scholarly journals A deletion mutation causes hemophilia B in Lhasa Apso dogs

Blood ◽  
1996 ◽  
Vol 88 (9) ◽  
pp. 3451-3455 ◽  
Author(s):  
AE Mauser ◽  
J Whitlark ◽  
KM Whitney ◽  
CD Jr Lothrop
Keyword(s):  
Molecular Basis ◽  
Genetic Test ◽  
Premature Termination Codon ◽  
Deletion Mutation ◽  
Factor Ix ◽  
Hemophilia B ◽  
Heteroduplex Analysis ◽  
Clotting Factor ◽  
Plasma Factor ◽  
Sequence Encoding

Hemophilia B is a bleeding disorder caused by a deficiency of clotting factor IX (FIX). A colony of FIX deficient Lhasa Apso dogs has been established and the molecular basis of hemophilia B has been determined. The plasma factor IX levels were < 1% of normal canine levels in affected dogs. A complex deletion mutation at nucleotides 772–777 was found when hepatocyte cDNA from a hemophilia B dog was sequenced. The sequence was identical to the normal canine sequence except for a deletion including nucleotides 772–776 and a C-->T transition at nucleotide 777. The mutation results in mRNA instability and a premature termination codon in the nucleotide sequence encoding the activation peptide. The mutation was verified by sequencing genomic DNA from an FIX-deficient dog. A genetic test for the detection of heterozygous animals was established using heteroduplex analysis. Although hemophilia B has been described in many dog breeds, this is only the second mutation to be sequenced. The Lhasa Apso dog model should be valuable for evaluating novel strategies for treating hemophilia B such as gene therapy.

scholarly journals Production of human factor IX in animals by genetically modified skin fibroblasts: potential therapy for hemophilia B

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 438-445
Author(s):  
TD Palmer ◽  
AR Thompson ◽  
AD Miller
Keyword(s):  
Human Factor ◽  
Normal Skin ◽  
Human Fibroblasts ◽  
Retroviral Vectors ◽  
Factor Ix ◽  
Skin Fibroblasts ◽  
Hemophilia B ◽  
Clotting Factor ◽  
Human Factor Ix

Inherited diseases might be treated by introducing normal genes into a patient's somatic tissues to correct the genetic defects. In the case of hemophilia resulting from a missing clotting factor, the required gene could be introduced into any cell as long as active factor reached the circulation. We previously showed that retroviral vectors can efficiently transfer genes into normal skin fibroblasts and that the infected cells can produce high levels of a therapeutic product in vitro. In the current study, we examined the ability of skin fibroblasts to secrete active clotting factor after infection with different retroviral vectors encoding human clotting factor IX. Normal human fibroblasts infected with one vector secreted greater than 3 micrograms factor IX/10(6) cells/24 h. Of this protein, greater than 70% was structurally and functionally indistinguishable from human factor IX derived from normal plasma. This suggests that infected autologous fibroblasts might provide therapeutic levels of factor IX if transplanted into patients suffering from hemophilia B. By transplanting normal diploid fibroblasts infected with the factor IX vectors, we showed that human factor IX can be produced and is circulated at readily detectable levels in rats and mice.

EXPRESSION IN MAMMALIAN CELLS OF FUSION PROTEINS BETWEEN HUMAN FACTORS IX AND VII

1987 ◽  
Author(s):  
K L Berkner ◽  
S J Busby ◽  
J Gambee ◽  
A Kumar
Keyword(s):  
Fusion Protein ◽  
Fusion Proteins ◽  
Factor Ix ◽  
Biologically Active ◽  
Factor Vii ◽  
Clotting Factor ◽  
Clotting Factors ◽  
Mammalian Expression ◽  
Plasma Factor ◽  
Gla Domain

