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At least 667 records · Page 37Linked to original sources
PubMed1984

Carrier detection of Hemophilia B by using a restriction site polymorphism associated with the coagulation Factor IX gene.

The cloned complementary DNA for coagulation Factor IX (FIX) detects a frequent restriction fragment length polymorphism (RFLP) in human genomic DNAs digested with the restriction endonuclease Taq I. This genetic marker was used, in parallel with coagulation and immunological assays, to follow the segregation of an abnormal FIX gene in a large Hemophilia B family. Among the six potential female carriers, functional assays showed that four had a high probability, and two a low probability of being carriers. Analysis at the DNA level with the cDNA probe was informative in five of the six cases, and in all these five the diagnosis of carrier state was definitively confirmed. This demonstrates the feasibility of using linkage analysis at the DNA level for the genetic screening of Hemophilia B. This method has the advantages over conventional assays of giving a diagnosis of certainty, and of being applicable to early prenatal diagnosis using biopsies of trophoblast villi. At present, the single known polymorphism associated with the FIX gene restricts the application of linkage analysis to informative cases (40%), but findings of additional RFLPs in this region should improve this figure.

Adolescent↗
PubMed1995

Age-specific regulation of clotting factor IX gene expression in normal and transgenic mice.

Factor IX (FIX), a circulating serine protease that serves as an essential component of the blood coagulation pathway, has been shown to increase with age in humans. We show here that murine FIX mRNA and activity levels also increase with age. Furthermore, one form of hemophilia B, hemophilia B Leyden, which is caused by mutations within the promoter region of the FIX gene, has a distinct age-dependent phenotype. To determine the source of the age-related increases in FIX gene expression, we have analyzed the regulation of the normal FIX gene promoter and FIX Leyden gene promoter with the +13 mutation during aging by generating transgenic mice that contain the -189 to +21 bp promoter segment ligated to a chloramphenicol acetyltransferase reporter gene. We have established that the normal FIX promoter and the Leyden promoter transgenes are expressed in a tissue-specific manner in vivo. The normal FIX promoter transgene does not show any differences in the pattern of expression with age or sex of the organism, whereas the Leyden promoter transgene showed age-dependent male-specific expression. This is the first demonstration of the FIX Leyden phenotype in a transgenic mouse model.

Age Factors↗
PubMed1993

Expression of human factor IX by microencapsulated recombinant fibroblasts.

Deficiency of clotting factor IX (FIX) causes hemophilia B in humans. We propose a novel approach to its treatment by engineering FIX-secreting cell lines suitable for implantation in different allogeneic hosts. To prevent graft rejection following implantation, the recombinant cells can be protected with biocompatible membranes that permit exit of FIX but not entry of cellular immune mediators. To explore the feasibility of this approach, we now report on the creation of mouse Ltk- fibroblast cell lines that can deliver FIX through such immune-protective membranes. Mouse fibroblasts (Ltk-) were transfected with the cDNA for human FIX and clones secreting high levels of FIX were isolated. About 70% of the secreted FIX was biologically active. Over 98% of the recovered biological activity was precipitable by barium citrate, indicating appropriate. gamma-carboxylation of the secreted FIX. The secreted FIX was similar in molecular weight and immunoreactivity to plasma-derived human FIX. Upon enclosure in microcapsules fabricated from the biocompatible polymers, alginate-polylysine-alginate, the cells survived the encapsulation procedure with about 70-90% viability, proliferated within the microcapsules to twice their original number within 2 weeks, and continued to secrete FIX into the culture medium at similar rates as the unencapsulated cells. The biological activity, degree of post-translational gamma-carboxylation, and immunoreactivity of the FIX recovered from the culture media of the encapsulated cells were identical to those of the FIX secreted by the unencapsulated cells. In conclusion, fibroblasts engineered to secrete recombinant human FIX can proliferate and continue to secrete biologically active FIX through the alginate microcapsules. This demonstrates the feasibility of using microencapsulated recombinant cells to deliver human FIX and the potential for allogeneic somatic gene therapy for hemophilia B.

Animals↗
PubMed1994

Regulatory mechanism of human factor IX gene: protein binding at the Leyden-specific region.

