Genetic mapping of DNA segments relative to the locus for the fragile-X syndrome at Xq27.3.

We have tested linkage between the locus for the fragile-X [fra(X)] syndrome at Xq27.3 and five polymorphic restriction sites identified by four DNA probes mapping distal to Xq26.1. A maximum distance of approximately 15 centimorgans (cM) between Xq27.3 and the marker loci mapping to this region was predicted based on the physical chromosome length. Close linkage between the disease and marker loci was excluded for probes DXS19 and DXS37 (theta = .05, Z = -2.94 and Z = -4.17, respectively). These marker loci were estimated to be less than five cM apart but approximately 40 cM proximal to the fragile site, indicating that there is a significantly greater frequency of recombination in this region of the X chromosome than expected from the physical length. Linkage results for the other marker loci and the fra(X) syndrome were inconclusive. However, the pX45d probe locus appears very closely linked to the factor IX locus (Z = 1.94 at theta = 0) and is approximately 20 cM proximal to Xq27.3. A relative map of the polymorphic restriction sites, fra(X) syndrome locus, and factor IX locus was constructed by maximizing lod scores over the Xq26.1----q27.3 region.     

Factor VIII and factor IX in a twin population. Evidence for a major effect of ABO locus on factor VIII level.

In order to establish the relative importance of genetic factors on the variation in plasma concentration of coagulation factors VIII and IX, these parameters were determined in 74 monozygotic and 84 like-sexed dizygotic twin pairs. The twins belonged to two age groups: 33-39 years and 57-62 years. Factor VIII was determined as factor VIII coagulant antigen (VIIICAg) and as factor VIII-related antigen (VIIIRAg). Factor IX was determined as factor IX antigen (IXAg). A higher value for each coagulation factor was found in the older-age group compared to the younger group, whereas no difference was found between the sexes. A significant correlation was found between values for VIIIRAg and VIIICAg (r = .56). For VIIICAg, it could be demonstrated that the age effect was secondary to the age effect on VIIIRAg. The concentration of VIIICAg and VIIIRAg varied among ABO blood types, being lowest in type O individuals, higher in A2 individuals, and highest in A1 and B individuals. The effect of the ABO locus on VIIICAg was secondary to an effect on VIIIRAg. Analysis of variance revealed a significant genetic influence on the variance of VIIICAg and VIIIRAg with a heritability estimate of .57 for VIIICAg and .66 for VIIIRAg. This is in agreement with a previous hypothesis of an effect of several autosomal genes on factor VIII concentration. Thirty percent of the genetic variance of VIIIRAg was due to the effect of ABO blood type. The ABO locus is therefore a major locus for the determination of factor VIII concentration. No significant genetic effect on the variation in plasma concentration of IXAg could be detected.     

A complete deletion of the factor IX gene and new TaqI variant in a hemophilia B kindred

Summary We report a hemophilia B kindred in which the proband has a complete deletion of the factor IX gene extending a minimum of 80 kilobase pairs (kb) 3 of the gene. This individual has severe factor IX deficiency with no detectable circulating factor IX protein. In common with one previous report, despite a total deletion of the factor IX gene, this patient has not developed antibodies to factor IX. The mother of the proband was found to have a new TaqI variant of the factor IX gene on the nondeletion-bearing X chromosome. The location of the altered TaqI site was found to be 5 of exon IV between residues 9731-9734 and does not affect the function of the factor IX protein. The familial natures of both the variant allele and the deletion were established. In addition a study of this kindred at the DXS99 locus demonstrated the first reported recombination event between this site and the factor IX gene.     

