Factor IX variants improve gene therapy efficacy for hemophilia B

Intramuscular injection of adeno-associated viral (AAV) vector to skeletal muscle of humans with hemophilia B is safe, but higher doses are required to achieve therapeutic factor IX (F.IX) levels. The efficacy of this approach is hampered by the retention of F.IX in muscle extracellular spaces and by the limiting capacity of muscle to synthesize fully active F.IX at high expression rates. To overcome these limitations, we constructed AAV vectors encoding F.IX variants for muscle- or liver-directed expression in hemophilia B mice. Circulating F.IX levels following intramuscular injection of AAV-F.IX-K5A/V10K, a variant with low-affinity to extracellular matrix, were 2-5 fold higher compared with wild-type (WT) F.IX, while the protein-specific activities remained similar. Expression of F.IX-R338A generated a protein with 2- or 6-fold higher specific activity than F.IX-WT following vector delivery to skeletal muscle or liver, respectively. F.IX-WT and variant forms provide effective hemostasis in vivo upon challenge by tail-clipping assay. Importantly, intramuscular injection of AAV-F.IX variants did not trigger antibody formation to F.IX in mice tolerant to F.IX-WT. These studies demonstrate that F.IX variants provide a promising strategy to improve the efficacy for a variety of gene-based therapies for hemophilia B.     

Factor IX gene mutations in haemophilia B: a New Zealand population-based study

Summary. Haemophilia B (Christmas disease) is an X-linked bleeding disorder resulting from an inherited deficiency of coagulation factor IX activity. Due to the heterogeneity of mutations within the factor IX gene there is a marked clinical variability in disease severity. By applying techniques of mutational analysis and direct sequencing of PCR products it is now potentially possible to determine the pathogenic gene defect in entire haemophilia B populations. We report here characterization of the factor IX gene defect in all the haemophilia B patients in New Zealand as part of a nationwide approach towards providing efficient and cost-effective haemophilia B genetic counselling services for these families. Twenty-six different mutations were identified in 32 unrelated haemophilia B families. Three defects at nucleotide positions +8,6659 and 17696 are novel mutations which have not been reported by other laboratories. A PCR-based diagnostic screening test for direct mutational analysis could be performed in most cases; 17 of the 26 mutations altered a restriction enzyme recognition sequence and, with the exception of the total gene deletion, base changes not affecting a restriction enzyme site could be detected by allele-specific PCR.     

Heteroduplex analysis in hemophilia B: Detection of two novel factor IX gene mutations

Heteroduplex analysis of polymerase chain reaction (PCR)-amplified factor IX (FIX) sequences in eight hemophilia B pedigrees localized the causative hemophilia mutation to a single exon in each case. Subsequent PCR-based direct DNA sequence analysis identified two novel FIX mutations and six recurrent mutations. Three of the eight pedigrees represent sporadic hemophilia B, and direct mutation analysis facilitated hemophilia carrier diagnosis in each case. © 1996 Wiley-Liss, Inc.     

Intragenic Factor IX restriction site polymorphism in hemophilia B variants

This study includes 47 normal subjects and 25 hemophilia B patients without inhibitor(s), showing different factor IX coagulant activity and antigen levels. Genomic DNA, digested with various restriction endonucleases, was hybridized with two different factor IX probes, ie, the cDNA and the subgenomic probe for the intragenic TaqI polymorphic site. cDNA restriction patterns suggest absence of gross rearrangements and/or deletions in all hemophilic patients. The frequency of the X chromosome bearing the TaqI polymorphic site is 0.32 +/- 0.09 in hemophilic subjects v 0.36 +/- 0.06 in normal control subjects, the latter value being comparable to that reported for the normal British population. No association between this polymorphism and hemophilia B variants has been observed, thus indicating that a wide spectrum of mutations underlies this blood-clotting disorder and particularly each of its variants.     

