Hemophilia B (factor IXSeattle 2) due to a single nucleotide deletion in the gene for factor IX.

To understand the molecular basis for hemophilia B in patients with little or no circulating Factor IX antigen, a patient who had less than 0.2% circulating Factor IX antigen (Factor IXSeattle 2) was selected for analysis of his Factor IX gene. Genomic DNA fragments from the abnormal gene were cloned into bacteriophage lambda vectors and recombinant phage were identified using radiolabeled genomic probes obtained from the normal Factor IX gene. The exons and flanking regions of the abnormal gene were sequenced by the dideoxy chain-termination method and this sequence was compared with that of the normal gene. Only one significant difference was observed, the deletion of a single adenine nucleotide in exon V. This resulted in a frameshift that converted an aspartic acid at position 85 in the protein to a valine and the formation of a stop signal at position 86. These data indicate that the gene for Factor IXSeattle 2 codes for an 85 residue polypeptide that terminates after the first epidermal growth factor domain. Thus, the putative Factor IXSeattle 2 polypeptide lacks the second epidermal growth factor domain, the activation peptide, and the catalytic domain present in the normal protein. This provides an explanation for the coagulation disorder in this patient and represents the first report of a single nucleotide deletion and frameshift resulting in hemophilia B.     

变性梯度凝胶电泳检测到四例新的FⅨ基因突变

目的：研究凝血因子Ⅸ的基因突变。方法：应用聚合酶链反应（ＰＣＲ）和变性梯度凝胶电泳（ＤＧＧＥ）筛选技术，分析了１２例血友病Ｂ患者ＦⅨ基因的全部８个外显子，剪接区及３′，５′端部分非编码区，并对ＤＧＧＥ行为异常片段进行双脱氧链终止法ＤＮＡ序列分析。结果：发现４例新的基因突变，分别位于ＦⅨ基因第１号外显子Ｔ（ＴＧＴ）１１１Ｇ（ＧＧＴ），导致Ｃｙｓ－１９Ｇｌｙ；第３号内含子剪接位点Ｃ１０３８０Ｔ和第８号外显子Ａ（ＴＡＣ）３０９１８Ｇ（ＴＧＣ），导致Ｔｙｒ２６６Ｃｙｓ；第８号外显子Ａ（ＡＣＧ）３１００７Ｃ（ＣＣＧ），导致Ｔｈｒ２９９Ｐｒｏ。结论：阐明血友病Ｂ的点突变有助于进一步了解ＦⅨ蛋白各功能域的精细功能，并为血友病Ｂ家系携带者检测和产前诊断提供了一个重要的遗传信息。     

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

A partial gene product was identified in a pedigree with hemophilia B due to a partial deletion of the Factor IX gene (Chen, S.-H.,S. Yoshitake, P.F. Chance, G.L. Bray, A.R. Thompson, C.R. Scott, and K. Kurachi, 1985, J. Clin. Invest., 76:2161-2164). Levels of this mutant protein in plasma of affected family members studied ranged from 24 to 36 ng/ml (0.6-0.9 U/dl or percent of normal) by a solid-phase immunoassay which is sensitive and specific for the calcium-dependent conformation of human Factor IX. No Factor IX antigen could be detected in patients' plasmas by a non-calcium-requiring monoclonal anti-Factor IX antibody (less than 2 ng/ml). The unconcentrated urine from the five affected family members and four obligate heterozygotes the five affected family members and four obligate heterozygotes tested contained calcium-dependent Factor IX antigen levels ranging from 64 to 160 ng/ml (1.6-4.0 U/dl) and from 10 to 68 ng/ml (0.25-1.7 U/dl), respectively. Of nine normal volunteers screened, three had detectable calcium-dependent antigen in unconcentrated first morning-voided urines with 9.6-16.8 ng/ml (0.24-0.42 U/dl), while the remaining six had detectable urinary antigen only after a 10-fold concentration. Abnormal and normal urinary Factor IX antigen species were concentrated, immunoaffinity purified, electrophoresed, immunoblotted, and distinguished by autoradiography after incubation with 125I-polyclonal calcium-requiring anti-Factor IX. After reducing purified or concentrated samples, a single abnormal 36,000-mol-wt band was identified in the urines from the four affected family members and four obligate heterozygotes tested. Electrophoresis of the reduced urinary Factor IX antigen from the one normal subject tested showed a broad 15,000-20,000-mol-wt band. This normal band was smaller than the species in patients' urines, and was seen as a minor component in the samples from the heterozygotes. No abnormal antigen could be detected in urine from the two other female family members tested. Thus, abnormal urinary Factor IX antigen represents a marker for the presence of the hemophilic Factor IX gene in this family.     

