A domain mutations in 65 haemophilia A families and molecular modelling of dysfunctional factor VIII proteins

A variety of mutations are found in haemophilia A families. Those with circulating, dysfunctional protein can provide insights into structural determinants of factor VIII function. A molecular model based upon the crystal structure of the homologous A domains in caeruloplasmin enables predictions of molecular consequences of mutations. To identify haemophilic mutations in coding regions for three A domains of factor VIII and predict amino acid substitutions important for coagulant cofactor function, amplified DNA fragments from 188 unrelated haemophilia A families were screened for heteroduplex formation. Exons 1–19 were examined. 65 families were positive for 58 distinct mutations (39 novel) on DNA sequencing. 12 were non-missense mutations. 38 missense mutations were found in patients that circulate or potentially circulate dysfunctional factor VIII protein and are in an A domain molecular model. Of these 38, 12 have identical residues among all known species of factors V, VIII and caeruloplasmin. These 38 mutations have been localized onto a factor VIII A domain molecular model. Of these, 19 are in coiled, 15 in β-pleated sheet, and two each in turns and α-helical structures. 15 substituted residues are on the surface, nine are partially on the surface and 14 are buried within the model structure. Mutant side-chain substitutions were inserted to predict changes in surface groups or, for buried residues, potential surface areas whose structure is probably disrupted by the substitution.     

Prenatal diagnosis in haemophilia A: experience of the genetic diagnostic laboratory

The paper describes the experience of the Genetic Diagnostic Laboratory in prenatal testing for haemophilia A, an X‐linked recessive disease caused by mutations in the F8 gene. Knowledge of a familial mutation prior to pregnancy can benefit prenatal diagnosis and decrease wait time for molecular testing during pregnancy. This is a retrospective review of a series of pregnant women who pursued F8 gene testing from December 1997 through May 2012, highlighting three cases, which demonstrate the technical complexities of analysis and the implications of not knowing carrier status prior to pregnancy. Mutations of the F8 gene were detected in affected males, obligate female carriers and suspected female carriers by DNA sequencing, inverse‐PCR, qRT‐PCR, Southern blot and exonic dosage analysis. The same methods were used to analyse prenatal samples from obligate or suspected female carriers upon request. Maternal cell contamination studies were performed for all prenatal samples analysed. Ninety‐nine women pursued F8 testing during pregnancy, either for carrier status alone or carrier status and prenatal diagnosis. Ninety‐one women (91%) requested carrier testing because they did not know their F8 mutation carrier status prior to pregnancy. Eight women requested prenatal diagnosis only, and only 4 of these were aware of their mutation status. Thirty‐seven individuals were found to be mutation carriers. Forty‐two prenatal samples were received for prenatal diagnosis. In total 21 foetuses were identified as mutation carriers. Mutation detection was complex and increased the turnaround time in some cases. Only four of 99 women who submitted samples for F8 testing were aware of their F8 mutation status prior to pregnancy. Knowledge of F8 mutation status prior to pregnancy allows for efficient prenatal diagnosis, when desired. Thus, preconception genetic counselling is required to inform patients of the available options and the complex and time‐consuming nature of F8 testing.     

Some factor VIII exon 14 frameshift mutations cause moderately severe haemophilia A

Factor VIII's exon 14 codes for its B domain that includes nearly one-third of its amino acid sequence that is not necessary for function. Frameshift mutations appear to occur more frequently within exon 14 than in other exons. To assess exon 14 frameshift mutations and their clinical correlates, a series of unrelated, severe or moderately severe haemophilia A patients were screened for heteroduplex formation in amplified exon 14 fragments. In 25 families, a frameshift mutation was identified by sequencing. Occurrence of haemophilia was isolated in 18 of these families. Moderate severity was noted in at least six out of 13 families with an A insertion or deletion at one of two sequences where the frameshift resulted in a sequence of 8-10 As. Inhibitors occurred in five of the other 12 families including one with an A insertion within a sequence of six As. Recurrent insertions into an A(8) (codons 1439-1441) or an A(9) (codons 1191-1194) sequence or of an A deletion from the A(9) sequence are common, recurrent causes of haemophilia A that may have a moderately severe phenotype.     

