Identification of factor IX mutations in Iranian haemophilia B patients by SSCP and sequencing

Different kinds of mutations, mostly point mutations, in the coagulation factor IX (FIX) gene F9 result in a recessive X-linked bleeding disorder known as haemophilia B. In this study, molecular analysis of 76 unrelated Iranian haemophilia B patients was performed by PCR, single strand conformational polymorphism (SSCP) on important functional regions of the F9 gene followed by sequencing on samples with different migration pattern. Using this approach we found mutation in 52 out of 76 patients. Our data showed that the pathologic mechanisms are heterogeneous as recorded for patients in haemophilia B mutation database and seven of the mutations are previously undescribed.     

Factor IX gene analysis in 70 unrelated patients with haemophilia B: description of 13 new mutations

Seventy unrelated patients suffering from haemophilia B have been screened for determining the molecular defect and for evaluating the spectrum of factor IX mutations in the Rh?ne Alpes region in France. Most patients were characterized with respect to factor IX antigen and factor IX coagulant activity. We have used denaturing gradient gel electrophoresis to obtain a full scanning of the whole coding, promoter, and exon flanking sequences of the factor IX gene. This technique enabled us to determine the molecular defect in 68 out of 70 families (97%), and the mutation was further identified in the two last patients with a direct sequencing of the gene. A total of 2 complete gene deletions in patients with antifactor IX inhibitor, 6 small insertions/deletions and 62 point mutations were found. Two of these nucleotide substitutions (Arg145His and Ala233Thr) were detected in 21 patients (30%) suggesting the existence of a local founder effect. Thirteen mutations were previously undescribed, including 7 missense mutations. The detection of mutations in patients affected with haemophilia B may shed some light in the structure-function relationship of factor IX molecule within the coagulation system.     

Factor IX activity and factor IX antigen in haemophilia B carriers

Examinations performed on the female relatives of haemophilia B patients suggest that factor IX activity is significantly reduced in the carriers of haemophilia B. In the carriers of haemophilia B^? the amount of factor IX antigen is in correlation with factor IX activity, while in the carriers of the B⁺ variant the amount of factor IX antigen is appreciably higher in relation to factor IX activity. Since the occurence of the B⁺variant appears to be rare, family studies and statistical analysis of the results suggest that in the majority of cases haemophilia B carriers can most probably be detected on the basis of factor IX activity.     

Three distinct point mutations in the factor IX gene of three Japanese CRM+ hemophilia B patients (factor IX BMNagoya 2, factor IX Nagoya 3 and 4)

Enzymatic DNA amplification and complete sequence analysis were used to investigate human factor IX coding sequences in three CRM+ hemophilia B patients. In a patient with severe hemophilia B and a markedly prolonged ox-brain prothrombin time, a C to T transition in exon VI changed the codon for Arg180 to Trp (factor IX BMNagoya 2). This mutation would impair the cleavage by factor XIa required for activation of the zymogen. In a patient with mild hemophilia B, a G to A transition in exon VI changed the codon for Arg145 to His(factor IX Nagoya 3). This substitution also would be predicted to preclude the cleavage of factor IX by factor XIa at this peptide bond (Arg145-Ala146). Furthermore, this point mutation creates a new NlaIII restriction site which provides a quick and reliable method for carrier detection in the affected family members. A patient with severe hemophilia B (factor IX Nagoya 4) had a G to A transition in exon II changing the codon for Glu21 to Lys. This novel point mutation is assumed to impair the function of factor IX by disrupting the calcium binding of factor IX.     

Incidence of factor IX inhibitor development in severe haemophilia B patients treated with only one brand of high purity plasma derived factor IX concentrate.

