scholarly journals Partial factor IX protein in a pedigree with hemophilia B due to a partial gene deletion.

1986 ◽  
Vol 77 (4) ◽  
pp. 1194-1200 ◽  
Author(s):  
G L Bray ◽  
A R Thompson
Keyword(s):  
Gene Deletion ◽  
Factor Ix ◽  
Hemophilia B ◽  
Partial Factor ◽  
Partial Gene Deletion

Genetic Basis and Carrier Detection of Hemophilia B of Chinese Origin

1993 ◽  
Vol 69 (03) ◽  
pp. 247-252 ◽  
Author(s):  
Shu-Wha Lin ◽  
Ming-Ching Shen
Keyword(s):  
Amino Acid ◽  
Gene Deletion ◽  
Direct Sequencing ◽  
Screening Method ◽  
Point Mutations ◽  
Factor Ix ◽  
Hemophilia B ◽  
Sscp Analysis ◽  
Amino Acid Residues ◽  
Chinese Origin

SummaryWe have characterized the genetic defects of 17 hemophilia B patients of Chinese origin by means of the polymerase chain reaction (PCR) and direct sequencing. The single-strand conformation polymorphism (SSCP) was used as an initial screening method to analyze the entire coding region and the flanking introns of each individual’s factor IX gene. The abnormal exons were subsequently amplified and the nucleotide sequence determined. Of the 17 patients studied, 16 had single point mutations and one had a gross gene deletion of exons VII and VIII of factor IX. Among these 16 factor IX variants with point mutations 13 were missense and two were nonsense mutations. The remaining one had a nucleotide deleted, resulting in frame shifting at amino acid residue 97. A total of ten novel mutations, including the one with gross gene deletion, are reported in this study which have not been described previously. Five of the remaining seven variants were missense mutations with novel amino acids substituted for residues 127, 132, 180, 207, and 215, respectively. Mutations containing different amino acid residues at those positions have been reported. The last two are variants that have already been described to contain mutations at amino acid residues 333 and 365, respectively. To evaluate the efficiency of SSCP analysis in assessing the mutated exons and to further confirm our results we sequenced the entire exons of all 17 factor IX genes. The mutations detected by SSCP method were indeed the only mutation identified in each factor IX variant. The SSCP analysis and direct sequencing have also allowed us to circumvent the difficulties of carrier determination for Chinese by direct detection of the abnormal factor IX alleles inherited by the females.

Hepatic Gene Transfer of Factor IX Reverses Inhibitors and Protects From Anaphylaxis in a Murine Hemophilia B Model

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 669-669 ◽  
Author(s):  
David Markusic ◽  
Roland W Herzog
Keyword(s):  
At Risk ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Antibody Response ◽  
B Cell ◽  
Gene Deletion ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
High Dose