The vitamin K-dependent plasma proteins demonstrate remarkable similarities in their structures: all have multiple domains in common and extensive homology is observed within many of these domains. In order to investigate the structure-function relationship of these proteins, we have interchanged domains of one protein (factor IX) with that of another (factor VII) and have compared the expression of these fusion proteins with recombinant and native factors IX and VII. Oligonucleotide-directed mutagenesis was used to generate four fusion proteins: factor IX/VII-1, which contains the factor IX leader and gla domain fused to the growth factor and serine protease of factor VII; factor VII/IX-1, a reciprocal fusion protein of factor IX/VII-1; factor IX/VII-2, which contains the factor IX leader adjoined to the mature factor VII protein sequence; and factor VII/IX-2, the reciprocal fusion protein of factor IX/VII-2. The cDNAs encoding all four proteins were cloned into mammalian expression vectors, and to date three of these (factors IX/VII-1, 2 and VII/IX-1) have been transfected into baby hamster kidney (BHK) cells or 293 cells and characterized. Factors IX/VII-1 and VII/IX-1 were both secreted at levels comparable to recombinant factors IX and VII. The factor IX/VII-1 was identical in molecular weight to native or recombinant factor VII (i.e., 53 K). Factor VII/IX-1 was expressed as two proteins with molecular weights around 68 kd, as observed with recombinant factor IX. The factor IX/VII-1 protein has been purified to homogeneity and has been found to possess factor VII biological activity, but at a specific activity approximately 20% that of plasma factor VII. Thus, the gla domain of one clotting factor is capable of directing the activation of another and of generating biologically active protein. In contrast, no activity was observed with the factor IX/VII-2 fusion protein, indicating that there are limits to the interchanges which can generate functional blood clotting factors.

scholarly journals Production of human factor IX in animals by genetically modified skin fibroblasts: potential therapy for hemophilia B

Blood ◽  
1989 ◽  
Vol 73 (2) ◽  
pp. 438-445 ◽  
Author(s):  
TD Palmer ◽  
AR Thompson ◽  
AD Miller
Keyword(s):  
Human Factor ◽  
Normal Skin ◽  
Human Fibroblasts ◽  
Retroviral Vectors ◽  
Factor Ix ◽  
Skin Fibroblasts ◽  
Hemophilia B ◽  
Clotting Factor ◽  
Human Factor Ix

Abstract Inherited diseases might be treated by introducing normal genes into a patient's somatic tissues to correct the genetic defects. In the case of hemophilia resulting from a missing clotting factor, the required gene could be introduced into any cell as long as active factor reached the circulation. We previously showed that retroviral vectors can efficiently transfer genes into normal skin fibroblasts and that the infected cells can produce high levels of a therapeutic product in vitro. In the current study, we examined the ability of skin fibroblasts to secrete active clotting factor after infection with different retroviral vectors encoding human clotting factor IX. Normal human fibroblasts infected with one vector secreted greater than 3 micrograms factor IX/10(6) cells/24 h. Of this protein, greater than 70% was structurally and functionally indistinguishable from human factor IX derived from normal plasma. This suggests that infected autologous fibroblasts might provide therapeutic levels of factor IX if transplanted into patients suffering from hemophilia B. By transplanting normal diploid fibroblasts infected with the factor IX vectors, we showed that human factor IX can be produced and is circulated at readily detectable levels in rats and mice.

Efficient transfer and expression of human clotting factor IX cDNA in neonatal hemophilia B mice mediated by VSV-G pseudotyped retrovirus

2001 ◽  
Vol 46 (18) ◽  
pp. 1534-1538
Author(s):  
Hongwei Wang ◽  
Chenbo Ye ◽  
Li Chen ◽  
Xuefeng Wang ◽  
Xinfang Qiu ◽  
...  
Keyword(s):  
Factor Ix ◽  
Hemophilia B ◽  
Clotting Factor ◽  
B Mice ◽  
Human Clotting Factor ◽  
Efficient Transfer

scholarly journals Christmas disease in a Hovawart family resembling human hemophilia B Leyden is caused by a single nucleotide deletion in a highly conserved transcription factor binding site of the F9 gene promoter