Study of mutations in Jordanian patients with haemophilia A: identification of five novel mutations

Summary.  Haemophilia A (HA) is an X-linked recessive bleeding disorder caused by mutations in the factor VIII gene ( F8 ), which encodes factor VIII (FVIII) protein, a plasma glycoprotein, that plays an important role in the blood coagulation cascade. In the present study, our aim was to identify F8 gene mutations in HA patients from Jordan. One hundred and seventy-five HA patients from 42 unrelated families were included in this study. Among these patients, 117 (67%) had severe HA, 13 (7%) had moderate HA and 45 (26%) had mild HA. Severe patients were first tested for intron-22 inversion using long range polymerase chain reaction (PCR), then negative patients were tested for intron-1 inversion using PCR. Sequencing for the entire F8 gene was performed for all severe HA patients who were found negative for intron-22 and -1 inversions and it was also performed for moderate and mild HA patients. HA causative mutations were identified in all patients. Intron-22 and -1 inversions were detected in 52% and 2% of families respectively. Beside these two mutations, 19 different mutations were identified, which include 15 missense and four frameshift mutations. Five novel mutations were identified including one frameshift and four missense mutations. No large deletions or nonsense mutations were detected in patients who participated in this study. Only 17 patients with severe HA were found positive for FVIII inhibitors. The data presented will play an important role for genetic counselling and health care of HA patients in Jordan.     

Identification of factor VIII gene mutations in patients with severe haemophilia A in Venezuela: identification of seven novel mutations

Summary. Haemophilia A is caused by mutations in the gene encoding coagulation factor VIII (FVIII). In severe Haemophilia A (sHA), two inversions are responsible for approximately 50% of the genetic alterations (intron 22 and intron 1 inversions). The other mutations are extremely diverse and each affected family generally has its own mutation. Our aim was to detect the genetic alterations present in the FVIII gene (F8) in 54 unrelated male patients with sHA in Venezuela. We initially detected the presence of the intron 22 inversion by performing inverse PCR, and the negative patients for this inversion were analysed for the intron 1 inversion by PCR. Patients negative for both inversions were analysed using Conformation Sensitive Gel Electrophoresis for mutations in all exons, promoter region and 3′‐UTR. sHA causative mutations were identified in 49 patients. Intron‐22 and ‐1 inversions were detected in 41% and 0% of patients respectively. Besides these two mutations, 25 different mutations were identified, including nine nonsense, four small deletions, two small insertions, four missense, three splicing mutations and three large deletions. Seven novel mutations were identified, including two nonsense mutations, two small deletions, one small insertion, one missense mutation and one splicing mutation. Thirty one percent of the patients with identified mutations developed inhibitors against exogenous FVIII. This is the first report of F8 mutations in patients with sHA in Venezuela; the data from this study suggests that the spectrum of gene defects found in these patients is as heterogeneous as reported previously for other populations.     

A rapid multifluorescent polymerase chain reaction for genetic counselling in Chinese haemophilia A families

Linkage analysis is a widely used strategy for genetic counselling in haemophilia A (HA) families. We attempted to develop more informative markers closely linked to factor VIII (FVIII) gene and establish a rapid multifluorescent polymerase chain reaction (PCR) method with these markers. Five extragenic (DXS15, DXS9901, G6PD, DXS1073 and DXS1108) and one intragenic (F8Civs13) markers were examined in 118 healthy individuals and 12 HA families which had been diagnosed before. Five extragenic markers were within an interval of about 1.5 Mb to FVIII gene and located on each side of the gene. The expected heterozygote rate (HR) of DXS15, DXS9901, G6PD, DXS1073, DXS1108 and F8Civs13 were 74.97%, 79.77%, 56.06%, 59.92%, 39.97% and 47.61%, while the observed HR were 88.24%, 82.35%, 21.57%, 62.75%, 35.29% and 52.94%. When six polymorphic markers were combined together, all the studied females were informative in at least one of these markers and 29.41% of them were detected informative in three markers with the highest frequency. The diagnostic rates of DXS15, DXS9901, G6PD, DXS1073, DXS1108 and F8Civs13 in 12 haemophilia families were 75.00%, 91.67%, 41.67%, 75.00%, 33.33% and 66.67% respectively. All the genetic diagnosis was consistent with the result we analysed before and no recombination was observed. Family 1 was given as an example in this study and was found to be informative in three polymorphic markers DXS15, DXS9901 and DXS1073. The patient's sister was detected the same allele as the proband, but her male fetus did not inherit the affected allele from her, which was consistent with the result of sequencing. It was demonstrated that the multifluorescent PCR method established in this study was convenient and efficient and can be applied to carrier detection and prenatal diagnosis in HA families.     