DNA sequence analysis of three inhibitor-positive hemophilia B patients without gross gene deletion: identification of four novel mutations in factor IX gene

Three hemophilia B patients with anti-factor IX antibodies who had no detectable gross deletion of the factor IX gene by Southern blotting analysis were investigated at the molecular level. All eight exons, accompanied by their splicing junction sites and presumptive promoter regions of the factor IX gene in these patients (total 5.5 kb in length) were amplified with the use of the polymerase chain reaction, followed by complete nucleotide sequence analysis. Three different types of novel single base substitutions and a 2 base-pair nucleotide deletion were identified. Patient HB-5 had two point mutations in his factor IX gene. One was located at the promoter region at nucleotide -793 and the other (C-to-T transition) was found in exon VI of the gene changing Gln-191 to a stop codon. Patient HB-6 had a point mutation (G-to-A) in the splice acceptor site, which interrupted the normal splicing of the last intron G. A small two-nucleotide deletion in exon III was detected in patient HB-7 and yielded frameshifted amino acids and terminated by a stop codon. These resuslts suggest that not only the gross gene deletion of factor IX gene but also the point mutations or small nucleotide deletion that may cause the interruption of coding informations for mature protein synthesis is predisposed to development of anti-factor IX inhibitors in patients with hemophilia B.     

Hemophilia B (Christmas disease) variants and carrier detection analyzed by DNA probes.

We have used two strategies to study 14 hemophilia B families from 11 kindreds for possible carrier detection and prenatal diagnosis. First, we sequentially used the Factor IX probes (sequentially with restriction enzymes Taq I, Xmn I, and Dde I), and the linked probes p45h (Taq I), p45d (Pst I), and 52a (Taq I) for restriction fragment length polymorphism (RFLP) analysis. Second, we searched for useful variant Taq I digestion fragments using the Factor IX complementary DNA. Two separate new Taq I variants in exon VIII were identified. Using both strategies, 11 of 14 families (from 9 of 11 kindreds) were informative for further studies. In five kindreds studied in detail, the carrier status of all 11 at risk females was determined and prenatal diagnosis could be offered to the offsprings of each of the six carriers identified. Thus, in this study, we have identified a higher proportion of informative families than has previously been reported.     

Potential role of recombinant factor VIIa as a hemostatic agent

Recombinant factor VIIa (rFVIIa) has been shown to induce hemostasis in hemophilia patients with inhibitors against factor VIII or factor IX independent of factor VIII/factor IX. Factor VIIa binds to tissue factor (TF) exposed at the site of injury and generates, through factor X activation on the TF-bearing cells, enough thrombin to activate factors VIII, V, and XI, as well as platelets. The thrombin-activated platelets provided a perfect template for binding of activated factors VIII, IX, and V, further activation of factor X, and thrombin generation. Factor VIIa in high concentrations binds to thrombin-activated platelets and is capable of activating factor X, thereby generating thrombin independent of the presence of factor VIII or factor IX. Accordingly, rFVIIa has been shown to initiate hemostasis in severe hemophilila patients with inhibitors subjected to major surgery and suffering from serious limb- and life-threatening bleeding. Since rFVIIa enhances thrombin generation-thereby providing the formation of tight, stable fibrin hemostatic plugs resistance to premature lysis-it should be hemostatic in other situations characterized by impaired thrombin generation. A hemostatic effect has been reported in patients with various platelet disorders and factor XI deficiency. Further, a hemostatic effect of rFVIIa has been reported in patients subjected to trauma and extensive surgery who have developed profuse, excessive bleeding resulting in hemodilution and changes in coagulation patterns. rFVIIa was developed to treat bleeding in hemophilia patients with inhibitors against factor VIII or factor IX and has been shown to induce effective hemostasis in most such patients and also in life- and limb-threatening bleeding. It has also been used successfully to stop bleeding in patients who do not have hemophilia but who do have acquired antibodies against factor VIII (acquired hemophilia). rFVIIa initiates hemostasis by forming a complex with TF exposed as a result of vessel wall injury. Pharmacologic doses of rFVIIa can enhance thrombin generation on platelets that are already thrombin-activated, resulting in the formation of full thrombin burst. By enhancing thrombin generation, rFVIIa helps to form tight, stable, fibrin plugs resistant to premature fibrinolysis. This also maintains hemostasis in the absence of factor VIII or factor IX. Pharmacologic doses of rFVIIa may accordingly be of benefit in producing hemostasis in situations other than hemophilia characterized by profuse bleeding and impaired thrombin generation. There is now clinical experience indicating a hemostatic effect in patients with thrombocytopenia and functional platelet defects. rFVIIa has also been successfully used in acute trauma patients with profuse bleedings and in other bleeding situations.     