Abstract Abstract 669 Hemophilia B is a X-linked bleeding disorder caused by loss of coagulation factor IX (F.IX) function. Under current treatment protocols, approximately 2–4% of hemophilia B patients develop inhibitory antibodies to F.IX protein, and those with F.IX gene deletions are at risk for anaphylaxis. We work with a murine model for this pathogenic antibody response, namely C3H/HeJ hemophilia B (HB) mice with a F9 gene deletion. Anaphylactic reactions to intravenously administered F.IX protein are the result of a strong Th2-driven antibody response, comprised of IgG1 and IgE. Interestingly, we find that IgE levels correlate with the dose of recombinant F.IX protein. HB mice treated with weekly intravenous doses of 0.1 or 0.3 IU F.IX were free from allergic/anaphylactic reactions during the course of treatment (1 IP and 5 weekly IV injections of F.IX protein) and had very low to undetectable IgE against F.IX. In contrast, a dose of 1 IU induced IgE formation and caused fatal anaphylactic reactions after repeated administration. These results suggest that prophylaxis with lower F.IX doses may be recommended in individuals with F.IX gene deletion (Mice with missense or nonsense F.IX mutations did not show inhibitor formation or anaphylaxis at the high dose). In an attempt to reverse the response and desensitize the gene deletion HB animals, F.IX-treated mice with inhibitors, including those with detectable circulating IgE, received hepatic AAV8-F.IX gene transfer (1e11 vg/mouse). Within one month, these mice lost detectable IgG and IgE against F.IX and showed a level of correction of coagulation comparable to gene transfer in naive mice. Subsequent intravenous injections of 1 IU F.IX (weekly for 1 month) did not cause anaphylaxis, demonstrating successful desensitization. Control mice (no gene transfer) maintained their IgE levels during the course of the experiment. We are currently investigating if a lower AAV8 F.IX vector dose (1e10 vg) is similarly capable of reversing existing F.IX inhibitors/anaphylaxis. In order to determine the fate of F.IX antibody-secreting cells (ASC), we performed a B cell ELISPOT assay on bone marrow cells and splenocytes in F.IX immunized HB mice left untreated or treated with AAV8 F.IX vector. Control immunized mice showed predominantly ASC in the spleen with a few detected cells from bone marrow. ASC were nearly undetectable in vector-treated mice. These results suggest that AAV8 liver gene transfer not only suppresses F.IX-specific ASC, but may also prevent the re-activation of memory B cells. Experiments are ongoing to explore the role of induced regulatory T cells in modulating ASC. To characterize T cell responses against F.IX, we isolated splenocytes isolated from control immunized and vector treated mice, stimulated these cells in vitro with F.IX protein, and collected RNA for analysis with RT-PCR array. Control mice showed an upregulation of Th2 cytokines IL-4 and IL-13, which are known to induce B cell class switching to IgG1 and to IgE, corresponding to the observed antibody formation. Vector treated immunized mice did not show up regulation of any cytokines representative of Th1 or Th2 responses, indicating down-regulation of T help required for the antibody response. In summary, our data show that liver directed AAV8 F.IX gene transfer may not only induce tolerance to those at risk of developing inhibitors, but may offer an alternative treatment approach to expensive and long-term immune tolerance induction (ITI) protocols in those with existing pathogenic antibody responses. This concept may also apply to other genetic diseases, in which antibodies complicate protein replacement therapy. Disclosures: Herzog: Genzyme Corporation: Patents & Royalties.

Severe haemophilia B due to a 6 kb factor IX gene deletion including exon 4: Non-homologous recombination associated with a shortened transcript from whole blood

2007 ◽  
Vol 97 (02) ◽  
pp. 176-180 ◽  
Author(s):  
Ting-Chang Hsu ◽  
Shelley Nakaya ◽  
Arthur Thompson
Keyword(s):  
Growth Factor ◽  
Whole Blood ◽  
Gene Deletion ◽  
Factor Ix ◽  
Severe Haemophilia ◽  
Haemophilia B ◽  
French Family ◽  
Exon 4 ◽  
Intron 4 ◽  
Partial Gene Deletion

SummaryIn genotyping a severe hemophilia B subject, exons 1–3 and 5–8 were normal. Exon 4 did not amplify, suggesting a partial gene deletion. Previously, a French family with an exon 4 deletion had severe haemophilia B with a circulating, dysfunctional factor IX protein missing its first growth factor-like domain; breakpoints were not analyzed. Using a 5’ primer for exon 3 and a 3’ primer for exon 5 fragments, the subject’s factor IX gene amplified a 5 kb fragment whereas 11 kb was predicted, indicating a 6 kb deletion. Restriction endonucleases localized the 3’ intron 4 deletion breakpoint to 1.2 kb 5’ to exon 5. Sequencing through the breakpoints revealed a 5,969 bp deletion that included exon 4 and was accompanied by a 13 bp duplication inserted near the 3’ breakpoint site. Haemophilia was familial; on testing, his mother was confirmed as a heterozygous carrier, whereas his sister was homozygous for the normal, larger fragments. As exons 4 and 5 of the factor IX gene are in frame, this deletion should produce a shortened transcript, missing 114 bp (38 codons from the first growth factor-like domain). Reverse transcription of mRNA prepared from whole blood and PCR identified the shorter cDNA fragment. Western blotting demonstrated a smaller factor IX protein.