Linkage heterogeneity and fragile X

Summary A multipoint test of heterogeneity on published data from 57 families with the fragile X syndrome has been undertaken. The hypothesis being tested was that there are two loci coding for fragile X expression, mutations at either of which can produce the phenotype. No predivision of the families was undertaken, as the test used an admixture parameter. Maximum likelihoods of the hypothesis have been calculated and compared with those produced on assuming a single locus for fragile X. The data do not suggest that there are two such loci within the interval between probes 52a and St14. In particular, the large kindred published by Camerino et al. (1983) does not supply convincing evidence of heterogeneity under this test. It is argued that the observed heterogeneity between factor IX and fragile X must have another explanation. There is some evidence for a second locus for fragile X outside the interval noted above; this locus being most probably proximal to these probes. The majority of the data suggesting this result comes from a family published by Davies et al. (1985).     

Identification of amino acids in the factor XI apple 3 domain required for activation of factor IX

Activated coagulation factor XI (factor XIa) proteolytically cleaves its substrate, factor IX, in an interaction requiring the factor XI A3 domain (Sun, Y., and Gailani, D. (1996) J. Biol. Chem. 271, 29023-29028). To identify key amino acids involved in factor IX activation, recombinant factor XIa proteins containing alanine substitutions for wild-type sequence were expressed in 293 fibroblasts and tested in a plasma clotting assay. Substitutions for Ile(183)-Val(191) and Ser(195)-Ile(197) at the N terminus and for Ser(258)-Ser(264) at the C terminus of the A3 domain markedly decreased factor XI coagulant activity. The plasma protease prekallikrein is structurally homologous to factor XI, but activated factor IX poorly. A chimeric factor XIa molecule with the A3 domain replaced with A3 from prekallikrein (FXI/PKA3) activated factor IX with a K(m) 35-fold greater than that of wild-type factor XI. FXI/PKA3 was used as a template for a series of proteins in which prekallikrein A3 sequence was replaced with factor XI sequence to restore factor IX activation. Clotting and kinetics studies using these chimeras confirmed the results obtained with alanine mutants. Amino acids between Ile(183) and Val(191) are necessary for proper factor IX activation, but additional sequence between Ser(195) and Ile(197) or between Phe(260) and Ser(265) is required for complete restoration of activation.     

Comparison of factor IX methylation on human active and inactive X chromosomes: implications for X inactivation and transcription of tissue-specific genes.

Maintenance of dosage compensation for housekeeping genes on the human X chromosome is mediated through differential methylation of clustered CpG nucleotides associated with these genes. To determine if methylation has a role in maintaining inactivity of X-linked genes which show tissue-specific expression, we examined the locus for blood clotting Factor IX. The analysis encompassed 91% of the HpaII and HhaI sites in the 41-kb region that includes the presumed promoter region, 5 kb of 5'- and 4 kb of 3'-flanking sequences. Although there are sex differences in methylation of the locus in leukocytes, the methylation pattern in liver, where the gene is expressed, is essentially the same for loci on the active and inactive X chromosome. The lack of differences in methylation of active and inactive genes makes it unlikely that methylation within the locus has a role in expression of the Factor IX gene. These findings, along with the absence of clustered CpG dinucleotides within the Factor IX locus, suggest that functional differences in DNA methylation related to X chromosome dosage compensation may be limited to CpG clusters. In any event, dosage compensation seems to be maintained regionally, rather than locus by locus.     

Anticoagulant mechanism of factor IX/factor X-binding protein isolated from the venom of Trimeresurus flavoviridis

Anticoagulant mechanism of the coagulation factor IX/factor X-binding protein (IX/X-bp) isolated from the venom of Trimeresurus flavoviridis was investigated. IX/X-bp had no effect on the amidase activity of factor Xa measured with a synthetic peptide substrate Boc-Leu-Gly-Arg-pNA. Prothrombin activation by factor Xa without cofactors, such as factor Va and phospholipids, was only slightly influenced by IX/X-bp. However, prothrombin activation by factor Xa in the presence of factor Va resulted in IX/X-bp inhibiting the increase of k(cat) of thrombin formation through inhibition of interaction between factor Xa and factor Va. IX/X-bp also inhibited the decrease of K(m) for thrombin formation through interaction with phospholipids. Thus, IX/X-bp appears to act as an anticoagulant protein by inhibiting the interaction between factor Xa and its cofactors in the prothrombinase complex by binding to the Gla domain of factor Xa.     