Genotyping of resistance to thyroid hormone in South American population. Identification of seven novel missense mutations in the human thyroid hormone receptor β gene

Thyroid Hormone Receptor β (THRB) defects, typically transmitted as autosomal dominant traits, cause Resistance to Thyroid Hormone (RTH). We analyzed the THRB gene in thirteen South American patients with clinical evidence RTH from eleven unrelated families. Sequence analysis revealed seven novel missense mutations. Four novel mutations were identified in exon 9. The first, a c.991A>G transition which originates a substitution of asparagine by aspartic acid (p.N331D). The second nucleotide alteration consists of a guanine to cytosine transversion at position 1003 (c.1003G>C) and results in substitution of the alanine at codon 335 by proline (p.A335P). The third mutation, a c.1022T>C transition produces a change of leucine by proline (p.L341P). The fourth mutation detected in exon 9 was a c.1036C>T transition which replaces the leucine at codon 346 by phenylalanine (p.L346F). The sequencing of the exon 10 detected three novel missense mutations. The first, a c.1293A>G transition changing isoleucine 431 for methionine (p.I431M). The second, the cytosine at position 1339 was replaced by adenine (c.1339C>A) resulting in the replacement of proline by threonine (p.P447T). The third mutation detected in exon 10 was a c.1358C>T transition resulting in the substitution of proline at codon 453 by leucine (p.P453L). Finally, sequencing analysis of the THRB gene revealed three substitutions previously described (p.A268G, p.P453T and p.F459C). The p.P453T was found in two patients.In conclusion, we report thirteen patients with RTH caused by heterozygous mutations of the THRB gene. Seven of the identified mutations correspond to novel substitutions.     

Heterogeneity of the factor IX locus in nine hemophilia B inhibitor patients.

DNA from nine hemophilia B patients who produce anti-factor IX inhibitors (antibodies), including two brothers, was analyzed by the Southern blotting method and hybridization with factor IX cDNA, intragenomic, and 3'-flanking probes. Two inhibitor patients were shown to have total deletions of the factor IX gene. Two other inhibitor patients, the brothers, were shown to have a presumably identical complex rearrangement of the factor IX gene involving two separate deletions. The first deletion is of approximately 5.0 kb and removes exon e. The second deletion is between 9 and 29 kb and removes exons g and h but leaves exon f intact. An abnormal Taq I fragment at one end of the deletion junctions acted as a marker for the inheritance of hemophilia B in the patients' family. Five other inhibitor patients have a structurally intact factor IX gene as detected by this method. Our studies indicate that whereas large structural factor IX gene defects predispose hemophilia B patients to developing an anti-factor IX inhibitor, the development of an inhibitor can be associated with other defects of the factor IX gene.     