Fifteen previously untreated patients (Pups) with severe haemophilia B (factor IX activity < or = 2 U/dl) only treated with one brand of plasma-derived high purity factor IX concentrate (FIX LFB) were studied. Age at first injection varied from 1 to 137 months and follow-up since this first injection from 21 to 86 months (median: 35). Cumulative exposure days (CED) were from 4 to over 100 (median: 26). Among these 15 Pups only one developed an inhibitor. Mutation analysis performed in all patients showed total gene deletion in the patient with inhibitor, partial gene deletion in another one, and missense mutations in 9 families. Mutation was not found in one patient. Actually, according to the data already published, only two patients were at high risk for inhibitor development in our population. Our study, although rather small, confirms the previously reported low incidence of inhibitors in haemophilia B. Large studies on incidence of FIX inhibitors are indeed difficult to perform, due to both the overall small number of severe haemophilia B patients and the low incidence of FIX inhibitors. Consequently, the impact of bias, such as prevalence of different types of gene defects in a given population, is major. Therefore, any study, dealing with incidence of FIX inhibitors in severe haemophilia B should report, for each patient, the type of gene defect.     

Characterisation of factor IX with a glycine-to-valine missense mutation at residue 190 in a patient with severe haemophilia B

A patient with severe haemophilia B with a glycine-to-valine missense mutation at residue 190 (c25, chymotrypsin numbering) in factor IX (FIX; FIX-G190V or FIX-FuChou) had <1% of normal FIX clotting activity and 36% of normal FIX antigen levels (cross-reacting material- reduced, CRMr). Residue 190 in the C-terminal protease domain of human FIX is highly conserved in mammalian species and the serine protease family, suggesting that it has an indispensable role in protein function. To explore the pathological mechanism by which this mutation contributes to dysfunction of the FIX molecule, we functionally characterised FIX-G190V in vitro and in vivo. Liver-specific FIX-G190V gene expression following hydrodynamic plasmid delivery into haemophilia B mice revealed a 5.7-fold reduction in specific clotting activity compared with FIX-WT (wild type) and a two-fold decrease in plasma FIX-G190V concentration. Pulse-chase analysis demonstrated that FIX-G190V was secreted at a significantly slower rate than was FIX-WT. Purified FIX-G190V and FIX-WT displayed normal calcium-dependent conformational changes as shown by intrinsic fluorescence quenching. The in vivo half-lives of FIX-G190V and FIX-WT were indistinguishable. FIX-G190V was, however, more readily degraded than FIX-WT, especially after being activated by the active form of FXI. The vulnerable sites were mapped to the peptide bonds at Arg11?-Leu11?, Lys2??-Tyr2??, Arg32?-Val32?, and Arg33?-Ser33?, which are in the exposed loops of the FIX molecule. Also, failure of FXIa-activated FIX-G190V to bind p-aminobenzamidine indicated an abnormal conformation of the active-site pocket. Thus, the mutation at residue 190 of FIX may result in protein misfolding that affects secretion, clotting function, and hydrolysis.     

Hemophilia B with mutations at glycine-48 of factor IX exhibited delayed activation by the factor VIIa-tissue factor complex

Gly-48 is in the conserved DGDQC sequence (residues 47-51 of human factor IX) of the first EGF (EGF-1)-like domain of factor IX. The importance of the Gly-48 is manifested by two hemophilia B patients; factor IXTainan and factor IXMalmo27, with Gly-48 replaced by arginine (designated IXG48R) and valine (IXG48V), respectively. Both patients were CRM+ exhibiting mild hemophilic episodes with 25% (former) and 19% (latter) normal clotting activities. We characterize both factor IX variants to show the roles of Gly-48 and the conservation of the DGDQC sequence in factor IX. Purified plasma and recombinant factor IX variants exhibited approximately 26%-27% normal factor IX's clotting activities with G48R or G48V mutation. Both variants depicted normal quenching of the intrinsic fluorescence by increasing concentrations of calcium ions and Tb3+, indicating that arginine and valine substitution for Gly-48 did not perturb the calcium site in the EGF-1 domain. Activation of both mutants by factor XIa appeared normal. The reduced clotting activity of factors IXG48R and IXG48V was attributed to the failure of both mutants to cleavage factor X: in the presence of only phospholipids and calcium ions, both mutants showed a 4 to approximately 7-fold elevation in Km, and by adding factor VIIIa to the system, although factor VIIIa potentiated the activation of factor X by the mutants factor IXaG48R and factor IXaG48V, a 2 to approximately 3-fold decrease in the catalytic function was observed with the mutant factor IXa's, despite that they bound factor VIIIa on the phospholipid vesicles with only slightly reduced affinity when compared to wild-type factor IXa. The apparent Kd for factor VIIIa binding was 0.83 nM for normal factor IXa, 1.74 nM for IXaG48R and 1.4 nM for IXaG48V. Strikingly, when interaction with the factor VIIa-TF complex was examined, both mutations were barely activated by the VIIa-TF complex and they also showed abnormal interaction with VIIa-TF in bovine thromboplastin-based PT assays. Taken together, our results suggest that mutations at Gly-48 altered the interaction of factor IX with its extrinsic pathway activator (VIIa-TF complex), its macromolecular substrate (factor X), and its cofactor (factor VIIIa).     