Evidence supporting tight linkage of X-linked Emery-Dreifuss muscular dystrophy to the factor VIII:C gene.

In a large German family with Emery-Dreifuss muscular dystrophy (EDMD) linkage analysis was performed using the factor IX gene (F9), the factor VIII:C gene (F8), the anonymous DNA probe DXS52, and DXS15 as markers. Tight linkage was found between the EDMD locus and the F8 probe (Zmax = 1.19; theta max = 0.00), DXS15 (Zmax = 1.75; theta max = 0.00) and DXS52 (Zmax = 2.26; theta max = 0.00). Weak linkage was found to F9 (Zmax = 0.02; theta max = 0.43). The data from the literature and our results suggest that the gene locus of EDMD is close to F8 (confidence interval theta = 0-0.07). The new linkage data are useful for carrier detection and diagnosis of EDMD patients before onset of major clinical signs.     

The endothelial cell binding determinant of human factor IX resides in the gamma-carboxyglutamic acid domain.

The blood coagulation factor IX(a) binds specifically to a site on endothelial cells with a Kd of 2.0-3.0 nM. A number of previous studies have attempted to define the region(s) of factor IX(a) that mediate this interaction. These studies suggested that there are two regions of factor IX(a), the gamma-carboxyglutamic acid (Gla) domain and the epidermal growth factor like (EGF-like) domains, that mediate high-affinity binding to endothelial cells. Recently, however, the participation of the EGF1 domain has been excluded from the interaction. This indicated that if there was an EGF component of factor IX contributing to the binding affinity, then it must be in the second EGF-like domain. In order to further evaluate this relationship, we performed competitive binding experiments between 125I plasma factor IX and a set of six chimeric proteins composed of portions of factor VII and factor IX. Our data suggest that the high-affinity interaction between factor IX and the endothelial cell binding site is mediated by the factor IX Gla domain and that the factor IX EGF domains are not involved in binding specificity.     

Binding of iron by factor IX. Possible role for beta-hydroxyaspartic acid

In concentrations exceeding 1 mg/ml, bovine factor IX exhibits a pink color that arises from a broad absorption band with a lambda max = 500 nm. Analysis by x-ray fluorescence reveals the presence of iron but no other transition metals in the factor IX preparation. Quantitative analysis by atomic absorption spectroscopy indicates that 1 g atom of iron is bound tightly to 1 mol of factor IX. The iron is removed slowly (t1/2 = 3 h) by EDTA. In contrast, prothrombin binds no detectable iron, and factor X binds less than 0.2 g atom/mol. alpha-Hydroxybutyrate chelates Fe3+ with sufficient stability to preclude formation of [Fe(OH)3]n. It is proposed that factor IX binds iron with physiologically significant affinity and that the beta-hydroxyaspartate residue in factor IX is a chelator for the bound metal.     

Characterization of the functional defect in factor IX Alabama. Evidence for a conformational change due to high affinity calcium binding in the first epidermal growth factor domain