Pharmacokinetics and pharmacodynamics of a humanized monoclonal antibody to factor IX in cynomolgus monkeys

The pharmacokinetics and pharmacodynamics (PK/PD) of a humanized anti-Factor IX IgG1 monoclonal antibody (SB 249417, FIX mAb) were studied in Cynomolgus monkeys. Single i.v. bolus doses of 1, 3, or 10 mg/kg of FIX mAb were administered. The total FIX mAb concentration, activated partial thromboplastin time (aPTT), and Factor IX activity were monitored for up to 4 weeks after dosing. In the monkey, FIX mAb had a plasma clearance of 0.6 ml/h/kg and a steady-state volume of distribution of approximately 70 ml/kg. The elimination phase half-life (3.8 days) was considerably less than other humanized IgG1 mAbs in the monkey, for which there is no binding to endogenous antigen. The suppression of Factor IX activity and the prolongation of aPTT were rapid and dose dependent. The time for aPTT values to return to basal levels (25-170 h) increased with increasing dose. A mechanism-based PK/PD model consistent with the stoichiometry of binding (2:1) was developed to describe the Factor IX activity and aPTT response time course. The model incorporated Factor IX synthesis and degradation rates that were interrupted by the sequestration of Factor IX by the antibody. aPTT values were related to free Factor IX activity. This model was able to describe the PD profiles from the three dose levels simultaneously. The estimated Factor IX half-life was 11 h and the third-order association rate constant was 3.96 x 10(3) microM(-2) h(-1). The PK/PD modeling was useful in summarizing the major determinants (endogenous and antibody-ligand binding) controlling FIX mAb-related effects.     

Technology evaluation: AAV factor IX gene therapy, Avigen Inc

Avigen has developed a recombinant adeno-associated viral vector expressing human blood-coagulation Factor IX (F.IX) for the potential treatment of hemophilia B. In a phase I clinical trial being conducted at The Children's Hospital of Philadelphia and Stanford University Medical Center, the vector, AAV-CMV-hF.IX (Coagulin-B), was injected at a low dose into three patients with severe hemophilia B. No evidence of toxicity, germline transmission of vector sequences, or formation of inhibitory antibodies against F.IX was observed, and in two of the three patients there was an indication of a modest clinical response.     

Characterization of the defect in activation of factor IX Chapel Hill by human factor XIa.

Factor IXChapel Hill (Factor IXCH), an abnormal Factor IX molecule isolated from the plasma of a patient with mild hemophilia B, has previously been shown to exhibit delayed activation by Factor XIa and calcium. In this study, we have found that Factor IXCH is cleaved upon incubation with human Factor XIa and calcium; however, cleavage of this protein is not observed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis under nonreducing conditions. Under reducing conditions, the rate of disappearance of the zymogen parallels both the appearance of the heavy chain and the generation of clotting activity. In addition, a protein band that migrates with an apparent molecular weight of 45,000 also increases in parallel with clotting activity. Factor IXCH and normal Factor IX (Factor IXN), after incubation with Factor XIa and calcium, were subjected to amino terminal sequence analysis. Activated Factor IXN is cleaved at an arginine-alanine (Arg-Ala) bond and an arginine-valine (Arg-Val) bond as demonstrated by formation of the three amino terminal sequences corresponding to the amino terminal of the light chain, heavy chain, and activation peptide. However, activated Factor IXCH has only two amino terminal sequences, corresponding to the original amino terminal sequence and the heavy chain (formed by cleavage at the Arg-Val bond). It is concluded that the major defect in Factor IXCH is the inability of Factor XIa to cleave the Arg-Ala bond at a significant rate. The rate of formation of clotting activity of Factor IXCH is approximately 60% of the rate of formation of clotting activity of Factor IXN. The specific clotting activity of activated Factor IXCH is between 20 and 33% of activated Factor IXN.     

Purification and characterization of an abnormal factor IX (Christmas factor) molecule. Factor IX Chapel Hill.

Human Factor IX (Christmas factor) was isolated from the plasma of a patient with mild hemophilia B. The patient's plasma contained 5% Factor IX clotting activity but 100% Factor IX antigenic activity as determined by immunological assays, which included inhibitor neutralization and a radioimmunoassay for Factor IX. This abnormal Factor IX is called Factor IX Chapel Hill (Factor IXCH). Both normal Factor IX and Factor IXCH have tyrosine as the NH2-terminal amino acid. The two proteins have a similar molecular weight, a similar amino acid analysis, the same number of gamma-carboxyglutamic acid residues (10 gamma-carboxyglutamic acid residues), and a similar carbohydrate content. Both exist as a single-chain glycoprotein in plasma. The major difference between normal Factor IX and Factor IXCH is that the latter exhibits delayed activation to Factor IXa in the presence of Factor XIa and Ca2+. Thus, Factor IXCH differs from other previously described abnormal Factor IX molecules.     

