Molecular characterization of hemophilia B in North Indian families: identification of novel and recurrent molecular events in the factor IX gene.
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BACKGROUND AND OBJECTIVES: Hemophilia B is an X-linked recessive, bleeding disorder caused by mutations in the factor IX gene. A wide range of mutations, showing large molecular heterogeneity, has been described in hemophilia B patients. Our study was aimed at characterizing mutations in the factor IX gene in a cohort of North Indian hemophilia B patients. DESIGN AND METHODS: Polymerase chain reaction (PCR) amplification and direct sequencing of all regions of likely functional significance- the coding regions, promoter, the 5' UTR, the splice junctions and parts of the 3' UTR of the factor IX gene was done in 18 families carrying a severe form of hemophilia B. RESULTS: We identified 10 point mutations (including 2 novel ones); one novel deletion and one donor splice site mutation. Recurrence of a nonsense and a missense mutation was observed. The mutation in 3 families could not be characterized. None of the 14 polymorphic positions reported in the Haemophilia B Mutation database in the regions sequenced were polymorphic; herein we report four novel synonymous single base mismatches. One mutation reported to be causative in the database was found to be more likely a non-causal polymorphism. INTERPRETATION AND CONCLUSIONS: Our data confirm the remarkable heterogeneity of the mutational spectrum in hemophilia B among affected families. This is the first mutation report on the disease in the Indo-Aryan population from the Indian subcontinent. Identification of a causative mutation leads to more precise carrier detection than does conventional polymorphism-based linkage analysis. This can effectively be used to establish genotype/ phenotype relationships. (+info)
Successful treatment of severe bleeding in hemophilic target joints by selective angiographic embolization.
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Bleeding into the joints is common in patients with hemophilia. After total knee or elbow replacement, profuse intraarticular bleeding unresponsive to high-dose clotting factor replacement sometimes occurs. In some patients who have severely damaged elbow or knee joints the same profuse bleeding pattern can be seen. To control bleeding in these patients, selective catheterization with a microcatheter and therapeutic embolization with microcoils was performed whenever a severe blush or microaneurysm was observed on angiography. Over 12 years, in 23 cases of massive joint bleeding in 18 patients with hemophilia selective catheterization was performed. In 15 cases the bleeding was postoperative and in 8 spontaneous. Results of angiographic imaging revealed vascular blush, false aneurysm, true aneurysm, and arteriovenous shunt in combination with an aneurysm as cause of bleeding. In 2 patients, the cause of bleeding was not found. In 21 cases an embolization procedure was performed, in which the bleeding was completely controlled by a single procedure in 14 cases. Recurrence of the bleeding occurred in 7 cases and required a second embolization procedure; in one patient even a third embolization was required to stop the bleeding completely. No difference in the outcome, that is, clinical end of bleeding and joint range of motion, was observed, when comparing postoperative and spontaneous bleeding. (+info)
Sustained correction of disease in naive and AAV2-pretreated hemophilia B dogs: AAV2/8-mediated, liver-directed gene therapy.
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Adeno-associated virus 8 (AAV8), a new member of the AAV family isolated from nonhuman primates, is an attractive candidate for hepatic gene transfer applications because of 10- to 100-fold improved transduction efficiency in mouse liver models. Additionally, AAV8 has lesser frequency of pre-existing immunity in humans. These properties could solve some of the problems associated with AAV2 vectors. The benefits of AAV8 demonstrated in mouse models, however, have not been confirmed in larger animals. In this study, we evaluate the efficacy and safety of AAV2/8 vector in both naive and AAV2-pretreated hemophilia B dogs. Two naive hemophilia B dogs that received a single intraportal administration of AAV2/8 vector have achieved sustained expression of 10% and 26% of normal levels of canine factor IX (cFIX) for more than a year. In an AAV2-pretreated hemophilia B dog, cFIX expression increased from less than 1% to 16% of normal levels when treated with an AAV2/8 vector, and a high level of expression has lasted for more than 2 years. No significant liver toxicity or cFIX-specific antibodies have been detected in these animals. Studies here have demonstrated the safety and improved efficacy of AAV2/8 vector in large-animal models for liver-directed gene therapy. (+info)
Correction of factor IX deficiency in mice by embryonic stem cells differentiated in vitro.
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Murine embryonic stem (ES) cells are pluripotent, but significant functional engraftment does not occur when they are introduced into the liver. However, here we demonstrate that functional liver engraftment does occur if the ES cells (from strain 129 mice) are first differentiated in vitro for 7 days in the presence of FGF. Strikingly, when these differentiated cells, termed putative endodermal precursors (PEPs), were injected into their livers, two of six C57BL/6 and four of eight BALB/c factor IX (F-IX)-deficient mice survived for >7 days, even though the recipients were of a different strain and, in the case of the BALB/c recipients, had a complete MHC mismatch. F-IX was detected in all six of the PEP-injected survivors. Two mice subsequently died of causes unrelated to F-IX; the others survived until death at 38 or 115 days after the transplantation. No uninjected control F-IX-deficient mice survived for >7 days. Large confluent regions of sinusoidal PEP engraftment were demonstrated by immunofluorescence in the long-term BALB/c survivors. The PEP engraftment was not associated with detectable cell fusion, and the transplantation was accompanied with only a low incidence of teratoma formation. (+info)
Molecular genotyping of the Italian cohort of patients with hemophilia B.