Two mutations of the factor IX gene including a donor splice consensus deletion and a point mutation in a Dutch patient with severe hemophilia B

The abnormal factor IX gene of a patient with severe hemophilia B (hemophilia B Ursem) was selected for study. All of the coding and their flanking regions and parts of the 5'- and 3'-untranslated regions of the factor IX gene were amplified from the patient's genomic DNA by using the polymerase chain reaction (PCR). By analyzing the nucleotide sequence of the PCR products we have identified two mutations in the patient's factor IX gene, viz. a tetranucleotide deletion (GAGT, nt 6492 to 6495) or (TGAG, nt 6491 to 6494) in the 5'-donor splice site consensus at the exon 2-intron B boundary, and a point mutation at nucleotide 31103 in the catalytic domain (exon 8) of factor IXa, which changes the codon for valine 328 (GTT) to one for isoleucine (ATT). PCR-amplified exon 8 from 45 normal males and 55 normal females had the codon for valine-328. We propose that the deletion within the donor splice-site consensus is the cause of the disease in this individual, whereas the substitution of valine-328 by isoleucine may be a neutral variant which is, at least, very rare in the normal population. In a family study the DNA sequence of the patient's mother shows both the G to A transition in exon 8 and the 5'-donor splice consensus deletion in intron B in one allele.     

An important role for the activation peptide domain in controlling factor IX levels in the blood of haemophilia B mice

The factors responsible for the removal of injected factor IX (fIX) from the blood of individuals with haemophilia B are only partly understood, and may include binding to endothelial or subendothelial sites, passive extravasation related to size or charge, or interactions requiring fIX activation. To investigate these issues, we have produced and characterised recombinant fIX proteins with amino acid changes: delta155-177, an internal deletion which removes most of the activation peptide while retaining the activation cleavage sites; S365A, which inactivates the serine protease activity of fIXa; and K5A, previously shown to eliminate fIX binding of endothelial/subendothelial collagen IV. All proteins were expressed in stably transfected HEK 293 cells, purified by immunoaffinity chromatography, and compared to the wild type HEK 293-derived protein (fIX (WT)). Mutant fIX proteins K5A and delta155-177 exhibited 72 and 202% of the specific activity of fIX (WT), respectively; S365A was without activity. Following intravenous injection in haemophilia B (fIX knockout) mice, recoveries did not differ for fIX (WT) and delta155-177, but were higher for K5A and S365A.The terminal catabolic half-life of delta155-177, alone among the mutants, was increased, by 45% versus fIX (WT). Nine hours post-injection, the observed areas under the clearance curve (AUCs) of delta155-177 and K5, but not S365A, were elevated 2-fold. delta155-177 was equally effective as fIX (WT) in reducing blood loss following tail vein transection in haemophilia B mice. Our results suggest that deletion of the multiple sites of fIX post-translational modification found within the activation peptide eliminated important fIX clearance motifs.     

A simple method for analyzing factor IX activation in the patients with hemophilia B variants

A simple method for analyzing the activation mechanism of FIX in patients with hemophilia B variants is described. The procedure consists of rapid partial purification of FIX by BaCl2 adsorption-elution from only 3 ml of plasma, incubation with FXIa/Ca2+, SDS-PAGE, western blotting and subsequent autoradiography using monoclonal anti-FIX antibody. Abnormal FIX from the plasma of 7 unrelated patients with hemophilia BR, B+ or BM was investigated. A time course study showed that FIX in the patient with hemophilia BM (Nagoya I), BM (Nagoya II) and B Kawachinagano seemed not to be cleaved by FXIa, FIX in the patient with hemophilia B Kashihara was partially cleaved, FIX in the patient with hemophilia BM (Takatsuki) showed delayed cleavage, and that FIX in the patient with hemophilia BM (Niigata) and BM (Kiryu) was cleaved completely at a rate similar to normal FIX. These findings were identical to those previously observed for the respective factors in a purified system. The procedure used here is useful for screening for a defective activation mechanism of abnormal FIX.     

