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

Abstract 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.

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

Blood ◽

10.1182/blood.v67.5.1508.bloodjournal6751508 ◽

1986 ◽

Vol 67 (5) ◽

pp. 1508-1511

Author(s):

CW Hay ◽

KA Robertson ◽

SL Yong ◽

AR Thompson ◽

GH Growe ◽

...

Keyword(s):

Human Factor ◽

Factor Ix ◽

Hemophilia B ◽

Carrier Status ◽

X Chromosomes ◽

Human Factor Ix ◽

History Of

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.

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

Blood ◽

10.1182/blood.v73.2.438.bloodjournal732438 ◽

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.

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Development of Monospecific Antibodies to Human Factor IX

10.1055/s-0039-1680660 ◽

1977 ◽

Author(s):

Cheryl Y. Tiarks ◽

Chin-Hai Chang ◽

Liberto Pechet

Keyword(s):

Neutralizing Antibodies ◽

Human Factor ◽

Human Albumin ◽

Factor Ix ◽

Hemophilia B ◽

Red Cross ◽

Factor X ◽

Normal Plasma ◽

Human Factor Ix ◽

Monospecific Antibodies

The purpose of this research was to develop neutralizing and precipitating antibodies to factor IX. Human factor IX, purified by the method of Rosenberg et.al. (J. Biol. Chem. 250:8883, 1975), was electrophoresed on acrylamide gel. Two major bands migrating adjacently were eluted. They contained factor IX activity only. The eluates and their homogenized gel segments 7 and 8 were injected separately into two rabbits, Rl and R2, respectively. On immunodiffusion the antiserum Rl showed one precipitating line with normal plasma. It neutralized human factor IX (20 Bethesda units) and also slightly neutralized factor X. It had no effect on factors II and VII. Following absorption of this antiserum with purified factor X it neutralized factor IX only. With continuous immunization, however, this antiserum revealed two new precipitating contaminants. The antiserum R2 neutralized only factor IX; it reached 220 Bethesda inhibitory units. On immunodiffusion it showed two precipitating lines, one of which disappeared after absorption with human albumin. On immunodiffusion and Laurell immunoelectrophoresis, the albumin-absorbed R2 antiserum showed one precipitin line of identity, or one rocket, with normal plasma, a Red Cross factor IX preparation (rich in factors IX, II and X), the original eluates 7 and 8, and a Hemophilia-B antigen-positive plasma. No line or rocket developed with normal plasma absorbed with aluminum hydroxide or with antigen-negative Hemophilia-B plasma. We conclude that the antisera Rl and R2 contain factor IX neutralizing antibodies and that albumin-absorbed R2 has monospecific precipitating antibodies to human non-activated factor IX.

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210. Neonatal Gene Therapy with a Human Factor IX-Expressing Retroviral Vector Prevents Antibody Responses in Dogs and Hemophilia B Mice

Molecular Therapy ◽

10.1016/s1525-0016(16)40652-0 ◽

2003 ◽

Vol 7 (5) ◽

pp. S83

Keyword(s):

Gene Therapy ◽

Human Factor ◽

Retroviral Vector ◽

Factor Ix ◽

Antibody Responses ◽

Hemophilia B ◽

Human Factor Ix ◽

B Mice

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Permanent partial phenotypic correction and tolerance in a mouse model of hemophilia B by stem cell gene delivery of human factor IX

Gene Therapy ◽

10.1038/sj.gt.3302638 ◽

2005 ◽

Vol 13 (2) ◽

pp. 117-126 ◽

Cited By ~ 38

Author(s):

B W Bigger ◽

E K Siapati ◽

A Mistry ◽

S N Waddington ◽

M S Nivsarkar ◽

...

Keyword(s):

Stem Cell ◽

Gene Delivery ◽

Mouse Model ◽

Human Factor ◽

Factor Ix ◽

Hemophilia B ◽

Human Factor Ix ◽

Cell Gene ◽

Stem Cell Gene

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Delivery of human factor IX in mice by encapsulated recombinant myoblasts: a novel approach towards allogeneic gene therapy of hemophilia B

Blood ◽

10.1182/blood.v87.12.5095.bloodjournal87125095 ◽

1996 ◽

Vol 87 (12) ◽

pp. 5095-5103 ◽

Cited By ~ 114

Author(s):

G Hortelano ◽

A Al-Hendy ◽

FA Ofosu ◽

PL Chang

Keyword(s):

