OR) 1.

Kinetic parameters of monoclonal antibodies ESH2, ESH4, ESH5, and ESH8 on coagulation factor VIII and their influence on factor VIII activity.

The murine monoclonal antibodies ESH2, ESH4, ESH5, and ESH8 specifically bind and inhibit the procoagulant activity of human coagulation factor VIII (FVIII). They are frequently used as a model of inhibitors which are raised against injected FVIII in about 25% of hemophiliacs as a serious side effect of substitution therapy. However, binding kinetics of the interaction of these antibodies with FVIII and their influence on FVIII activity (inhibition) have not yet been examined systematically.

Efficient factor VIII affinity purification using a small synthetic ligand.

Background: Hemophilia A is currently treated by infusions of the coagulation factor (F) VIII, of which production and purification remain a challenging task. Current purification procedures using immunoaffinity chromatography are cumbersome, expensive, and suffer from the instability of the applied antibody ligands, which elute along with the product and contaminate it. Recently, FVIII was purified using octapeptide ligands, but their use is limited due to the low resistance to proteases.

Modified expression of coagulation factor VIII by addition of a glycosylation site at the N terminus of the protein.

Recently, it was shown that glycoproteins with N-glycans close to the NH(2) terminus can directly enter the calnexin/calreticulin cycle and bypass BiP binding. This should allow efficient secretion of glycoproteins such as factor VIII (FVIII) whose secretion is negatively affected by BiP interaction. Examination of the glycosylation pattern of the NH(2) terminus of FV and FVIII revealed N-glycans at positions 23 and 27 in FV and at position 41 in FVIII.

Gender specific differences in levels of DNA methylation at selected loci from human total blood: a tendency toward higher methylation levels in males.

Abnormal patterns of DNA methylation are observed in many diseases such as tumors and imprinting disorders. Little is known about inter-individual and gender specific variations. Here, we report on accurate and sensitive quantitative measurements of methylation in DNA from total blood in 96 healthy human males and 96 healthy human females.

Lack of F8 mRNA: a novel mechanism leading to hemophilia A.

Hemophilia A (HA) is caused by partial or total deficiency of F8 protein activity. In a small group, about 1.8% of patients with HA, no mutation is found in the F8 gene.

Haemophilia A: from mutation analysis to new therapies.

Haemophilia is caused by hundreds of different mutations and manifests itself in clinical conditions of varying severity. Despite being inherited in monogenic form, the clinical features of haemophilia can be influenced by other genetic factors, thereby confounding the boundary between monogenic and multifactorial disease. Unlike sufferers of other genetic diseases, haemophiliacs can be treated successfully by intravenous substitution of coagulation factors.

Analysis of mRNA in hemophilia A patients with undetectable mutations reveals normal splicing in the factor VIII gene.

Background: haemophilia A (HA) is characterized by partial or total deficiency of factor VIII (FVIII) protein activity. It is caused by a broad spectrum of mutations in the FVIII gene. Despite tremendous improvements in mutation screening methods, in about 2% of HA patients no DNA change could be found, even after sequencing the whole coding part of the FVIII gene including the flanking splice sites, as well as the promotor and the 3' UTR regions.

Recombinant expression of mutations causing von Willebrand disease type Normandy: characterization of a combined defect of factor VIII binding and multimerization.

Von Willebrand disease type Normandy (VWD 2N) is caused by mutations at the factor VIII (FVIII) binding site of VWF, located at the amino-terminus of mature VWF. It is inherited in a recessive fashion and both homozygous and compound heterozygous mutations have been identified. Homozygous mutations are correlated with a clinical phenotype indistinguishable from mild hemophilia A by conventional laboratory tests, whereas compound heterozygosity with a quantitative defect may appear as VWD type 1 (VWD1).

Environmental and genetic factors influencing inhibitor development.

Inhibitor formation occurs at a frequency of 20% to 30% in severe hemophilia A, and 3% in hemophilia B. Today, it represents the major complication in patient care and renders classical substitution therapy ineffective. Genetic factors, such as factor VIII (FVIII) gene mutations and immune response genes, particularly the major histocompatibility complex, have been shown to constitute decisive risk factors for the development of inhibitors.

Regulation of human factor IX expression using doxycycline-inducible gene expression system.

Following substitution therapy with human factor IX (hFIX) concentrate, therapy of haemophilia B by viral gene transfer has become an attractive alternative therapy in recent years. However, high doses of expressed hFIX, which can already be achieved in animal studies, may cause thrombosis in humans (van Hylckama Vlieg et al., 2000).

A novel tetracycline-controlled transactivator-transrepressor system enables external control of oncolytic adenovirus replication.

The use of restricted replication-competent adenoviruses (RRCAs) inducing tumor cell-specific lysis is a promising approach in cancer gene therapy. However, the use of RRCAs in humans carries considerable risk, since after injection into the patient, further regulation or inhibition of virus replication from the outside is impossible. Therefore, we have developed a novel system allowing external pharmacological control of RRCA replication.

[Gathering and evaluation of phenotype data of haemophilia A patients for correlation with genotype data].

Haemophilia A is caused by a genetic defect of the factor VIII gene resulting in complete or considerable functional loss of factor VIII molecule within blood. The high bleeding risk of patients can be prevented by intravenous injections of factor VIII protein. However, 25% of patients affected with severe haemophilia, develop factor VIII antibodies against the concentrate substituted.

[The national GTH haemophilia registry as database within the scope of the German human genome project].

The Committee of Haemophilia of the GTH has established a central registry for all German centers treating patients with haemophilia. The intention was to establish a suitable system for collecting and analyzing epidemiological data relevant to bleeding disorders. The registry provides the database within the scope of the German Human-Genome-Project.

[Characterization of factor VIII antibody epitopes from haemophilia A patients using cellulose bound FVIII peptide libraries].

Approximately 30% of patients suffering from severe haemophilia A develop antibodies against factor VIII (FVIII) neutralizing the effect of the pro-coagulant activity of intravenously injected FVIII as a complication of replacement therapy. Generally, various epitopes on the FVIII molecule are bound by these antibodies. The detailed structure of such epitopes is unknown.

[Significance of mutation analysis in patients with haemophilia A].

Haemophilia A represents the most frequent hereditary bleeding disorder in humans. The disease is caused by mutations within the factor VIII gene leading to decreased or absent factor VIII activities with a bleeding tendency depending on the degree of factor VIII deficiency. Nowadays, the causative mutations can be routinely detected and have substantially improved diagnostic and understanding of the pathophysiology of haemophilia A.

[2% Haemophilia A patients without mutation in the FVIII gene].

In Germany, approximately 6,000 patients are suffering from haemophilia A. Screening methods cover 97% of the mutations. For the other patients the coding sequences of the FVIII gene have to be sequenced in total.

Quality management and quality assurance in haemophilia care: a model at the Bonn haemophilia centre.

The severe clotting defects associated with the diagnosis of severe haemophilia A and B require a quality management and quality assurance system designed to avoid both bleeding sequelae (such as damaged joints) through early on-demand or prophylactic treatment in a home-care setting, and side-effects such as infectious diseases (hepatitis A-G and human immunodeficiency virus), allergic reactions, haemolysis and if possible inhibitor formation, by using highly purified, virus-inactivated or recombinant products in which the factor VIII and IX proteins are as natural as possible. As the intravenous injection of the required clotting factor is entrusted to the patients in home treatment, the haemophilia centre has to check treatment protocols and, when necessary, joint and muscle status. In addition, it is imperative to ensure the safety of the product, and checks must be carried out to make sure that batch numbers are recalled as soon as possible if side-effects are observed.