Hemophilia B-Leyden is characterized by the gradual amelioration of bleeding after the onset of puberty. All Leyden phenotype mutations found to date lie within the Leyden-specific region, which spans roughly nt-40 to +20 in the 5' end of the human factor IX gene. With HepG2 cell nuclear extracts, the Leyden-specific region and its immediate neighboring region of the normal factor IX gene showed five DNase I footprints: FP-I (nt +4 to +19), FP-II (nt -16 to -3), FP-III (nt -27 to -19), FP-IV (nt -67 to -49), and FP-V (nt -99 to -77). Protein binding affinities of short oligonucleotides containing sequences of FP-I, FP-II, or FP-III were substantially reduced in the presence of Leyden phenotype mutations in these areas, correlating well with the negative effects of these mutations on factor IX gene expression. A Leyden phenotype mutation at nt -20 (T to A) caused a loss of both footprints FP-III and FP-II but generated a new footprint, FP-III' (nt -34 to -23), partially overlapping with FP-III, indicating mutation-dependent competitive protein binding at these sites. Although the FP-III' area contains an androgen responsive element-like sequence, the nuclear protein that binds at FP-III' is not androgen receptor. The protein was not recognized by anti-androgen receptor antibody and, furthermore, was present not only in liver but also in both androgen receptor-positive and androgen receptor-negative cells in electrophoretic mobility shift assays. The nuclear concentration of this protein increased significantly upon treatment of the HepG2 cells with testosterone. Its binding affinity to an oligonucleotide (-32sub) containing the FP-III' sequence was greatly reduced in the presence of exogenous androgen receptor, suggesting a possible interaction of this protein with androgen receptor. The affinities of both this protein and a protein which binds to FP-III (presumably HNF-4) to -32sub with a mutation at nt -26 were grossly lowered. These findings suggest that the amelioration of hemophilia B-Leyden with a mutation at nt -20 after puberty involves binding of a specific non-androgen receptor nuclear protein at FP-III' and it is able to substitute for the function of a protein bound at FP-III in the normal gene optimally through its elevated interaction with androgen receptor upon a surge of testosterone.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

T296----M, a common mutation causing mild hemophilia B in the Amish and others: founder effect, variability in factor IX activity assays, and rapid carrier detection.

By direct genomic sequencing, we have delineated the causative mutation in 64 families of European decent with hemophilia B. Six (9%) had a C----T transition at base 31008, which substitutes methionine for threonine 296 (T296----M) in the catalytic domain of factor IX. Five of the patients had the same haplotype (frequency of 16% in the northern European population). These individuals are of Amish/German descent and they are likely to share a common ancestor. The sixth patient had a different haplotype, which indicates that his mutation had an independent origin. The data highlight the importance of clinical criteria for the classification of hemophilia B. All six patients had clinically mild disease and their factor IX coagulant activities were in the range of 3%-6% when tested simultaneously in one laboratory, yet the factor IX activities provided with patient records varied 40-fold. Due to the high frequency of this mutation, we have utilized the technique of polymerase chain reaction amplification of specific alleles (PASA) to perform rapid and inexpensive carrier diagnoses in the families with this mutation. This is of particular importance for the Amish since the mutation should account for much of, if not all, the mild hemophilia B that is commonly found in this population.

Base Sequence↗
PubMed2006

Delayed diagnosis of congenital factor IX deficiency (Christmas disease) in a girl with Turner's Syndrome.

Patients with Turner's syndrome are at risk of X-linked recessive disorders. We report a case of a young girl with Turner's syndrome with persistent mildly abnormal coagulation studies associated with a mild to moderate bleeding diathesis. The abnormalities were initially attributed to intrahepatic cholestasis and were partially responsive to vitamin K. After an interval of several years an episode of unexplained iron deficiency anaemia prompted re-investigation of the mild coagulopathy. Disproportionate reduction in the factor IX concentration and restoration of haemostasis with factor IX concentrate lead to a revised provisional diagnosis of mild haemophilia B which was subsequently confirmed by sequencing the factor IX gene.

Anemia↗
PubMed1993

Haemophilia B due to a de novo insertion of a human-specific Alu subfamily member within the coding region of the factor IX gene.

A de novo insertion of an Alu repeated DNA element was found within exon V of the factor IX gene in a patient with severe haemophilia B. The element interrupts the reading frame of the mature factor IX at glutamic acid 96 resulting in a stop codon within the inserted sequence. The Alu repeat is 322 bp long, and the 5' region is shortened by 38 bp. The insertion created a target site duplication of 15 bp consistent with retroposition, and contains a pure polyadenine tract of at least 78 resides at the 3' end. The nucleotide sequence agrees with a consensus for an Alu subfamily which is evolutionarily the most recently inserted, suggesting that it is an exact copy of a putative source gene. These observations indicate that retroposition of Alu elements is a continual process and a mechanism for generating human genetic defects.