Humoral and Anaphylactic Responses to Factor IX In Murine Hemophilia B Are Genotype Dependent and Can Be Reversed by Hepatic Gene Transfer

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2202-2202
Author(s):  
David Markusic ◽  
Sushrusha Nayak ◽  
Roland W Herzog
Keyword(s):  
Gene Transfer ◽  
Antibody Response ◽  
Missense Mutation ◽  
Gene Deletion ◽  
Stop Codon ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
Tail Vein ◽  
Wide Range

Abstract Abstract 2202 Hemophilia B is the X-linked bleeding disorder caused by loss of coagulation factor IX (F.IX) function. Current treatment relies on infusion of plasma derived or recombinant F.IX protein. Approximately 2–4% of hemophilia B patients develop inhibitory antibodies to F.IX protein, and those with F.IX gene deletions are at risk for anaphylaxis. We recently described a murine mode for this pathogenic antibody response, namely C3H/HeJ hemophilia B (HB) mice with a F9 gene deletion (PNAS 107:7101). Anaphylactic reactions were the result of a strong Th2-driven antibody response, comprised of IgG1 and IgE. In new experiments, we again found that repeated exposure to F.IX protein from weekly intravenous injections of recombinant human F.IX (1 IU/mouse) resulted in inhibitors, with 53% of the treated mice experiencing fatal anaphylaxis following the third administration. Surviving animals were given three additional intravenous F.IX protein injections (the fifth and sixth injections were given with antihistamine) and resulted in an average inhibitor titer of 6.2 BU/ml corresponding to an IgG1 titer of 5 mg/ml. Previously, we generated C3H/HeJ HB mice expressing non-functional crm- hF.IX variants due a missense mutation analogous to the UNC-Chapel Hill strain of hemophilia B dog (HB-CH) or due to a stop codon at amino acid residue 338 (HB-338). These mice, in contrast to the gene deletion mutation, failed to develop CD8+ T cell responses to hF.IX, but still formed inhibitors (albeit at reduced titers) upon muscle-directed gene transfer with AAV vector (Mol. Ther 17:1733). Furthermore, HB mice transgenic for a crim+ hF.IX missense mutation (HB-180) were entirely unresponsive to functional hF.IX. When challenged by a total of 6 weekly injections of hF.IX protein, all three strains (HB-338, HB-CH, and HB-180) tolerated the hF.IX antigen without any indication of inhibitors/IgG formation or anaphylaxis, which was in sharp contrast to the gene deletion mice. These results indicate that anaphylaxis in the murine model is F.IX genotype dependent, as it is in humans. In other experiments, naïve gene deletion mice were successfully tolerized to hF.IX by hepatic gene transfer. AAV8 or AAV2 (Y444/500/730F) capsid vectors containing a livers-specific expression cassette were injected into the tail vein. AAV8 vector induced tolerance over a wide range of vector doses. For optimized AAV2, hF.IX levels of 8–14% of normal were achieved with a dose of 2×10^11 vg/mouse, which protected from inhibitor formation and anaphylaxis upon subsequent challenge with F.IX protein. Next, we attempted to reverse the inhibitor response that had formed after protein therapy in gene deletion mice. These were repeatedly treated with hF.IX protein resulting in an average inhibitor of 6 BU/ml. Shortly afterwards, AAV8 vector expressing hF.IX (1×10^11 vg/mouse, n=4) was injected via the tail vein for hepatic gene transfer. Strikingly, this resulted in a complete reversal of antibody titers and systemic hF.IX levels of >40% of normal and aPTTs in the normal range. There was no evidence for an amnestic response, and the animals could be challenged with intravenous hF.IX protein without anaphylaxis. These results demonstrate that inhibitor formation resulting from F.IX replacement therapy can be reversed by liver gene transfer. Moreover, gene therapy in pre-immune mice corrected coagulation and protected from subsequent anaphylactic reactions after gene transfer. We are currently testing whether the protocol is safe for even higher pre-existing inhibitor titers. Disclosures: Herzog: Genzyme Corp: Patents & Royalties.