Electroimmunoassay of factor IX in hemophilia B.

In order to detect genetic variants of hemophilia B an electroimmunoassay of factor IX antigen was established by means of a precipitating rabbit antiserum against human factor IX. Plasma cross-reacting material was determined in 13 patients with hemophilia B. Seven patients had no detectable cross-reacting material whereas six patients had values from 18 to 108%. The values obtained were compared with values for cross-reacting material as determined by an antibody-neutralization test giving a coefficient of correlation of 0.81. A reaction of identity was observed between normal factor IX antigen and cross-reacting material from the patients. Our results confirm earlier observations of genetic heterogeneity in hemophilia B and indicate a close antigenic relationship between normal factor IX antigen and cross-reacting material.     

Absence of correlation between X chromosome inactivation pattern and plasma concentration of factor VIII and factor IX in carriers of haemophilia A and B

Haemophilia A and B are X-linked disorders which are due to a reduced activity of coagulation factor VIII or IX, respectively. Female carriers have a wide range of plasma concentration of factor VIII or factor IX, and may in rare cases have an affected phenotype. In order to investigate if this variation is related to X chromosome inactivation, we determined the X inactivation pattern in 31 haemophilia A and 15 haemophilia B carriers, using a PCR in the androgen receptor locus in blood DNA. Seven of the haemophilia A carriers and none of the haemophilia B carriers had a skewed pattern (> or =80:20). One of the skewed haemophilia A carriers had a low plasma concentration of factor VIII (0.15 U/ml), but the remaining 6 carriers did not differ in factor VIII concentration from that of carriers with a random X inactivation pattern. One carrier with a high factor VIII concentration (2.0 U/ml) did not have a skewed pattern. Similarly, for the haemophilia B carriers, there was no tendency to a more skewed X inactivation pattern in the carriers with low or high factor IX concentrations. In addition, we analysed a female with haemophilia B who was heterozygous for the mutation R180W in the factor IX gene. She had a random X chromosome inactivation pattern. We conclude that the wide range in plasma concentration of factor VIII and factor IX in haemophilia A and B carriers cannot in general be explained by the X chromosome inactivation pattern in peripheral blood cells.     

Novel ATP7B gene mutations in Chinese Han patients with hepatolenticular degeneration

BACKGROUND: ATP7B gene exon 8 Arg778Leu and exon 12 Arg952Lys are gene mutation hot spots in Chinese Han patients with hepatolenticular degeneration, or Wilson's disease (WD). However, the gene fragments are too short for detection and the mutation detection rate remains low.OBJECTIVE: To analyze DNA sequences of ATP7B gene exon 8-exon 9 and exon 10-exon 12 sections. DESIGN, TIME AND SETTING: A concurrent, non-randomized, controlled, genetic polymorphism study was performed at the Anhui Medical Genetics Center, Anhui, China from March to July in 2009.PARTICIPANTS: Fifty patients, who were admitted to the Department of Neurology at the First Affiliated Hospital of Anhui Traditional Chinese Medical College between March and July in 2009, were diagnosed with WD. The WD group comprised 32 males and 18 females, with an average age of (18.8 ± 8.3) years. WD was confirmed by clinical observation, as well as physical, imaging, and biochemical examinations, including testing for serum copper, ceruloplasmin, and copper oxidase. The control group comprised 20 normal subjects, who underwent physical examination at the First Affiliated Hospital of Anhui Traditional Chinese Medical College, and included 13 males and 7 females, with an average age of (27.9 ± 2.4) years. All subjects were Chinese Han population.METHODS: Genomic DNA was extracted from 50 WD patients and 20 normal controls. Polymerase chain reaction amplification of ATP7B gene exon 8-exon 9 (about 1 100 bp) and exon 10-exon 12 (about 850 bp) segments was performed. DNA exon-intron amplification products from all subjects were processed through direct bidirectional sequencing, and sequencing results were analyzed. MAIN OUTCOME MEASURES: Sequence changes of ATP7B gene exon 8-exon 9 and exon 10-exon 12 segments.RESULTS: In the 50 included WD patients, ATP7B gene intron 8 nt53592A → G with nt53671G → A homozygous mutation was detected between exon 8-exon 9 in seven cases; exon 8 Arg778Leu mutations with Leu770Leu synonymous mutation was detected in four cases; exon 11 Gly790Arg heterozygous missense mutation between exon 10-exon 12 was found in four cases; exon 12 Arg952Lys heterozygous missense mutation was seen in 11 cases; and two additional cases were associated with exon 12IIe929Val polymorphism.CONCLUSION: ATP7B gene intron 8 mutation is a possible pathogenic mutation that is associated with WD pathogenesis. The exon 11 mutation rate accounts for 8% of all WD patients, and the very few previously reported cases deserve further study.     