Factor IX Alabama is a factor IX variant in which a glycine has been substituted for Asp47 in the first epidermal growth factor (EGF) domain. The structural defect in factor IX Alabama results in a molecule with 10% of normal coagulant activity. The interactions of immunoaffinity-purified factor IX Alabama with its activator, cofactors, and substrate have been investigated to determine the functional defect in the variant. Factor IX Alabama is activated by factor XIa/calcium at near normal rates. Calcium fluorescence-quenching experiments indicate that high affinity calcium binding in the first EGF domain is not altered in factor IX Alabama. The active site of factor IXa Alabama is fully competent to activate factor X in the absence of calcium when using polylysine as a surface to catalyze the reaction. Factor IXa Alabama has only 64% of normal factor IXa activity in the presence of 300 microM CaCl2 in the polylysine-catalyzed system although apparent high affinity calcium binding constants are similar. Factor IXa Alabama has 52-60% of normal activity in a calcium/phospholipid vesicle system. The addition of factor VIIIa to the phospholipid vesicle system decreases the relative rate of factor IXa Alabama to 18-19% of normal. Three-dimensional computer-aided models of the first EGF domain of normal factor IX and factor IX Alabama indicate no major structural alterations resulting from the glycine substitution for Asp47. The model of the first EGF domain of normal factor IX predicts a calcium-binding site involving Asp47, Asp49, Asp64, and Asp65. Our binding data, however, indicate that Asp47 is not necessary to form the high affinity binding site. We conclude that Asp47 in normal factor IX coordinates to the bound calcium, inducing a conformational change in the molecule essential for proper interaction with factor X and factor VIIIa.     

Two sisters with a distal deletion at the Xq26/Xq27 interface: DNA studies indicate that the gene locus for factor IX is present

Summary Two sisters with premature menopause and a small deletion of the long arm of one of their X chromosomes [del (X)(pterq26.3:)] were investigated with polymorphic DNA probes near the breakpoint. The deleted chromosome retained the factor IX (F9) locus and the loci DXS51 (52A) and DXS100 (pX45h), which are proximal to F9. However, the factor VIII (F8) locus was not present, nor were two loci tightly linked to this locus, DXS52 (St14) and DXS15 (DX13) This deletion refines the location of the F9 locus to Xq26 or to the interface Xq26/Xq27, thus placing it more proximally than has been previously reported. The DNA obtained from these patients should be valuable in the mapping of future probes derived from this region of the X chromosome.     

Physical mapping of probes proximal to the fragile X locus (FRAX) confirms the order F9-DXS105 (cX55.7)-DXS98 (4D8)-FRAXA

A woman with an abnormal karyotype, (46,X,der(X) (pter----q27::q27----q21), was analyzed using DNA probes in the region Xq27----qter. The results indicate that she is trisomic for the Factor IX locus, disomic for the locus DXS105 (cX55.7) and monosomic for the loci DXS98 (4D8), DXS52 (St14) and Factor VIII. This confirms the absence of the region Xq28 in the abnormal chromosome. Furthermore, the presence of only one copy of 4D8 and two copies of cX55.7 places the DXS98 locus distal to Factor IX and closer to the fragile X locus than DXS105.     

Identification and chemical synthesis of a substrate-binding site for factor IX on coagulation factor XIa.

We have previously used monoclonal antibodies to identify an epitope on the heavy chain of factor XIa that is a substrate-binding site for factor IX (Sinha, D., Seaman, F.S., and Walsh, P.N. (1987) Biochemistry 26, 3768-3775; Baglia, F.A., Sinha, D., and Walsh, P.N. (1989) Blood 74, 244-251). To define the factor XIa domain that binds factor IX, we have now screened a panel of factor XI heavy chain-derived synthetic peptides for their capacity to inhibit the formation of an activation peptide reflecting factor IX activation by factor XIa. Peptide Asn145-Ala176 (which is located in the second tandem repeat or A2 domain of the factor XI heavy chain) is a competitive inhibitor of factor IX activation by factor XIa with a Ki of 30 nM, whereas structurally similar peptides in the A1, A3, and A4 domains were required at 10-1000-fold higher concentrations for similar effects, and a synthetic peptide identical with a highly homologous region of the heavy chain A2 domain of prekallikrein (Tyr143-Ala176) had no effect on factor IX activation by factor XIa. Because detailed structural information is lacking, a potential three-dimensional structure for the factor XI A2 domain was calculated based on its sequence information in conjunction with previously determined structural constraints. The resulting structure depicted three juxtaposed beta-stranded stem-loops that, based on biological information, constitute a candidate surface for contact with factor IX. The A2 model was therefore used as a template in the rational design of three synthetic peptides (Ala134-Ile146 (peptide a), Leu148-Arg159 (peptide b), and Ile160-Leu172 (peptide c]. When peptides a and b or a and c were added together and the activation of factor IX by factor XIa was examined, a synergistic inhibitory effect was observed, compared with each peptide added individually, whereas peptides b and c showed additive effects. Our data suggest that the sequence of amino acids from Ala134 through Leu172 of the heavy chain of factor XI contains three antiparallel beta-strands connected by beta-turns that together comprise a continuous surface utilized for the binding of factor IX.     