Base Sequence↗
PubMed1997

Haemophilia B in female twins caused by a point mutation in one factor IX gene and nonrandom inactivation patterns of the X-chromosomes.

Haemophilia B is a X-linked recessive bleeding disorder with an incidence of 1:25,000-30,000 male birth. Usually female carriers are clinically normal. Phenotypic expression of the disease in female carriers is extremely rare. We describe cytogenetically inconspicuous female identical twins both with factor IX levels below 2%, prolonged bleeding after venipuncture as well as haematomas after intramuscular injections. The father, suffering from a severe haemophilia B, is deceased. By sequencing one point mutation was characterized in heterozygote condition in the factor IX gene of the probands at nt 17678. This mutation leads to the substitution cystein 88 to tyrosine in the growth factor domain of the factor IX. Investigation of the X-chromosomal inactivation by comparison of methylation patterns of genomic DNA at locus DXS255 after digestion with Pst I and Pst I +Hha I and hybridisation with the probe M27beta indicated a nonrandom pattern of X-chromosomal inactivation in the twins. In both girls, only the paternal X-chromosome was the active one leading to the phenotypic expression of haemophilia in the female carriers.

Blood Coagulation Tests↗
PubMed1985

Localization by in situ hybridization of the coagulation factor IX gene and of two polymorphic DNA probes with respect to the fragile X site.

The coagulation factor IX gene and two other polymorphic loci corresponding to DNA probes 52 A and St 14 have been previously localized in the q27 to qter region of the human X chromosome. In order to study their localization with respect to the fragile site at Xq27-28, we have hybridized the three DNA probes to metaphase chromosomes of a boy with fragile X mental retardation. We show that probe 52 A is located in the proximal part of the Xq27 band, while the coagulation factor IX gene is on the distal part of this band, but proximal to the fragile site. The very polymorphic St 14 probe is located in the distal part of the Xq28 band, on the other side of the fragile site.

Chromosome Banding↗
PubMed1987

Determination of factor IX allotypes for carrier identification in haemophilia B.

The existence of two genetic variants (allotypes) of normal human factor IX is used for carrier detection in three families with severe and one with mild haemophilia B. By analysis of IX:Ag with two different monoclonal antibodies in 93 members of the families, allelic assignment is shown to be a complement in carrier diagnosis to genotypic DNA studies. Allelic assignment makes possible a reliable diagnosis based on phenotypic studies, though its usefulness is limited due to ethnic variation in allelic frequency. Determination of factor IX allotypes should be useful for carrier detection in many Swedish families with haemophilia B.

Alleles↗
PubMed1979

A test method for the absence of thrombogenicity in factor IX complex.

Consumption coagulopathy was developed in three cases of liver cirrhosis and a case of haemophilia B upon administration of Factor IX preparations which had been tested by the manufacturers for the absence of active clotting factors according to the Japanese Minimum Requirements for Biological Products. By employing the TGT determination, however, active clotting factor(s) could be detected in some of the preparations used in these cases. Accordingly, it was found necessary to modify the current test method in the Minimum Requirements by introducing calcium ion into the test medium. There was no correlation between the clotting activitiy detected by our modified test method and the Factor IX potency of the product. Addition of heparin did not significantly influence the results in our modified method.

Adult↗
PubMed2003

Prophylactic dosing of factor VIII and factor IX from a clinical pharmacokinetic perspective.

The high cost and limited availability of factor concentrates make dosing of factor VIII (FVIII) or factor IX (FIX) a crucial issue in the prophylactic treatment of haemophilia. It has often been recommended that this treatment should aim to maintain a minimum plasma level of 1% of normal coagulation factor activity (FVIII:C or FIX:C). The dosage needed is commonly given as 25-40 U kg(-1) three times weekly for FVIII or twice weekly for FIX. However, these guidelines are valid only with several qualifications. First, the actual trough levels required may vary considerably between patients. The clinical severity of haemophilia may depend on more factors than the endogenous level of FVIII:C or FIX:C. Secondly, interindividual variations in dose requirements are also due to variance in the pharmacokinetics of the coagulation factors. Pharmacokinetic calculations are useful to design optimal dosing schedules to achieve required trough levels of FVIII:C or FIX:C. Moreover, tailoring of the dosing of FVIII or FIX according to their disposition in the individual patient can markedly improve the cost-effectiveness of prophylactic treatment. However, the usefulness of in vivo recovery as a guide for prophylactic dosing seems questionable. It should be clearly understood that maintaining a certain trough level of FVIII:C or FIX:C is not an end in itself. Clinical outcome, not the achieved trough level, determines whether a dosage is adequate. Chiefly for economic reasons, the minimum effective dosage of coagulation factor should be determined and used in every patient. The dose requirement should also be re-evaluated at appropriate times.