Factor IX antigen by radioimmunoassay. Abnormal factor IX protein in patients on warfarin therapy and with hemophilia B.

Factor IX, isolated from normal human plasma, was homogenous by polyacrylamide gel electrophoresis in urea and sodium dodecyl sulfate. On the latter, it migrated as a single polypeptide chain with or without reducing agents and had an apparent mol wt of 62,000. After iodination by chloramine-T, a single peak of 125I was found on gels. Immunoelectrophoresis in agarose with rabbit antifactor IX sera gave a single arc against both isolated and partially purified factor IX preparations. The rabbit antibody was specific as it failed to inhibit the activities of prothrombin or factors VII or X in normal plasma. At an additional 20-fold dilution, factor IX activity was inhibited 50%. In a double-antibody radioimmunoassay, excess rabbit anti-human factor IX precipitated 90-95% of the 125I-human factor IX. Control without specific antibody gave 6-8%. Dilutions of a pool of normal human plasma paralleled dilutions of the isolated preparation and were used for the standard curve. Of 39 plasma samples from normal donors, the mean factor IX antigen level was 93% of that of a separate normal pool. The radioimmunoassay detected the abnormal factor IX produced in patients on warfarin therapy. After Al(OH)3 adsorption of warfarin treated patient's plasma, factor IX antigen, but not activity, was present in the supernate. Samples from 28 patients on warfarin gave a mean factor IX clotting activity of 27% with a mean antigen of 69%. The antigen level from the warfarin group was significantly lower than the antigen level of the normal group (P less than 0.001). The factor IX antigen level was then assessed in 36 patients from 29 pedigrees with hemophilia B. The median antigen level was 17% of normal. The distribution of the antigen level was wide with two patients around 100% of normal; only two had levels below the limits of resolution of the radioimmunoassay as currently performed (less than 2%). Within each of the five pedigrees in which more than one affected member was tested, activity and antigen levels were the same. The degree of neutralization of the antibody's inhibition of normal plasma by patient's plasma was highly correlated. Additional evidence for the detection of abnormal protein was provided by immunodiffusion of plasmas concentrated by lyophilization. Reactions of complete identity occurred between normal, a warfarin treated and a hemophilia B subject's plasmas.     

Replacing the first epidermal growth factor-like domain of factor IX with that of factor VII enhances activity in vitro and in canine hemophilia B.

Using the techniques of molecular biology, we made a chimeric Factor IX by replacing the first epidermal growth factor-like domain with that of Factor VII. The resulting recombinant chimeric molecule, Factor IXVIIEGF1, had at least a twofold increase in functional activity in the one-stage clotting assay when compared to recombinant wild-type Factor IX. The increased activity was not due to contamination with activated Factor IX, nor was it due to an increased rate of activation by Factor VIIa-tissue factor or by Factor XIa. Rather, the increased activity was due to a higher affinity of Factor IXVIIEGF1 for Factor VIIIa with a Kd for Factor VIIIa about one order of magnitude lower than that of recombinant wild-type Factor IXa. In addition, results from animal studies show that this chimeric Factor IX, when infused into a dog with hemophilia B, exhibits a greater than threefold increase in clotting activity, and has a biological half-life equivalent to recombinant wild-type Factor IX.     

Abnormality of helper/suppressor T cell ratio in patients with hemophilia

Lymphocyte subpopulations in patients with hemophilia were analyzed. The results were compared with those in age- and sex-matched controls. The patients had been receiving blood and blood products including Factor VIII or Factor IX for a number of years as required at the time of the onset of the disease. Heparinized peripheral blood was used in direct tests for cell marker analysis with OKT4, OKT8 and OKT11 monoclonal antibodies. There were less OKT4+ cells in the hemophiliac blood as compared with the controls, while both the groups had the same number of OKT8+ cells. As a result, the OKT4/OKT8 ratio was markedly depressed in the hemophiliac group and the conversion of the OKT4/OKT8 ratio was observed in the group of patients receiving long-term therapy with anti-hemophiliac products. Alteration of lymphocyte subpopulations in patients with hemophilia in relation to antihemophiliac therapy was discussed.     