The putative factor IX gene promoter in hemophilia B Leyden

Hemophilia B Leyden is characterized by low levels of factor IX antigen and activity before the age of 15, whereas after puberty factor IX levels rise at a rate of about 5% per year. A single base substitution (-A----T) at position -20 was identified in the putative promoter of the gene cloned from a patient with hemophilia B Leyden. This nucleotide change was confirmed in a second patient from the same pedigree and was also found in a patient from a second Dutch pedigree with the same hemophilic phenotype. The results indicate that the two Dutch kindreds are related and point to the functional significance of the -20 position for the expression of the human factor IX gene.     

Factor VIII gene mutations profile in 148 Chinese hemophilia A subjects

Background: Hemophilia A (HA) is a common X‐linked recessive bleeding disease caused by mutations in FVIII gene. The identification of mutation in HA subjects can lead to more accurate diagnosis and contribute to the genetic counseling/prenatal diagnosis. Objectives: Our objective is to identify the FVIII defects in 148 unrelated Chinese HA subjects and to analyze the potential consequence of novel mutations. Methods: FVIII: C was assayed using one‐stage method, and FVIII inhibitor was tested using Bethesda method. Intron 22 and 1 inversions were identified by PCR technique. Non‐inversion mutations of FVIII gene were identified by direct sequencing. Novel mutations were further analyzed based on a B‐domain deleted FVIII crystallographic structure and bioinformatics tools. Results: The intron 22 and 1 inversions affected 57 and three severe subjects, respectively. Sixty‐seven different mutations were identified in non‐inversion subjects including 35 novel mutations that were not reported previously. Novel mutations include five nonsense mutations, 15 missense mutations, three insertions, eight small deletions, two splice site mutations and two partial gene deletions. The potential deleterious effects of these novel missense mutations include disruption of the protein core, impairment of inter‐domain interaction and FVIII binding with other proteins. Conclusion: Similar to other races, intron 22 and one inversions are also recurrent mutation in severe HA subjects monitored in our centre. Sixty‐seven mutations (52% novel reported) among 88 non‐inversion subjects represent the high degree of heterogeneity of FVIII gene mutations causing HA. Characteristic of HA FVIII gene mutations extend our insight into structure–function relationship of the FVIII molecule.     

Novel mutations in the Factor VII gene of Taiwanese Factor VII-deficient patients

The genetic defects of four Taiwanese patients with factor VII (FVII) deficiency were studied. FVII activity and antigen levels were < 1 u/dl and 125.7 u/dl (patient I), < 1 u/dl and < 1 u/dl (patient II), 3.4 u/dl and 5.9 u/dl (patient III), and 1.2 u/dl and 30.4 u/dl (patient IV) respectively. The 5' flanking region, and all exons and junctions were amplified using polymerase chain reaction and sequenced. Patient I was homozygous for a 10824C-->A transversion with Pro303-->Thr mutation in exon 8. In patient II, a heterozygous transversion, 9007+1G-->T at the IVS6, a heterozygous decanucleotide insertion polymorphism at -323 (both mutations present in his father) and a heterozygous deletion, del TC (26-27) in exon 1A (originating from his mother) were identified. Patient III had a homozygous 10961T-->G transversion with His348-->Gln mutation in exon 8. Patient IV had a heterozygous 10902T-->G transversion with Cys329-->Gly mutation in exon 8 (transmitted to her second son) and a heterozygous decanucleotide insertion polymorphism at -323 (transmitted to her third son). All but one of the FVII gene mutations detected in the four patients have not been previously reported. In conclusion, four novel mutations of the FVII gene in Taiwanese, including two missense mutations in exon 8, one point mutation at the exon 6 splice site and one deletion in exon 1A, were identified.     

Factor IX inhibitor in hemophilia B presented with anaphylactoid symptoms: report of 3 cases

Three patient with hemophilia B who developed anti-factor IX antibodies were reported. All 3 had allergic and/or anaphylactoid symptoms when the antibodies were found. The antibodies were noted between 4 and 17 days after exposure to factor IX. It was suggested that the anaphylactoid symptoms were closely related to the occurrence of anti-factor IX antibodies.     