Gene Therapy ◽

Human Factor ◽

Ex Vivo ◽

Cost Effective ◽

Factor Ix ◽

Hemophilia B ◽

Therapy Strategy ◽

Human Factor Ix

A potentially cost-effective strategy for gene therapy of hemophilia B is to create universal factor IX-secreting cell lines suitable for implantation into different patients. To avoid graft rejection, the implanted cells are enclosed in alginate-polylysine-alginate microcapsules that are permeable to factor IX diffusion, but impermeable to the hosts' immune mediators. This nonautologous approach was assessed by implanting encapsulated mouse myoblasts secreting human factor IX into allogeneic mice. Human factor IX was detected in the mouse plasma for up to 14 days maximally at approximately 4 ng/mL. Antibodies to human factor IX were detected after 3 weeks at escalating levels, which were sustained throughout the entire experiment (213 days). The antibodies accelerated the clearance of human factor IX from the circulation of the implanted mice and inhibited the detection of human factor IX in the mice plasma in vitro. The encapsulated myoblasts retrieved periodically from the implanted mice up to 213 days postimplantation were viable and continued to secrete human factor IX ex vivo at undiminished rates, hence suggesting continued factor IX gene expression in vivo. Thus, this allogeneic gene therapy strategy represents a potentially feasible alternative to autologous approaches for the treatment of hemophilia B.

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

Blood ◽

10.1182/blood.v73.2.438.438 ◽

1989 ◽

Vol 73 (2) ◽

pp. 438-445 ◽

Cited By ~ 121

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.

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Factor IX New London: substitution of proline for glutamine at position 50 causes severe hemophilia B

Blood ◽

10.1182/blood.v75.5.1097.1097 ◽

1990 ◽

Vol 75 (5) ◽

pp. 1097-1104 ◽

Cited By ~ 19

Author(s):

JN Lozier ◽

DM Monroe ◽

S Stanfield-Oakley ◽

SW Lin ◽

KJ Smith ◽

...

Keyword(s):

Translation Termination ◽

Factor Ix ◽

Hemophilia B ◽

Molar Ratio ◽

Carrier Status ◽

Severe Hemophilia ◽

Base Pairs ◽

Human Factor Ix ◽

Restriction Endonuclease Cleavage Site ◽

Status Analysis

Abstract We describe a novel point mutation in the fourth exon of human factor IX (encoding the first EGF-like domain) in which cytosine is substituted for adenosine at position 10,401, resulting in the substitution of proline for glutamine at position 50 in the polypeptide chain. Sequence analysis of all eight exons, all exon-intron junctions, 160 base pairs (bp) of DNA 5′ to the proposed translation start site, and 60 bp 3′ to the translation termination site shows no other difference from the normal factor IX gene, with the exception of a previously described benign polymorphism at position 148 in the protein (Ala----Thr). The affected subject has severe hemophilia B with no detectable factor IX activity despite normal factor IX antigen levels. We purified the abnormal factor IX by immunoaffinity chromatography and demonstrated that its activation by factor Xla is markedly delayed compared with normal factor lX. Once activated, the abnormal factor lX binds antithrombin III in a 1:1 molar ratio, and the activated protein demonstrates catalytic activity, suggesting an intact active site. The mutation creates a new Bst Yl restriction endonuclease cleavage site. Restriction with Bst Yl shows the mutation in maternal DNA and offers the possibility of direct carrier status analysis and prenatal diagnosis in kindreds with this mutation. We designate this new mutation factor lXNew London. This is the only reported mutation in the first EGF-like domain that causes severe hemophilia B.

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Enzyme-Linked Immunosorbent Assay For The Detection Of Alloantibodies To Factor IX In Hemophilia B

10.1055/s-0038-1653008 ◽

1981 ◽

Author(s):

K H Örstavik ◽

I Örstavik

Keyword(s):

Immunosorbent Assay ◽

Solid Phase ◽

Factor Ix ◽

Hemophilia B ◽

Negative Reaction ◽

Enzyme Linked Immunosorbent Assay ◽

Nitrophenyl Phosphate ◽

Human Factor Ix ◽

Coagulation Assay

A solid phase enzyme-linked immunosorbent assay (ELISA) was developed for the detection and quantitative determination of acquired inhibitors to factor IX. Wells of polystyren Micro-ELISA plates were coated with the IgG fraction of a sheep antiserum to human factor IX. After incubation with pooled normal plasma as a factor IX source, the wells were incubated with test plasma. The binding of alloantibodies to the factor IX-sheep-anti-factor IX complexes was then detected by incubation with alkaline phophatase conjugated antiserum to human IgG. As substrate was used p-nitrophenyl phosphate.Plasma samples from five patients with severe hemophilia B and acquired inhibitors to factor IX were examined. All samples gave a positive reaction in the ELISA. The titers as determined in the ELISA were in good agreement with the titers as determined in a coagulation assay (0.1-800 U/ml). Plasma from 13 patients with hemophilia B and no detectable inhibitor in a coagulation assay all gave a negative reaction in the ELISA. A negative reaction was also found in plasma from four patients with hemophilia A and acquired inhibitors to factor VIII, and in plasma from 15 healthy persons.It is concluded that the ELISA is a simple and sensitive technique for the determination of acquired inhibitors to factor IX in hemophilia B.

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