Inversions of the factor VIII gene in Swedish patients with severe haemophilia A

Abstract: The series comprised 49 Swedish patients with severe haemophilia A [belonging to 49 families (21 with known and 28 with sporadic haemophilia)], of whom 12 had developed F. VIII inhibitors. Using Southern blotting, 45% (22/49) were found to have inversions, i.e., intrachromosomal rearrangements of the tip of the X-chromosome. Twenty patients had one or the other of the two variants of inversions recently published, whereas 2 patients manifested novel band patterns. Inversions were found in 50% of the families with sporadic haemophilia, and in 38% of those with known haemophilia. Fourteen families with sporadic haemophilia A had inversions, the proband carrying the de novo mutation in 4 cases and the proband's mother in 10 cases. Six inversions derived from a male and five from a female X-chromosome meiosis, the origin of the remaining three was not established. Genetic counselling of patients with severe haemophilia A and their families will be considerably improved, as inversions occur in half the severe cases and can be detected by a simple Southern blotting procedure.     

Mechanisms of action of immune tolerance induction against factor VIII in patients with congenital haemophilia A and factor VIII inhibitors

In its most severe form, haemophilia A is a life-threatening haemorrhagic bleeding disorder that is caused by mutations in the factor VIII (FVIII) gene. About 25% of patients who receive replacement therapy with intravenous FVIII products develop neutralising antibodies (FVIII inhibitors) that inhibit the function of substituted FVIII. Long-term application of high or low doses of FVIII has evolved as an effective strategy for eradicating antibodies and inducing long-lasting immune tolerance. Despite clinical experience with the therapy, little is known about the immunological mechanisms that cause the down modulation of FVIII-specific immune responses or the induction of long-lasting immune tolerance against FVIII. This review summarises current knowledge of the immunological mechanisms that might be involved in the induction of immune tolerance against FVIII in patients with haemophilia A who have FVIII inhibitors. In addition to data from patients with haemophilia A, data from patients who have had organ transplants or have immune-related disorders, such as autoimmune diseases, are considered as well as data from animal models.     

Spectrum of mutations in Albanian patients with haemophilia A: identification of ten novel mutations in the factor VIII gene

Genetic analysis was carried out in 37 Albanian patients with haemophilia A. The factor VIII intron 22 inversion was detected only in 2/19 (10.5%) apparently unrelated patients with severe haemophilia A, while the intron 1 inversion was absent. A total of 19 different gene mutations were identified. Ten mutations were novel: four null mutations in severe haemophilia A patients (Gln1090X, Cys1832X, 2374delT, 5676insT) and six missense mutations (five in severe haemophilia A) (Ile76Thr, Leu299Pro, Asp525Glu, Cys692Tyr, His1755Leu and Trp1835Cys). None of these novel mutations occurred at CpG hotspots. These results further emphasize the extreme heterogeneity of the molecular basis of haemophilia A. The low prevalence of intron 22 inversion in Albanian patients with severe haemophilia A should be addressed by further studies.     