Use of a BamHI polymorphism in the factor IX gene for the determination of hemophilia B carrier status

A BamHI polymorphism has been identified in the human factor IX gene. This polymorphism, which occurs in approximately 6% of X chromosomes, has been used to determine the carrier status of a female in a family with a history of hemophilia B. This family was uninformative for the previously reported TaqI and Xmnl polymorphisms in the factor IX gene.     

Molecular analysis of factor IX gene in an Iranian female with severe hemophilia B

Hemophilia B, a recessive X-linked coagulopathy, is rare in females, and only a few cases have been reported so far. In this report, we describe a 9-year-old female, offspring of a consanguineous marriage, with a clinically severe course of hemophilia B and a normal 46,XX karyotype. Polymerase chain reaction and conformation sensitive gel electrophoresis techniques have been applied to the important regions of the factor IX gene,and an abnormal conformation sensitive gel electrophoresis profile was identified in exon 5 of the gene. After sequencing, the mutation was found to be C17761T (R116X) in homozygous form. Then, polymerase chain reaction-restriction fragment length polymorphism using the EcoRV restriction enzyme was applied for confirmation of the homozygous mutation in the proband and for carrier testing in the relatives. In addition, haplotype analysis was informative at the HhaI polymorphic site for the female patient.     

AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B

Hemophilia B is an X-linked coagulopathy caused by absence of functional coagulation factor IX (F.IX). Previously, we established an experimental basis for gene transfer as a method of treating the disease in mice and hemophilic dogs through intramuscular injection of a recombinant adeno-associated viral (rAAV) vector expressing F.IX. In this study we investigated the safety of this approach in patients with hemophilia B. In an open-label dose-escalation study, adult men with severe hemophilia B (F.IX < 1%) due to a missense mutation were injected at multiple intramuscular sites with an rAAV vector. At doses ranging from 2 x 10(11) vector genomes (vg)/kg to 1.8 x 10(12) vg/kg, there was no evidence of local or systemic toxicity up to 40 months after injection. Muscle biopsies of injection sites performed 2 to 10 months after vector administration confirmed gene transfer as evidenced by Southern blot and transgene expression as evidenced by immunohistochemical staining. Pre-existing high-titer antibodies to AAV did not prevent gene transfer or expression. Despite strong evidence for gene transfer and expression, circulating levels of F.IX were in all cases less than 2% and most were less than 1%. Although more extensive transduction of muscle fibers will be required to develop a therapy that reliably raises circulating levels to more than 1% in all subjects, these results of the first parenteral administration of rAAV demonstrate that administration of AAV vector by the intramuscular route is safe at the doses tested and effects gene transfer and expression in humans in a manner similar to that seen in animals.     

A codon 338 nonsense mutation in the factor IX gene in unrelated hemophilia B patients: factor IX338 New York

Hemophilia B is an X-linked recessive bleeding disorder resulting from a deficiency of the coagulation factor IX (FIX) protein activity, a vitamin K-dependent serine protease active in both the intrinsic and extrinsic coagulation systems. DNA analyses of the factor IX gene in two unrelated patients with severe hemophilia B, with a IX coagulant activity less than 1% and undetectable FIX antigen, detected the loss of the second TaqI site in exon h (VIII) in both individuals. Polymerase chain reaction (PCR) amplification of 576 base pairs of exon h (VIII) with cloning and dideoxy sequencing of cloned DNA from one hemophiliac revealed a single C----T transition in codon 338 that changes an arginine residue codon CGA to a nonsense codon TGA. Allele- specific oligonucleotide probe hybridization with a mutant (C----T) and a wild-type allele confirmed the same mutation in amplified genomic DNA of the second hemophilia patient. The C----T transition represents another example of mutation at a CpG dinucleotide. DNA polymorphism analysis of the FIX gene in both individuals revealed each to be on a separate FIX haplotype; therefore, predicting each to be a separate mutation event.