2018 ◽  
Author(s):  
Bertram Brenig ◽  
Lilith Steingräber ◽  
Shuwen Shan ◽  
Fangzheng Xu ◽  
Marc Hirschfeld ◽  
...  
Keyword(s):  
Coagulation Factor ◽  
Bleeding Disorder ◽  
Factor Ix ◽  
Hemophilia B ◽  
Start Codon ◽  
Luciferase Reporter ◽  
Single Nucleotide ◽  
Single Nucleotide Deletion ◽  
Nucleotide Deletion ◽  
Plasma Factor

Hemophilia B is a classical monogenic X-chromosomal recessively transmitted bleeding disorder caused by genetic variants within the coagulation factor IX gene (F9). Although hemophilia B has been described in 32 dog breeds hitherto, it has not yet been reported in the Hovawart. Here we describe the identification of a Hovawart family transmitting typical signs of an X-linked bleeding disorder. Five males had been reported to suffer from recurrent hemorrhagic episodes, four of them had to be euthanized finally and one died due to severe blood loss. A blood sample of one of these males with only 2% of the normal concentration of plasma factor IX (FIX) together with samples of seven relatives including the mother and grandmother were provided for further analysis. Next generation sequencing of DNA of the mother and grandmother revealed a single nucleotide deletion in the F9 promoter (NC_006621.3:g.109,501,492delC; CanFam3.1). Genotyping of the deletion in 1,298 dog specimens (83 different breeds) including 720 Hovawarts revealed that the mutation was only present in the aforementioned Hovawart family. The deletion is located 73 bp upstream of the F9 start codon in the highly conserved overlapping DNA binding sites of hepatocyte nuclear factor 4α (HNF4α) and androgen receptor (AR). The deletion only abolishes binding of HNF4α as demonstrated by electrophoretic mobility shift assay (EMSA) using purified recombinant human HNF4α and a transient overexpression lysate of human AR with double-stranded DNA probes encompassing the mutated promoter region. Luciferase reporter assays using wild type and mutated promoter fragment constructs transfected into Hep G2 cells showed a 65.3% reduction in expression of the mutated promoter. The data presented here provide evidence that the deletion identified in the Hovawart family caused a rare type of hemophilia B resembling human hemophilia B Leyden.

scholarly journals Etranacogene dezaparvovec for hemophilia B gene therapy

2021 ◽  
Vol 2 ◽  
pp. 263300402110588
Author(s):  
Courtney D. Thornburg
Keyword(s):  
Gene Therapy ◽  
Clinical Trials ◽  
Continuous Production ◽  
Factor Ix ◽  
Hemophilia B ◽  
Clotting Factor ◽  
Plain Language ◽  
Long Term Follow Up

The treatment landscape for hemophilia has been rapidly changing with introduction of novel therapies. Gene therapy for hemophilia is a promising therapeutic option for sustained endogenous factor production to mitigate the need for prophylactic treatment to prevent spontaneous and traumatic bleeding. Etranacogene dezaparvovec is an investigational factor IX (FIX) gene transfer product that utilizes the adeno-associated virus (AAV) 5 vector with a liver-specific promoter and a hyperactive FIX transgene. Here, the development of etranacogene dezaparvovec and available efficacy and safety data from clinical trials are reviewed. Overall, etranacogene dezaparvovec provides sustained FIX expression for more than 2 years and allows for a bleed and infusion-free life in the majority of patients. Safety, efficacy, and quality-of-life data will inform shared decision-making for patients who are considering gene therapy. Long-term follow-up regarding duration of expression and safety are crucial. Plain Language Summary Factor IX Padua gene therapy to boost clotting factor and prevent bleeding for people living with hemophilia B People living with hemophilia have low or missing clotting factor, which can lead to bleeding that is unexpected or caused by a traumatic event (such as a sports injury or surgery). There are two main types of hemophilia: clotting factor (F)VIII deficiency (known as hemophilia A) and FIX deficiency (known as hemophilia B). People living with the severe or moderately severe forms of hemophilia (clotting factor levels below 3% of normal) need regular treatment, typically by infusions into the vein, to stop or prevent bleeding and damage to their joints. Gene therapy is currently being investigated as a new treatment option that introduces a working copy of the clotting factor gene to the liver. Following treatment, clotting factor is produced by the liver. Etranacogene dezaparvovec [Et-ra-na-co-gene dez-a-par-vo-vec] is a form of gene therapy for people living with hemophilia B. This form of gene therapy includes a modified form of FIX (FIX Padua) which produces high levels of FIX activity compared with normal FIX. It is being tested to see whether individuals will have low rates of bleeding and not need to treat themselves with clotting factor. In the clinical trials, participants with FIX levels below 2% (of normal) receive a single gene therapy infusion. The results of the trials have so far shown that patients given etranacogene dezaparvovec have continuous production of FIX, whereby they have reported much less bleeding and factor treatment. Questions relating to the safety of the gene therapy and how long it works will hopefully be answered through long-term follow-up of the patients once the trials are completed.