ANALYSIS OF FACTOR IX GENE IN NORMAL SUBJECTS AND HEMOPHILIA B PATIENTS IN JAPAN

1987 ◽  
Author(s):  
T Kojima ◽  
M Tanimoto ◽  
T Kamiya ◽  
Y Obata ◽  
K Kurachi ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Normal Subjects ◽  
Molecular Probes ◽  
Factor Ix ◽  
Hemophilia B ◽  
De Novo Mutation ◽  
Carrier Status ◽  
Normal Pattern ◽  
Normal Japanese ◽  
Japanese Subjects

We have examined DNA samples from 25 hemophilia B patients (21 B- patients, 2 BR patients and 2 B+ patients) and 51 normal subjects with molecular probes (pHFIX and 2 genomic fragments). By structural gene analysis, 4 out of 7 patients who developed anti-factor IX antibodies were detected to have gross factor IX gene deletion. Although these four patients showed normal pattern of HPRT gene detected by pCDHPRT, the gene deletions were found to expand more than 34kb including with entire factor IX exons. Quantitative Southern blot analysis of factor IX gene of the patient's family members indicated that the gene deletion was inherited in one family, establishing the carrier status of 2 aunts, 2 cousins and one sister. The 'de novo' mutation of factor IX gene was also established in 2 families. Three patients with anti-factor IX antibodies and 17 patients without antibody to factor IX had normal pattern of factor IX gene by several restriction enzyme digestions. Analysis of factor IX gene of three patients with anti-factor IX antibodies and two B+ patients are now underway to detect the unique gene defects which may be responsible for the disease Phenotypes. Common RFLPs in factor IX gene were studied in normal Japanese subjects. More than 80 X chromosomes were analysed with BamHI, Ddel, MspI, TaqI or XmnI digestion, followed by hybridization with pHFIX. RFLPs produced by these enzymes were found to be uncommon or possibly absent in normal Japanese subjects. These results imply that racial differences in the frequency of gene polymorphisms should be seriously considered before initiating the gene counseling by the genetic probes.

Markers of Hypercoagulability in Patients with Hemophilia B Given Repeated, Large Doses of Factor IX Concentrates during and after Surgery

1994 ◽  
Vol 71 (06) ◽  
pp. 737-740 ◽  
Author(s):  
E Santagostino ◽  
P M Mannucci ◽  
A Gringeri ◽  
G Tagariello ◽  
F Baudo ◽  
...  
Keyword(s):  
High Risk ◽  
Ex Vivo ◽  
Factor Ix ◽  
Hemophilia B ◽  
Coagulation System ◽  
Prolonged Treatment ◽  
Orthopedic Procedures ◽  
Factor X ◽  
Prothrombin Complex Concentrates ◽  
Increased Risk

SummaryPurer factor IX (FIX) concentrates have been produced for the treatment of hemophilia B in the attempt to reduce the risk of thrombotic complications associated with the use of prothrombin complex concentrates. To evaluate ex vivo whether or not FIX concentrates activate the coagulation system in conditions associated with a high risk for thrombosis, we measured markers of hypercoagulability in 10 patients with hemophilia B who underwent surgery, mainly orthopedic procedures, covered by multiple concentrate infusions (40-80 U/kg/day). Postinfusion plasma levels of prothrombin fragment 1+2 and factor X activation peptide did not differ significantly from the presurgical levels, neither before nor after each concentrate dose. Therefore, it appears that prolonged treatment of patients with hemophilia B undergoing high risk surgical procedures with high doses of FIX concentrate does not cause systemic activation of coagulation. This suggests that purified FIX concentrates are preferable to prothrombin complex concentrates for conditions associated with an increased risk of thrombosis.