Hydrolysis of factor VIII mediated by catalytic antibodies occurs in haemophilia A patients with or without factor VIII inhibitors

The major complication of the substitutive treatment of haemophilia A (HA) is the development of antifactor VIII (FVIII) antibodies. Most of these antibodies neutralize FVIII procoagulant activity, and are identified as FVIII inhibitor. A subgroup of these antibodies, ‘catalytic antibodies’, catalyses the FVIII hydrolysis. We investigated the frequency and the activity of catalytic antibodies, according to the phenotype of HA and the presence or absence of FVIII inhibitor. IgG from 16 patients with inhibitor and 17 patients without inhibitor were purified. Rates of FVIII hydrolysis and inhibitor titres were evaluated. Anti‐FVIII catalytic antibodies were detected in 63.6% of patients with HA, irrespective of the HA phenotype and the presence of FVIII inhibitor. The frequency was significantly higher for severe HA patients (73.3%) and patients with inhibitor (87.5%), but their FVIII‐proteolytic activity was not significantly different from patients with mild or moderate HA and patients without inhibitor. The evolution of both catalytic and inhibitory activities was studied for 11 patients with FVIII inhibitor. We observed two profiles. In the profile 1, 18.2% of patients, the catalytic activity and the inhibitor titre coevolved. In contrast, a dissociated evolution of these two parameters was observed in 72.8% patients in profile 2. These data confirm the importance of anti‐FVIII catalytic activity in patients with severe, moderate and mild HA. Interestingly, most of the patients presented a dissociated profile, suggesting that anti‐FVIII antibodies might not systematically act as FVIII inhibitors.     

Inhibitor development in haemophilia A: the role of von Willebrand factor/factor VIII concentrates

Summary.  The presence of inhibitors that neutralize the function of factor VIII (FVIII) decreases the haemostatic efficacy of replacement clotting factor concentrate and increases morbidity among patients with haemophilia A. Certain genetic and environmental variables have been linked to a higher incidence of inhibitors. Conversely, the presence of von Willebrand factor (VWF) in some plasma-derived FVIII products may provide some measure of protection against inhibitor development, although the evidence is not conclusive. Clinical trials are needed to resolve this issue and determine the appropriate role of VWF-containing FVIII concentrates in the treatment of haemophilia A patients.     

Persistent factor VIII inhibitors and orthopaedic complications in children with severe haemophilia A

Summary. Persistence of inhibitors against factor VIII (FVIII) may be a risk factor that increases physical disability in haemophilia A (HA) patients. This study aimed to evaluate prevalence of FVIII inhibitors in previously treated children with severe HA and the impact of persistent inhibitors on knee joint status and lumbar bone mineral density (BMD). Fifty children with severe HA, FVIII <1%; aged 5–16 years were enrolled in this study; they received plasma‐derived FVIII on‐demand treatment for 50–250 exposure days (EDs). Inhibitors were checked at basal visit and were followed up for 1 year, using Bethesda assay. Cross‐sectional clinical scoring and radiological evaluation of the knee joint (by Arnold‐Hilgartner staging and Pettersson score), along with lumbar BMD by Dual Energy X‐ray Absorptiometry (DEXA) were performed. Patients with persistent inhibitors for 1 to 5 years, median 2.5 years, were 10 (20%). Six had high titre and none of them had completely normal knees, seven had advanced knee arthropathy and six had low lumbar BMD in comparison to 2 and 8 of the 40 patients without inhibitors respectively (P < 0.05). Persistence of inhibitors for more than 2 years without immuno‐prophylaxis was a risk factor for joint damage. Low lumbar BMD was found in 88.9% of patients with stages four and five knee arthropathy and in 66.7% of patients with positive hepatitis C. Severe HA children in this Egyptian study had a relatively low prevalence of persistent FVIII inhibitors, which, if not treated, may increase the risk of knee arthropathy and lumbar osteopenia.     

Thirteen novel mutations in the factor VIII gene in the Nijmegen haemophilia A patient population

The development of neutralising antibodies to factor VIII (FVIII) is a major complication of haemophilia A (HA) therapy. We aimed to construct an individual risk profile for the development of inhibitors in HA and started by screening for the causative mutation in our HA patient population. A total of 109 patients and 28 carriers were screened. The analysis revealed 38 different mutations in the FVIII gene, of which 13 have not been described on the Haemophilia A Mutation, Search, Test and Resource Site (HAMSTeRS). Twenty-five mutations have been reported previously and all except two had a similar phenotype to what has been described. Three novel mutations were associated with severe HA: one non-missense mutation, a small insertion in the A2 domain, and two missense mutations, a H256R mutation in the A1 domain and a L2025P substitution in the C1 domain. One novel mutation, Y156C, was associated with moderate HA. Nine novel mutations caused mild HA. The P130R, D167E and V278M mutations are located in the A1 domain. R439C, Y511H, A544G and Q645H in the A2 domain, L1758F in the A3 domain and a S2157R mutation in the C1 domain. In conclusion, the genotypic profile of our HA population was not different from others described and is suitable to study inhibitor formation.     