Drug Administration Schedule↗
PubMed1990

An outbreak of non-A, non-B hepatitis associated with the infusion of a commercial factor IX complex during cardiovascular surgery.

An outbreak of non-A, non-B hepatitis was recognized among cardiovascular surgical patients from one hospital in June 1985. Illness was found to be significantly associated with a commercial brand of factor IX complex given to patients because of intraoperative bleeding. A change in the commercial brand of factor IX stocked by the hospital pharmacy had occurred in October 1984 and coincided with the onset of the outbreak. A retrospective study of cardiovascular surgery patients identified 23 cases and 7 probable cases of non-A, non-B hepatitis among patients who had received infusions of brands A and B factor IX complex. Three cases were in brand A recipients and 27 were in brand B recipients. Respective brand-specific attack rates were 5 and 42% (relative risk = 7.7; p less than 2 x 10(-5); chi 2 test). Nineteen of 30 case patients (63%) were jaundiced, including 2 brand A recipients and 17 brand B recipients. Median peak serum aminotransferase was 690.5 IU (range 27-2,824). The incubation period for cases in brand B recipients was a median of 7 weeks (range 2-17 weeks). Prevention of non-A, non-B hepatitis in this population requires adhering to strict indications for the use of clotting factor preparations and avoiding these products when at all possible. Heat treatment of clotting factor products may reduce the risk of viral disease transmission, but certain methods may not inactivate the causative agents of non-A, non-B hepatitis.

Arizona↗
PubMed1992

High efficient transfer and expression of human clotting factor IX cDNA in cultured human primary skin fibroblasts from hemophilia B patient by retroviral vectors.

To study the possibility of somatic gene therapy for hemophilia B via gene transfer to primary factor IX-deficient skin fibroblasts, we constructed four retroviral vectors containing factor IX cDNA driven by retroviral LTR promoter, SV40 early promoter and mouse MT-I promoter, respectively. These retroviral vectors were transfected into an amphotropic packaging cell line, PA317 cells, by electroporation, and a human fibrosarcoma cell line, HT1080 cells, was used to assay the factor IX-virus titers of these four virus-producing PA317 cells, which ranged from 2 x 10(4) to 5 x 10(5) cfu/ml. The factor IX proteins produced by bulk population of four virus-producing PA317 cells were determined by ELISA. Results showed that LTR promoter directed the highest production of factor IX at the rate of 584 ng/10(6) cells/24 h, while SV40 early promoter and MT promoter directed about 10 and 20 times less production of factor IX than LTR promoter. The highest expressed retroviral vector XL-IX was used to infect a line of factor IX-deficient human primary skin fibroblasts, FDIX cells. The factor IX secretion rate of the infected FDIX cells was about 549 ng/10(6) cells/24 h and over 75% of secreted factor IX was biologically active. We are convinced that this factor IX-deficient human primary skin fibroblast had been cured, or genetically corrected, by retroviral-mediated gene therapy in vitro.

Adult↗
PubMed1990

Two factor IX mutations in the family of an isolated haemophilia B patient: direct carrier diagnosis by amplification mismatch detection (AMD).

Rapid identification of gene defects allows definite carrier and prenatal diagnosis in virtually every family with haemophilia B. We report a study of the family of an isolated patient. Analysis of all the essential regions of the patient's factor IX gene (promoter, exons, transcript processing signals) revealed two mutations: one C----T transition at residue 17762 and another at residue 30890. The former created a translation stop at codon 116, and the latter caused substitution of His 257 by Tyr. The translation stop is an obvious detrimental mutation, while the His 257----Tyr substitution has uncertain functional consequences. From analysis of other family members, it was found that the first mutation had occurred at grandpaternal gametogenesis, in keeping with the negative family history, while the second was of more remote origin and did not reduce the maternal grandfather's factor IX coagulant and antigen level. This neutral mutation (His 257----Tyr) pinpoints a poorly conserved amino acid in factor IX and related serine proteases. Its coexistence with a detrimental mutation stresses the need to examine all essential regions of a gene before attempting to interpret the functional consequences of its sequence changes.

Antigens↗