A subpopulation of platelets responds to thrombin- or SFLLRN-stimulation with binding sites for factor IXa

Strong agonists cause platelets to expose a procoagulant surface supporting the assembly of two important coagulation enzyme complexes. Equilibrium binding has determined the density of high affinity saturable factor IXa binding sites to be 500-600 sites/platelet. We have now used flow cytometry to visualize the binding of factor IX and IXa to thrombin- or SFLLRN-activated platelets. Concentrations of these agonists that are half-maximal or maximal in kinetic studies resulted in only a small subpopulation (4-20%) of platelets binding factor IX or IXa with the density of binding sites for factor IX being about half of that for factor IXa, consistent with previous equilibrium binding studies. A small subpopulation (5 +/- 1.5%) of platelets stimulated with either agonist also exposed annexin V binding sites, and this subpopulation of platelets also bound factor IXa. Annexin V decreased factor IXa binding in the presence or absence of factor VIIIa, and factor IXa could also decrease annexin V binding on some platelets indicating a common binding site in agreement with previous studies. All platelets binding factor IXa were positive for glycoprotein IX, at the same glycoprotein IX surface density as seen in platelets negative for factor IXa binding. These studies refine the results from equilibrium binding studies and suggest that, on average, only a small subpopulation (approximately 10%) of PAR 1-stimulated platelets expose approximately 6000 factor IXa binding sites/platelet.     

gamma-Carboxyglutamic acids 36 and 40 do not contribute to human factor IX function.

The gamma-carboxyglutamic acid (Gla) domains of the vitamin K-dependent blood coagulation proteins contain 10 highly conserved Gla residues within the first 33 residues, but factor IX is unique in possessing 2 additional Gla residues at positions 36 and 40. To determine their importance, factor IX species lacking these Gla residues were isolated from heterologously expressed human factor IX. Using ion-exchange chromatography, peptide mapping, mass spectrometry, and N-terminal sequencing, we have purified and identified two partially carboxylated recombinant factor IX species; factor IX/gamma 40E is uncarboxylated at residue 40 and factor IX/gamma 36,40E is uncarboxylated at both residues 36 and 40. These species were compared with the fully gamma-carboxylated recombinant factor IX, unfractionated recombinant factor IX, and plasma-derived factor IX. As monitored by anti-factor IX:Ca (II)-specific antibodies and by the quenching of intrinsic fluorescence, all these factor IX species underwent the Ca(II)-induced conformational transition required for phospholipid membrane binding and bound equivalently to phospholipid vesicles composed of phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine. Endothelial cell binding was also similar in all species, with half-maximal inhibition of the binding of 125I-labeled plasma-derived factor IX at concentrations of 2-6 nM. Functionally, factor IX/gamma 36,40E and factor IX/gamma 40E were similar to fully gamma-carboxylated recombinant factor IX and plasma-derived factor IX in their coagulant activity and in their ability to participate in the activation of factor X in the tenase complex both with synthetic phospholipid vesicles and activated platelets. However, Gla 36 and Gla 40 represent part of the epitope targeted by anti-factor IX:Mg(II)-specific antibodies because these antibodies bound factor IX preferentially to factor IX/gamma 36,40E and factor IX/gamma 40E. These results demonstrate that the gamma-carboxylation of glutamic acid residues 36 and 40 in human factor IX is not required for any function of factor IX examined.     