Molecular basis of severe factor XI deficiency in seven families from the west of France. Seven novel mutations, including an ancient Q88X mutation

Summary.  Inherited factor (F)XI deficiency is a rare disorder in the general population, though it is commonly found in individuals of Ashkenazi Jewish ancestry. In particular, two mutations—a stop mutation (type II) and a missense mutation (type III)—which are responsible for FXI deficiency, predominate. The bleeding tendency associated with plasma FXI deficiency in patients is variable, with ∼ 50% of patients exhibiting excessive post-traumatic or postsurgical bleeding. In this study, we identified the molecular basis of FXI deficiency in 10 patients belonging to six unrelated families of the Nantes area in France and one family of Lebanese origin. As in Ashkenazi Jewish or in French Basque patients, we have identified a new ancient mutation in exon 4 resulting in Q88X, specific to patients from Nantes, that can result in a severely truncated polypeptide. Homozygous Q88X was found in a severely affected patient with an inhibitor to FXI and in three other unrelated families, either as homozygous, heterozygous or compound heterozygous states. Other identified mutations are two nonsense mutations in the FXI gene, in exon 7 and 15, resulting in R210X and C581X, respectively, which were identified in three families. A novel insertion in exon 3 (nucleotide 137 + G), which causes a stop codon, was characterized. Finally, sequence analysis of all 15 exons of the FXI gene revealed three missense mutations resulting in G336R and G350A (exon 10) and T575M (exon 15). Two mutations (T575M and G350A) with discrepant antigen and functional values are particularly interesting because most of the described mutations are associated with the absence of secreted protein.     

Whole blood clot formation phenotypes in hemophilia A and rare coagulation disorders. Patterns of response to recombinant factor VIIa

Summary. Until now, no routinely used clotting assay has demonstrated the power to reflect significantly a patient's response to recombinant factor (rF)VIIa. Adopting a thrombelastographic principle, profiles of continuous whole blood (WB) coagulation were studied in minimally altered WB activated with a small amount of tissue factor (TF). Investigation of the WB clotting profile was performed before and after ex vivo addition of rFVIIa 20 nm to WB from 26 patients with hemophilia A, two patients with severe hemophilia B, and individuals with deficiencies of FV, FX, FXI, and FXIII. In five patients with hemophilia plus inhibitors, the response to ex vivo added rFVIIa and to activated complex concentrate (APCC) was studied. Patients with severe and moderate hemophilia A demonstrated remarkable variance in the hemostatic characteristics at baseline, even in groups with the same FVIII:C activity levels. The response to rFVIIa at 20 nm also varied extensively, the effect correlating with the continuous WB coagulation phenotype at baseline. This indicates that the efficacy of rFVIIa may be optimized by tailoring the dose according to the hemostatic response to varying doses tested prior to in vivo administration. In patients with inhibitors against FVIII and factor IX, rFVIIa and APCC substitution resulted in quite similar response patterns that appeared to be dose dependent. In severe FV, FX, and FXIII‐deficient WB, rFVIIa addition induced minor changes only. In FXI deficiency, rFVIIa normalized the dynamic properties of clotting, although a reduced clot firmness remained unchanged. In conclusion, the thrombelastographic analysis of WB clotting, as activated with a minute amount of TF, seems an interesting method that detects phenotypic variation amongst hemophilia patients. The method appears useful for assessment of the hemostatic capacity and it seems a promising tool for evaluation of the individual response to rFVIIa or APCC before and during in vivo administration.     

Characterization of the clotting activities of structurally different forms of activated factor IX. Enzymatic properties of normal human factor IXa alpha, factor IXa beta, and activated factor IX Chapel Hill.