Intracellular accumulation of factor VIII induced by missense mutations Arg593-->Cys and Asn618-->Ser explains cross-reacting material-reduced haemophilia A

Patients with cross-reacting material (CRM)-reduced haemophilia A exhibit reduced levels of factor VIII antigen. In this study, we determined the molecular basis of the genetic defect in the factor VIII gene induced by either the Arg593-->Cys or the Asn618-->Ser missense mutation, identified in two CRM-reduced haemophilia A patients. We introduced either the Arg593-->Cys or the Asn618-->Ser mutation into a B-domain-deleted factor VIII cDNA and expressed the modified cDNAs in C127 cells. Reduced levels of factor VIII activity and factor VIII antigen in conditioned medium of transfected cells indicated that the secretion of both factor VIII variants was impaired. The ratio of factor VIII antigen present in cell extract to that in conditioned medium was 1.9 and 2.4 times higher for rFVIII-R593C and rFVIII-N618S, respectively, than for rFVIII. Metabolic labelling and immunoprecipitation of transfected cells revealed that rFVIII-R593C and rFVIII-N618S persisted somewhat longer inside the cell than factor rFVIII. Intracellular accumulation and subsequent degradation of factor VIII-R593C and factor VIII-N618S may explain the reduced levels of both factor VIII activity and antigen in plasma of mild haemophilia A patients with corresponding genetic defects.     

Immune regulatory gene polymorphisms as predisposing risk factors for the development of factor VIII inhibitors in Indian severe haemophilia A patients

Development of inhibitors to factor VIII, a serious complication of replacement therapy in haemophilia A patients, leads to increased bleeding, morbidity and mortality. There is no data on the risk factors for inhibitor development in Indian patients with severe haemophilia A. Our aim was to study the role of immune regulatory gene polymorphisms in the development of inhibitors. Fourteen immune regulatory gene polymorphisms (IL1β, IL4, IL10, TNFA and CTLA4) were analysed in 120 patients with severe haemophilia A, i.e. 50 inhibitor positive patients, and 70 inhibitor negative control patients, by PCR-RFLP, DNA sequencing and allele-specific PCRs. The IL10 promoter 'GCC' haplotypes overall (P: 0.002, OR: 3.452, 95% CI: 1.607-7.416), and 'GCC/ATA' (P: 0.011, OR: 3.492, 95% CI: 1.402-8.696) haplotype, associated with high and intermediate IL10 production, respectively, were significantly higher in inhibitor positive patients, whereas the 'non-GCC' haplotypes overall (P: 0.002,OR: 0.290, 95% CI 0.135-0.622) and 'ATA/ATA' haplotype (P: 0.025, OR: 0.278, 95% CI: 0.096-0.802), associated with low IL10 synthesis, were significantly higher among inhibitor negative patients. The TNFA rs1799724 C/T heterozygote prevalence was significantly higher in the inhibitor positive group (P: 0.021, OR: 3.190, 95% CI: 1.273-7.990), whereas the other polymorphisms showed no statistically significant association with the presence of inhibitors. Different immune regulatory gene polymorphisms play a significant role as possible risk factors for the development of inhibitors in severe haemophilia A patients.     

