Rainer Schwaab

Haemophilia B: database of point mutations and short additions and deletions, 7th edition

The seventh edition of the haemophilia B database lists in easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of <30 bp) identified in haemophilia B patients. The 1535 patient entries are ordered by the nucleotide number of their mutation. Where known, details are given on: factor IX activity, factor IX antigen in circulation, presence of inhibitor and origin of mutation. References to published mutations are given and the laboratories generating the data are indicated.

Immune Tolerance for the Treatment of Factor VIII Inhibitors ‐ Twenty Years' ‘Bonn Protocol’

Around 20 years ago, a mother admitted her hemophilic son, 1 % years of age, knowing that he had an inhibitor, to our center by ambulance. He showed severe bleeding episodes into his right shoulder, right upper arm and right chest. The inhibitor titer at that time was :>500 Bethesda units (BU). Some months earlier, in 1974, we had heard about a publication by Kurczinsky and Penner [ I ] about the successful treatment of bleeding episodes in patients with an inhibitor with activated prothrombin complex concentrates. When calling a manufacturer (Immuno), asking for a similar product, we were told that this was still in development, and not available at the moment. In this serious situation, we decided to combine high dosages of factor VIII with a regular prothrombin concentrate. This regimen was given twice a day to the patient, and the bleeding could be controlled. After 3 weeks the patient recovered completely, and he could be dismissed from the hospital. Astonishingly, during this treatment the inhibitor titer decreased to slightly more than 40 BU. Therefore, it appeared to be reasonable to evaluate this procedure also in other hemophilic patients with an inhibitor suffering from acute bleeding episodes. We observed that in some patients there was a booster effect on the inhibitor, and in some not, but the inhibitor titer decreased in all cases after some weeks of treatment. We decided not to stop the treatment, but to continue this procedure, and realized that the inhibitor finally disappeared. Subsequently, also the half-life of factor VIIl normalized. However, it took around 2 years to develop the dosage schedule that we are using today. Methods

Inhibitor development in correlation to factor VIII genotypes

Summary.  Alloantibodies (inhibitors) against factor VIII (FVIII) develop in 20–30% of patients with severe haemophilia A and render classical FVIII substitution therapy ineffective. Several studies have shown that genetic factors, the type of FVIII gene mutation and immune response genes (e.g. the Major Histocompatibility Complexes), influence the risk of inhibitor formation. In particular, the type of FVIII gene mutation has proven to be a decisive risk factor. Patients with severe molecular gene defects (e.g. large deletions, nonsense mutations, intron‐22 inversion) and no endogenous FVIII synthesis have a 7–10 times higher inhibitor prevalence than patients with milder molecular gene defects (e.g. missense mutations, small deletions, splice site mutations). To date, at least 10 distinct classes of mutations have been shown which have differing risks of associated inhibitor formation. A challenging observation in inhibitor patients is the heterogeneity of the antibody epitopes with respect to their number and their specifity. At least five epitopes in the FVIII molecule have been identified that constitute the targets for antibodies in most inhibitor patients. These epitopes are located in the ar3 region and the A2, A3, C1, C2 domains which correspond to the functional binding sites of the ligands of the FVIII protein. At present, the determinants of the characteristics of these epitopes and the subsequent inhibitor titre are unknown. A relationship of the mutation site and the epitope localization has been shown for some individual patients with mild haemophilia A. However, in severely affected haemophilia A patients, the influence of patient genetics on inhibitor titre and epitope specifity has yet to be elucidated.

Haemophilia B: Database of point mutations and short additions and deletions—eighth edition

The eighth edition of the haemophilia B database (http://www.umds.ac. uk/molgen/haemBdatabase.htm ) lists in an easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of <30 bp) identified in haemophilia B patients. The 1713 patient entries are ordered by the nucleotide number of their mutation. Where known, details are given on: factor IX activity, factor IX antigen in circulation, presence of inhibitor and origin of mutation. References to published mutations are given and the laboratories generating the data are indicated.

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. Haemophilia is also the most attractive model for developing gene-therapy protocols, as the normal life expectancy of haemophiliacs allows the side effects of gene therapy, as well as its efficiency, to be monitored over long periods.

Induction of immune tolerance in haemophilia A inhibitor patients by the 'Bonn Protocol': predictive parameter for therapy duration and outcome.

The treatment of inhibitors is one of the most challenging fields in haemophilia care. The present study reports the results of 60 haemophilia A inhibitor patients treated according to the ‘Bonn Protocol’ and evaluates predictors for the duration and outcome of therapy. Successful immune tolerance could be achieved in 52 patients (86.7%) while the therapy failed in eight patients (13.3%). The immune tolerance achieved was longlasting in all 52 patients, with no inhibitor relapse in up to 20–years follow–up. The course of ITT was influenced by several factors. Interruptions of treatment during the ITT course led to a substantial prolongation of ITT duration (median 39.9 months vs 14.1 months in continuously treated patients). Infections of intravenous central lines appeared to be frequently coincided with ITT prolongation and sometimes even ITT failure. Further negative predictors towards the ITT duration were high inhibitor titres at enrolment or during ITT. There was also a tendency towards longer ITT duration in patients exhibiting the prevalent intron 22 inversion. As a consequence of our data treatment interruptions and infections of intravenous central lines should be avoided during the course of ITT. Furthermore our data suggest, that ITT should be started at low inhibitor titres preferably with a high factor VIII dosage protocol.