Low Prevalence of the Factor V Leiden Mutation Among “Severe” Hemophiliacs with a “Milder” Bleeding Diathesis

1995 ◽  
Vol 74 (05) ◽  
pp. 1255-1258 ◽  
Author(s):  
Arnaldo A Arbini ◽  
Pier Mannuccio Mannucci ◽  
Kenneth A Bauer
Keyword(s):  
Hemophilia A ◽  
Factor V Leiden ◽  
Protein S ◽  
Factor Ix ◽  
Hemophilia B ◽  
Factor V ◽  
Bleeding Diathesis ◽  
Mutation Site ◽  
Spontaneous Bleeding ◽  
Factor V Leiden Mutation

SummaryPatients with hemophilia A and B and factor levels less than 1 percent of normal bleed frequently with an average number of spontaneous bleeding episodes of 20–30 or more. However there are patients with equally low levels of factor VIII or factor IX who bleed once or twice per year or not at all. To examine whether the presence of a hereditary defect predisposing to hypercoagulability might play a role in amelio rating the hemorrhagic tendency in these so-called “mild severe” hemophiliacs, we determined the prevalence of prothrombotic defects in 17 patients with hemophilia A and four patients with hemophilia B selected from 295 and 76 individuals with these disorders, respectively, followed at a large Italian hemophilia center. We tested for the presence of the Factor V Leiden mutation by PCR-amplifying a fragment of the factor V gene which contains the mutation site and then digesting the product with the restriction enzyme Mnll. None of the patients with hemophilia A and only one patient with hemophilia B was heterozygous for Factor V Leiden. None of the 21 patients had hereditary deficiencies of antithrombin III, protein C, or protein S. Our results indicate that the milder bleeding diathesis that is occasionally seen among Italian hemophiliacs with factor levels that are less than 1 percent cannot be explained by the concomitant expression of a known prothrombotic defect.

Extravascular Administration of Factor IX: Potential for Replacement Therapy of Canine and Human Hemophilia B

1997 ◽  
Vol 77 (05) ◽  
pp. 0944-0948 ◽  
Author(s):  
Darla Liles ◽  
Charles N Landen ◽  
Dougald M Monroe ◽  
Celeste M Lindley ◽  
Marjorie s Read ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Vitamin K ◽  
Absorption Rate ◽  
Venous Access ◽  
Factor Ix ◽  
Hemophilia B ◽  
Current Therapy ◽  
Home Therapy ◽  
Routes Of Administration ◽  
Plasma Compartment

SummaryCurrent therapy for hemophilia B requires large intravenous doses of factor IX (F.IX) given in the clinic or at home. Although home therapy is possible for many patients, it is often complicated by factors such as the lack of good venous access. Very little is known about extravascular routes for administering proteins like F.IX (57 kD) or other vitamin K-dependent procoagulant factors into the circulation. Questions about the absorption rate from extravascular administration as well as plasma recovery and bioavailability have arisen recently with the growing availibility of highly purified procoagulant proteins and increased interest in gene therapy of hemophilia B. Therefore, a group of studies were undertaken to determine the absorption rate, plasma recovery, and bioavailability of high purity, human plasma-derived F.IX concentrates administered via extravascular routes in hemophilia B dogs and in one human hemophilia B subject. Five hemophilia B dogs were given human F.IX via either a subcutaneous (SC), intramuscular (IM), intra- peritoneal (IP) or intravenous (IV) route. In a subsequent study, a single SC administration of human F.IX was compared to an identical IV dose of F.IX in the human hemophilia B subject. All extravascular routes of F.IX administration in both the canine and human gave lower levels of circulating plasma F.IX than the IV route, however all routes resulted in measurable F.IX activity. Of the extravascular routes, the IM injection in the canine resulted in a bioavailibility of 82.8%, while the SC injection resulted in a bioavailability of 63.5%. F.IX reached the plasma compartment by all extravascular routes used, confirming that F.IX can be absorbed extravascularly. The duration of measurable F.IX activity following extravascular administration is prolonged beyond that typically seen with IV administration. These data show that significant levels of F.IX may be obtained via SC injection in canine and ‘ human hemophilia B subjects and further highlight the potential of extravascular routes of administration for future experimental and clinical uses of F.IX and other procoagulant proteins.

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