Low-dose continuous infusion of factor VIII in patients with haemophilia A

BackgroundPatients with haemophilia A (HA) or B (HB) can be given prophylactic or on-demand treatment administered by continuous infusion or bolus injections of factor VIII (FVIII) or IX (FIX). In this study we evaluated the efficacy and safety of low-dose continuous infusion of FVIII or FIX.ResultsA total of 66 continuous infusions (40 in major surgery, 10 in minor surgery and 16 with bleeding episode) in 46 HA patients and 16 (15 in severe and 1 in mild HA) in eight HB patients were included in the study. During the first week of treatment, the median continuous infusion rates in HA patients undergoing major surgery, minor surgery and a bleeding event were 2.18 (0.75–3.68), 1.48 (1.0–2.54) and 2.24 (1.33–3.93) IU/kg/h, respectively. The median FVIII activities were 0.69 (0.37–1.19), 0.47 (0.39–0.84) and 0.52 (0.36–1.06) IU/mL. After the first week of treatment, even lower doses of FVIII were needed. Red blood cell transfusions had to be administered to three patients (2 with severe and 1 with moderate HA) during the continuous infusion and inhibitors developed in five patients. In HB patients, the median continuous infusion rate was 1.85 (1.07–2.94) IU/kg/h and the median FIX activity was 0.62 (0.30–1.04) IU/mL. Red blood cell transfusions were not required, and thrombophlebitis and inhibitors did not appear.DiscussionOverall, low-dose continuous infusion was shown to be an effective and safe way of treating patients with HA. The protocol used also proved efficient and safe in all HB patients.     

Efficacy and inhibitor development in previously treated patients with haemophilia A switched to a B domain-deleted recombinant factor VIII

There have been recent reports of unexpected poor efficacy of a B-domain-deleted recombinant factor VIII (BDD-rFVIII) in haemophiliacs, and inhibitor development in previously treated patients (PTPs) switched to BDD-rFVIII. The results of a 6-month prospective study of 25 PTPs and of a retrospective survey of 94 PTPs, all switched to BDD-rFVIII, were used to evaluate efficacy and inhibitor development. The prospective study showed that 89% of 362 bleeds were controlled by one to two infusions, reproducing the efficacy profiles of other recombinant products (rFVIIIs). One patient, previously treated with plasma-derived FVIII only, developed a high titre inhibitor (30 BU) after 5 days of exposure. The retrospective survey, carried out in the total Italian PTP population switched to BDD-rFVIII, involved 19 PTPs at higher inhibitor risk due to previous exposure of < or = 50 days and 75 PTPs at lower inhibitor risk due to previous exposure of > 50 days. One patient developed an inhibitor: he was a high-risk, severe PTP previously exposed to another rFVIII for 3 days only. Among the entire low-risk population of severe Italian PTPs switched to BDD-rFVIII (25 in the prospective study, 49 in the retrospective cohort) only one developed an inhibitor (1.3%). These data indirectly support the views that BDD-rFVIII is equivalent to other rFVIIIs in term of efficacy and inhibitor development.     

Undetected factor VIII in a patient with type 3 von Willebrands disease mistaken as severe haemophilia A

Summary.  von Willebrand disease (VWD) type 3 is a rare disorder characterized by absent or <0.1 UmL⁻¹ of ristocetin cofactor (VWF:RCo), and a very low level of factor VIII (FVIII:C). A total absence of FVIII:C has never been reported in type 3 VWD. This case illustrates the effect of severe von Willebrand factor (VWF) deficiency on the factor VIII level.     

von Willebrand factor in factor VIII concentrates protects against neutralization by factor VIII antibodies of haemophilia A patients

We investigated the neutralization activity of factor VIII (FVIII) antibodies of 12 haemophilia A patients, acquired during treatment with plasma-derived FVIII concentrates. All plasma samples, drawn in a clinically stable situation before any immunotolerance treatment, contained anti-A2 domain and anti-light-chain FVIII antibodies. In nine patients' plasmas, containing relatively high amounts of FVIII light-chain antibodies (53-96%), a higher neutralization activity was found against recombinant FVIII concentrate (Recombinate) than against plasma-derived von Willebrand factor (vWF)-containing concentrate (Haemoctin SDH). No difference in neutralization of the two concentrates was found in two patients' plasmas with almost equal content of FVIII light- and heavy-chain antibodies, or one plasma with predominantly heavy-chain antibodies. These results suggest that haemophilia A patients with relatively high amounts of FVIII light-chain antibodies in plasma might benefit by infusion of FVIII concentrates containing vWF because vWF appears to have some protective effect on FVIII. This hypothesis should be tested by a clinical study.