Missense mutations at ALA-10 in the factor IX propeptide: an insignificant variant in normal life but a decisive cause of bleeding during oral anticoagulant therapy.

Bleeding complications are the most common and unwanted side‐effect of oral anticoagulant therapy. We report three patients in whom mutations in the factor IX (FIX) propeptide were found to cause severe bleeding during coumarin therapy. Strikingly, the bleeding occurred within the therapeutic ranges of the prothrombin time (PT) and international normalized ratio (INR). In all three patients coumarin therapy caused an unusually selective decrease of FIX activity (FIX:C) to levels below 1–3%. Upon withdrawal of coumarin, FIX:C increased to subnormal or normal values of 55%, 85% and 125%, respectively. Analysis of the FIX gene revealed two different missense mutations affecting the Ala‐10 residue in the propeptide coding region: Ala[GCC] to Val[GTC] in two patients and Ala[GCC] to Thr[ACC] in one patient. No further mutation was detected by screening 195 random blood donors for mutations at Ala‐10, thus excluding a frequent polymorphism at this position. The mutation in the FIX propeptide at a position which is essential for the carboxylase recognition site causes a reduced affinity of the carboxylase enzyme to the propeptide. This effect leads to an impaired carboxylase epoxidase reaction which is decisively triggered by the vitamin K concentration. Determination of FIX and APTT in addition to PT and INR is therefore recommended in coumarin‐treated patients with an uncommon bleeding pattern.

Haemophilia A diagnosis by simultaneous analysis of two variable dinucleotide tandem repeats within the factor VIII gene

Summary. Haemophilia A is a bleeding disorder caused by defects in the gene coding for the co‐factor, factor VIII (FVIII). The few available intragenic restriction fragment length polymorphisms (RFLPs) currently used in carrier detection and prenatal diagnosis of haemophilia A are informative in only about 65% of cases. We earlier reported a multi‐allelic dinucleotide tandem repeat, (CA)n, specific to intron 13, which remains the single most informative marker within the FVIII gene. We here report a second informative dinucleotide repeat of the form (GT)n (AG)n, located to intron 22 of the FVIII gene. The polymerase chain reaction (PCR) method was used to examine the variability of the repeat in 60 individuals (75 X‐chromosomes) and revealed four alleles. The calculated heterozygosity rate is 45%, and family studies showed X‐linked mendelian inheritance. The intron 22 dinucleotide repeat is tightly linked with established RFLPs and tracks with haemophilia A in family studies. We now show that by simultaneous amplification of the intron 13 and 22 repeats using PCR all alleles for both markers are detectable on a single polyacrylamide gel. The information thus obtained from a single multiplexed analysis is greater than from multiple RFLP analyses. Hence, rapid haplotype determination by simultaneous amplification and detection of two intragenic dinucleotide repeats should supersede less informative RFLP analysis.

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. Our results show that no significant association with age was observed. However, the previously reported increase of LINE-1 methylation in males was reconfirmed. In females, although no correlation between LINE-1 or Alu methylation and hormone levels was observed, a significant stable individual specific level of methylation was noted. In vitro results largely confirmed these findings, as neither estrogen nor dihydrotestosterone affected LINE-1 or Alu methylation in Hek293T, HUVEC, or MDA-kb2 cell lines. In contrast, a decrease in methylation was observed in estrogen-treated T47-Kbluc cell lines strongly expressing estrogen receptor. The very low expression of estrogen receptor in blood cells could explain the observed insensitivity of methylation at LINE-1 to natural hormonal variations in females. In conclusion, neither natural cycle of hormones nor age has a detectable effect on the LINE-1 methylation in peripheral blood cells, while gender remains an important factor.

Characterization of mutations within the factor VIII gene of 73 unrelated mild and moderate haemophiliacs

Summary. To Screen for mutations within the factor VIII gene of 101 patients (85 unrelated), we used denaturing gradient gel electrophoresis (DGGE) after DNA amplification of target regions, including all coding regions except for the middle part (amino acid 757 to amino acid 1649) of the B domain. With this method, missense mutations were identified in 86% of unrelated patients. 41 different mutations were identified; 25 of them have not been described previously. Five of the genotypes are associated with CRM+ and 26 with CRMred status. Patients who are definitely related to each other showed no differences in DNA sequence. One patient showed two different base pair alterations, the first at amino acid 469 [ala(GCA→gly(GGA)] and the second at position 473 [tyr(TAT)→cys(TGT)]. One patient with an amino acid change at position 1689 [arg(CGC)→his(CAC)] has developed an inhibitor against factor VIII.