Two structurally different forms of activated human Factor IX (Factor IXa alpha and IXa beta) have been previously reported to have essentially identical clotting activity in vitro. Although it has been shown that activated Factor IX Chapel Hill, an abnormal Factor IX isolated from the plasma of a patient with mild hemophilia B, and normal Factor IXa alpha are structurally very similar, the clotting activity of activated Factor IX Chapel Hill is much lower (approximately fivefold) than that of normal Factor IXa beta. In the present study we have prepared activated Factor IX by incubating human Factor IX with calcium and Russell's viper venom covalently bound to agarose. Fractionation of the activated Factor IX by high-performance liquid chromatography demonstrated the presence of both Factors IXa alpha and IXa beta. On the basis of active site concentration, determined by titration with antithrombin III, the clotting activities of activated Factor IX Chapel Hill and IXa alpha were similar, but both activities were less than 20% of the clotting activity of Factor IXa beta. Activated Factor IX activity was also measured in the absence of calcium, phospholipid, and Factor VIII, by determination of the rate of Factor X activation in the presence of polylysine. In the presence of polylysine, the rates of Factor X activation by activated Factor IX Chapel Hill, Factor IXa alpha, and Factor IXa beta were essentially identical. We conclude that the clotting activity of activated Factor IX Chapel Hill is reduced when compared with that of Factor IXa beta but essentially normal when compared with that of Factor IXa alpha.     

Update on gene therapy for hereditary hematological disorders

The past 3 years have been characterized by a number of impressive advances as well as setbacks in gene therapy for genetic disease. Children with X-linked severe combined immunodeficiency disorder (SCID-X1) have shown almost complete reconstitution of their immune system after receiving retrovirally transduced autologous CD34+ hematopoietic stem cells (HSCs). However, two of 11 treated patients subsequently developed a leukemia-like disease probablydue to the undesired activation of an oncogene. Gene transfer to HSCs resulted in substantial correction of immune function and multi-lineage engraftment in two patients with adenosine deaminase (ADA)-SCID. Several Phase I clinical trials for treatment of hemophilia A and B have been initiated or completed. Partial correction of hemophilia A, albeit transient, has been reported by ex vivo gene transfer to autologous fibroblasts. Intramuscular injection of adeno-associated viral (AAV) vector to patients with severe hemophilia B resulted in evidence of Factor IX gene transfer to skeletal muscle and a separate trial based on hepatic infusion of AAV vector is ongoing. Sustained therapeutic levels of coagulation factor expression have been achieved in preclinical models using retroviral, lentiviral, AAV and high capacity adenoviral vectors. Efficient lentiviral gene transfer to HSC in murine models of beta-thalassemia and sickle cell disease demonstrated sustained phenotypic correction.     

Seven novel mutations in the factor XIII A-subunit gene causing hereditary factor XIII deficiency in 10 unrelated families.

BACKGROUND: Hereditary factor (F)XIII deficiency is a rare bleeding disorder mostly due to mutations in FXIII A subunit. OBJECTIVES: We studied the molecular basis of FXIII deficiency in patients from 10 unrelated families originating from Israel, India and Tunisia. METHODS: Exons 2-15 of genomic DNA consisting of coding regions and intron/exon boundaries were amplified and sequenced. Structural analysis of the mutations was undertaken by computer modeling. RESULTS: Seven novel mutations were identified in the FXIIIA gene. The propositus from the Ethiopian-Jewish family was found to be a compound heterozygote for two novel mutations: a 10-bp deletion in exon 12 at nucleotides 1652-1661 (followed by 22 altered amino acids and termination codon) and Ala318Val mutation. The propositus of the Tunisian family was homozygous for C insertion after nucleotide 863 within a stretch of six cytosines of exon 7. This insertion results in generation of eight altered amino acids followed by a termination codon downstream. The propositus from Indian-Jewish origin was found to be homozygous for G to T substitution at IVS 11 [+1] resulting in skipping of exons 10 and 11. In addition to the Ala318Val mutation, three of the novel mutations identified are missense mutations: Arg260Leu, Thr398Asn and Gly210Arg each occurring in a homozygous state in an Israeli-Arab and two Indian families, respectively. CONCLUSIONS: Structure-function correlation analysis by computer modeling of the new missense mutations predicted that Gly210Arg will cause protein misfolding, Ala318Val and Thr398Asn will interfere with the catalytic process or protein stability, and Arg260Leu will impair dimerization.