Two Distinct Mutations Cause Severe Hemophilia B in Two Unrelated Canine Pedigrees

1999 ◽  
Vol 82 (10) ◽  
pp. 1270-1275 ◽  
Author(s):  
Weikuan Gu ◽  
James Catalfamo ◽  
Jharna Ray ◽  
Kunal Ray ◽  
Marjory Brooks
Keyword(s):  
Catalytic Domain ◽  
Stop Codon ◽  
Spontaneous Mutation ◽  
Mammalian Species ◽  
Donor Site ◽  
Splice Junction ◽  
Factor Ix ◽  
Hemophilia B ◽  
Acceptor Site ◽  
Plasma Factor

SummaryThe molecular defects causing severe factor IX deficiency were identified in two distinct canine breed-variants. Both defects were associated with an absence of plasma factor IX coagulant activity and antigen. A large deletion mutation was found in 1 breed variant, spanning the entire 5’ region of the factor IX gene extending to exon 6. An approximately 5 kb insertion disrupted exon 8 of the second breed-variant. This insertion was associated with alternative splicing between a donor site 5’ and acceptor site 3’ to the normal exon 8 splice junction, with introduction of a new stop codon. The resultant transcript lacked most of the factor IX catalytic domain and 3’ untranslated region. Molecular analyses of canine hemophilia B define an experimental model for study of inhibitor formation and gene therapy strategies, and provide insight into spontaneous mutation mechanisms in the factor IX gene and on the X chromosome of mammalian species.

scholarly journals Monoclonal antibody to an epitope on the heavy chain of factor IX missing in three hemophilia-B patients

Blood ◽  
1983 ◽  
Vol 62 (5) ◽  
pp. 1027-1034
Author(s):  
AR Thompson
Keyword(s):  
Monoclonal Antibody ◽  
Heavy Chain ◽  
Polyclonal Antibody ◽  
Factor Ix ◽  
Hemophilia B ◽  
Structural Defects ◽  
Clotting Factor ◽  
Antibody Testing ◽  
Antibody Assay ◽  
Factor Ixa

A murine hybridoma cell line that produces a monoclonal IgG1 antibody to human factor IX was established to provide a conformational probe for the clotting factor and its genetic variants. The antibody inhibited factor IX procoagulant activity, but did not appreciably interfere with the cleavage of factor IX by factor XIa nor with the binding of antithrombin-III-heparin complex to factor IXa. The antigen- solid-phase-antibody complex could be readily dissociated by relatively low concentrations of guanidine or sodium dodecyl sulfate, but only partially by high concentrations of urea. After gel electrophoresis and blotting of reduced samples of factor IXa, the antibody bound exclusively to the heavy chain. Sensitive immunoradiometric assays were developed using insolubilized monoclonal or polyclonal antibodies. Bovine factor IX had little cross-reactivity with the monoclonal antibody. Of 55 patient samples representing different pedigrees with hemophilia-B, antigen levels by the two assays were in excellent agreement in 49. There were 2 severely affected patients whose levels were too low to quantitate in the monoclonal antibody assay. A third, who had the lowest level of all by polyclonal antibody testing, and 3 less severely affected patients had no detectable antigen in the monoclonal antibody assay system (less than 0.03 U/dl). The latter 3 had at least 100–500 times as much antigen by polyclonal antibody testing. It is proposed that these 3 individuals have structural defects involving the epitope recognized by the monoclonal antibody and that they are due to amino acid substitutions between residues 188 through 359. Furthermore, it is suggested the substitutions lead to abnormal kinetic properties.