Hydrophobic contact between the two epidermal growth factor-like domains of blood coagulation factor IX contributes to enzymatic activity

The three-dimensional structure of activated factor IX comprises multiple contacts between the two epidermal growth factor (EGF)-like domains. One of these is a salt bridge between Glu(78) and Arg(94), which is essential for binding of factor IXa to its cofactor factor VIII and for factor VIII-dependent factor X activation (Christophe, O. D., Lenting, P. J., Kolkman, J. A., Brownlee, G. G., and Mertens, K. (1998) J. Biol. Chem. 273, 222-227). We now addressed the putative hydrophobic contact at the interface between the EGF-like domains. Recombinant factor IX chimeras were constructed in which hydrophobic regions Phe(75)-Phe(77) and Lys(106)-Val(108) were replaced by the corresponding sites of factor X and factor VII. Activated factor IX/factor X chimeras were indistinguishable from normal factor IXa with respect to factor IXa enzymatic activity. In contrast, factor IXa(75-77)/factor VII displayed approximately 2-fold increased factor X activation in the presence of factor VIII, suggesting that residues 75-77 contribute to cofactor-dependent factor X activation. Activation of factor X by factor IX(106-108)/factor VII was strongly decreased, both in the absence and presence of factor VIII. Activity could be restored by simultaneous substitution of the hydrophobic sites in both EGF-like domains for factor VII residues. These data suggest that factor IXa enzymatic activity requires hydrophobic contact between the two EGF-like domains.     

Proteolytic activation of human factors IX and X by recombinant human factor VIIa: effects of calcium, phospholipids, and tissue factor

Previous studies indicated that factor VIIa, in complex with tissue factor, readily activates either factor X or factor IX in the presence of calcium ions. In order to assess the relative physiological importance of the activation of factor IX versus the activation of factor X by recombinant factor VIIa, we have obtained steady-state kinetic parameters for the factor VIIa catalyzed activation of factor IX and factor X under a variety of cofactor conditions that include calcium alone, calcium and phospholipids, calcium, phospholipids, and tissue factor apoprotein, and calcium and cell-surface tissue factor. Calcium alone stimulated the activation of factors IX and X by factor VIIa maximally at 1 and 2.5 mM, respectively. In the presence of 25 microM phospholipids, maximal rates of factor IX and factor X activation were achieved at 2.5-5 mM calcium. With calcium alone, or with phospholipid and calcium, the initial rates of factor IX activation by factor VIIa were significantly higher than that observed for factor X. Kinetic studies revealed that the Km for the factor VIIa catalyzed activation of factor IX was essentially constant in the presence of 5 mM calcium and 1-500 microM phospholipid, whereas the Km for factor X activation varied with phospholipid concentration, reaching a minimum at 7-20 microM phospholipid. At all concentrations of added phospholipid, the kcat/Km ratio for the activation of factor IX by factor VIIa appeared to be considerably greater than that observed for the activation of factor X.(ABSTRACT TRUNCATED AT 250 WORDS)     

The maternal DDK syndrome phenotype is determined by modifier genes that are not linked to Om

The DDK syndrome is a polar, early embryonic lethal phenotype caused by incompatibility between a maternal factor of DDK origin and a paternal gene of non-DDK origin. Both maternal factor and paternal gene have been mapped to the Om locus on mouse Chromosome (Chr) 11. The paternal contribution to the syndrome has been shown to segregate as a single locus. Although the inheritance of the maternal contribution has not been characterized in depth, it as been assumed to segregate as a single locus. We have now characterized the segregation of the DDK fertility phenotype in over 240 females. Our results demonstrate that females require at least one DDK allele at Om to manifest the syndrome. However, the DDK syndrome inter-strain cross-fertility phenotype of heterozygous females is highly variable and spans the gamut from completely infertile to completely fertile. Our results indicate that this phenotypic variability has a genetic basis and that the modifiers of the DDK syndrome segregate independently of Om. Received: 24 November 1998 / Accepted: 19 January 1999