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BACKGROUND AND OBJECTIVES: The aim of the study, funded by the Italian Ministry of Health, was to identify the causative mutation in all known patients with hemophilia B in Italy. DESIGN AND METHODS: Overall, 269 patients followed by 25 regional centers were considered in the study; after exclusion of the related individuals, 238 unrelated patients were analyzed (153 with severe, 59 with moderate and 26 with mild hemophilia B). Screening of the factor IX gene was performed using conformation sensitive gel electrophoresis (CSGE) followed by denaturing high performance liquid chromatography (dHPLC) or direct sequencing in negative cases, or by dHPLC/sequencing (36 cases). RESULTS: A mutation was identified in 236 of the 238 patients: 6 had large gene deletions (4 total and 2 partial), 14 small deletions, 1 combined deletion/insertion and 215 single nucleotide substitutions. A correlation was observed between the type of mutation and severity of hemophilia; however, a number of patients with the same genotype had varying severities of the disease. Eight of the 169 patients with severe hemophilia B (4.7%) developed inhibitors: 2 of these had a complete gene deletion, 1 had a large partial deletion (from exon A to part of exon H) while 5 had 3 different nonsense mutations. One patient with a nonsense mutation developed anaphylaxis. We also studied 65 families with hemophilia B involving 144 females (14 obligatory carriers, 85 carriers and 45 non-carriers) and performed 12 antenatal diagnoses. INTERPRETATION AND CONCLUSIONS: The data have been used to build the Italian mutation database to provide each family with knowledge of the disease-causing defect for genetic counseling. This Italian study confirms the marked heterogeneity of factor IX mutations in the population and the presence of a degree of genotype/phenotype discordance. The identification of the mutation can also be used to predict risk of inhibitor development. (+info)
Measurement of basal levels of factor VIIa in hemophilia A and B patients.
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Previous results, presented in abstract form, indicate that replacement of thromboplastin with a mixture of phospholipid and truncated soluble tissue factor apoprotein results in a coagulation assay that can directly measure plasma factor VIIa levels without interference from zymogen factor VII (Atherosclerosis Thromb 11:1544a, 1991 [abstr]). We have exploited the specificity and sensitivity of such a factor VIIa specific coagulation assay to directly assess the in vivo relationship of factor VIII and factor IX on the production of factor VIIa levels under nonthrombotic and nonstimulatory conditions. Normal individuals (n = 20) were found to possess an average circulating factor VIIa level corresponding to 4.34 +/- 1.57 ng/mL, or approximately 1% of their total factor VII antigen. Severe factor VIII deficient patients (n = 13) possessed a slightly lower but statistically significant (P less than .01) decrease in their basal factor VIIa levels (2.69 +/- 1.52 ng/mL), corresponding to approximately 60% of that observed in normal individuals. On the other hand, severe factor IX deficient patients (n = 7) were found to possess even lower levels of factor VIIa corresponding to 0.33 +/- 0.15 ng/mL, or less than 10% of that observed in normal individuals. Measurement of total factor VII antigen levels shows that the variation in basal factor VIIa levels stems from differences in the degree of factor VII activation as opposed to differences in factor VII antigen levels. Our present data are consistent with the hypothesis that factor IXa is the principal in vivo activator of factor VII under basal conditions. (+info)
Correlates of spontaneous clearance of hepatitis C virus among people with hemophilia.
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People with hemophilia were formerly at very high risk of infection with hepatitis C virus (HCV). Approximately 20% of HCV-infected patients spontaneously clear the virus. To identify correlates of spontaneous clearance of HCV, we studied a cohort of HCV-infected hemophilic subjects without human immunodeficiency virus infection who had never been treated with interferon. Plasma HCV RNA was persistently undetectable in 192 (27.0%) of 712 HCV-seropositive subjects. In multivariate analyses, HCV clearance was more likely in subjects infected with HCV at younger age, especially with infection before age 2 years (40.1%) compared with after age 15 years (14.9%, P(trend) < .0001), and with relatively recent infection, especially after 1983 (42.8%) compared with before 1969 (18.2%, P(trend) < .0001). HCV clearance was marginally reduced with African ancestry (19%) and greatly increased with chronic hepatitis B virus (HBV) infection (59.1%, P = .001). Resolved HBV infection, coagulopathy types and severity, types of clotting factor treatment, and sex were not associated with HCV clearance. In conclusion, hemophilic subjects coinfected with chronic HBV and those infected with HCV before age 2 years or after 1983 were significantly more likely to spontaneously clear HCV viremia. These data highlight and clarify the importance of nongenetic determinants in spontaneous recovery from HCV infection. (+info)
Human immunoglobulin inhibits liver transduction by AAV vectors at low AAV2 neutralizing titers in SCID mice.
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Long-term cures of hemophilia B have been achieved using AAV2 delivering the factor IX gene to the liver of adeno-associated virus (AAV)-naive hemophilic animals. However, the clinical success of this approach requires overcoming pre-existing AAV neutralizing antibodies prevalent in humans. To better define the inhibition of neutralizing antibodies on AAV2-mediated liver transduction, we developed an in vivo passive immunity model. SCID mice were first reconstituted to a defined neutralizing titer with pooled plasma-derived human immunoglobulin. AAV2-FIX vectors then were administered to the liver, and the transduction efficiency was measured by plasma FIX levels. Unexpectedly, AAV2 neutralizing titers lower than 1:10 were sufficient to neutralize 4 to 20 x 10(12) vg/kg of AAV2 vectors in vivo, a capacity that was underestimated by in vitro neutralizing assays. We also evaluated strategies to evade neutralization, including the use of alternative delivery routes, infusion parameters, empty capsids, and alternative AAV serotypes 6 and 8. The results indicate that low AAV2 neutralizing titers can be inhibitory to the tested human and primate AAV vectors delivered into the circulatory system. Therefore, novel nonprimate AAV vectors or compartmentalized delivery may offer more consistent therapeutic effects in the presence of pre-existing AAV neutralizing antibodies. (+info)