scholarly journals Real World Data Analysis of Coagulation Factor IX Costs in Specific Patients with Hemophilia B Switching from Standard Half-Life to Extended Half-Life Products

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4963-4963 ◽  
Author(s):  
Bartholomew J. Tortella ◽  
Patrick F. Fogarty ◽  
José Alvir ◽  
Margaret McDonald ◽  
Dean Spurden ◽  
...  
Keyword(s):  
Real World ◽  
Half Life ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Hemophilia B ◽  
Clotting Factor ◽  
Real World Data ◽  
Replacement Product ◽  
Traumatic Bleeding ◽  
Factor Concentrate

Abstract Introduction: Hemophilia B, an X-linked genetic disease characterized by low clotting factor IX (FIX) levels, leads to spontaneous and traumatic bleeding in affected individuals. Intravenous FIX replenishment is administered to maintain adequate levels. The recent introduction of an extended half-life (EHL) FIX replacement product provided the opportunity to compare costs of care associated with treatment with an EHL product versus a standard half-life (SHL) product. Methods: The Truven Marketscan US claims database (Oct 2010 - Apr 2016) was used to identify factor concentrate charges for patients who had claims data for at least 3 months prior to and after switching from SHL to EHL products. Additionally, available data for up to 12 months pre- and post-switch on these identified patients were analyzed. Results: Thirteen patients, ranging from 3-64 years of age (median 18) were included in the analyses. Factor concentrate costs were higher after switch from SHL to EHL in each of the time periods examined (Table). Median costs were used since they were less influenced by isolated outliers and were similar to means in most cases. The median costs were 238% higher in the 3 months just after switching to the EHL product compared to the 3 months just prior to switching from the SHL product. Median costs were $51,881, $79,654, $75,695 and $61,515 in the 12-10, 9-7, 6-4 and 3-1 months prior to switch respectively compared to $173,515, $147,061, $149,971 and $207,973 in the corresponding quarterly month intervals post switch. Conclusion: This analysis of real world administrative data following individual patients through the switch from SHL to EHL products continuum suggests that switching from an SHL to an EHL product is associated with increased median factor costs throughout the 12 months following the switch, and that further analysis with larger numbers of patients should be explored. Disclosures Tortella: Pfizer Inc: Employment. Fogarty:Pfizer Inc: Employment. Alvir:Pfizer Inc: Employment. McDonald:Pfizer Inc: Employment. Spurden:Pfizer Inc: Employment. Pleil:Pfizer Inc: Employment.

scholarly journals Molecular analysis of hemophilia B in Poland: 12 novel mutations of the factor IX gene.

1999 ◽  
Vol 46 (3) ◽  
pp. 721-726 ◽  
Author(s):  
K Wulff ◽  
K Bykowska ◽  
S Lopaciuk ◽  
F H Herrmann
Keyword(s):  
Direct Sequencing ◽  
Point Mutations ◽  
Factor Ix ◽  
Hemophilia B ◽  
Heteroduplex Analysis ◽  
Missense Mutations ◽  
Nonsense Mutations ◽  
Pcr Products ◽  
Factor Ix Deficiency ◽  
High Degree

We examined the molecular basis of factor IX deficiency in 53 unrelated Polish patients with hemophilia B. Heteroduplex analysis and direct sequencing of polymerase chain reaction (PCR) products were applied to identify the gene defect. Forty-three different point mutations were detected in the factor IX gene of 47 patients. There were 29 missense mutations, 9 nonsense mutations, 4 splice site mutations and 1 point mutation in the promoter region. Twelve mutations were novel. The results of this study emphasize a very high degree of heterogeneity of hemophilia B.

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