Purification and characterization of human factor IX.

Human Factor IX has been purified from plasma by a procedure involving DEAE-Sephadex chromatography, affinity chromatography on heparin-Sepharose gel and gel filtration on Sephadex G-200. The final preparation was homogeneous according to a number of criteria and the specific activity corresponded to a 12 000 fold purification from plasma. The molecular weight was 72000 and only tyrosine was found as the N-terminal amino acid. The amino acid composition resembled that of prothrombin. The carbohydrate content was 22.8 %. No change in molecular weight was observed upon polyacrylamide gel electrophoresis in 8 M urea containing mercaptoethanol indicating that Factor IX is a single polypeptide chain crosslinked by disulphide bonds.     

Detection of known haemophilia B mutations and carrier testing by microarray

The molecular basis of haemophilia B is heterogeneous and many mutations of the Factor IX (FIX) gene have been characterised. Using the allele-specific arrayed primer extension (AS-APEX) technology, we have designed a FIX array to simultaneously analyse 69 mutations found in British, Thai and Chinese patients. This technology overcomes the problem of multiple reverse dot-blot analysis and has a 100% accuracy in the detection of both affected subjects and carriers in families with known mutations. In seven unknown mutations from Thailand, the array could detect the specific mutation in five and in the remainders the normal primer at specific spots failed to extend due to a mutation a few nucleotides upstream, thus allowing their identification. Hence this FIX array can detect 53% of the 2891 mutation entries in the FIX database. Each of the microarray slide can be used for three different test samples and would be useful for carrier testing for common mutations and prenatal diagnosis. It is simpler and more cost effective than genome sequencing and would be particularly useful in laboratories with limited technical capabilities.     

Characterization of a mouse factor IX cDNA and developmental regulation of the factor IX gene expression in liver

A mouse factor IX cDNA was isolated and characterized. The cDNA was 1,837 bp in length and contained the coding region as well as short 5' and 3' untranslated sequences. Northern blot analysis of liver RNA showed two mRNA species of 3.2 kb (major) and 2.2 kb (minor) for the mouse factor IX. An antisense RNA probe prepared from the mouse cDNA was employed to determine the steady state level of factor IX mRNA in mouse liver at various developmental stages. The factor IX mRNA level was very low (2-5% of the adult level) during the gestational period until day -3 (gestational day 17) followed by a rapid increase at day -2 through birth. This phase of rapid increase was followed by a gradual increase before it reached the adult level at around 20 to 24 days. At birth, the factor IX mRNA level was found to be at about 43% of that of the adult. The mRNA levels in mouse liver agreed well with the plasma factor IX activity levels. These results indicate that reduced factor IX activity in newborns is due to the low levels of factor IX mRNA available for translation.     

Heterogeneity of factor IX in therapeutic factor IX concentrates

Rabbit antibody to human factor IX was used to investigate the factor IX antigenic content and electrophoretic mobility of commercial products as well as experimental “activated products”. Rocket immunoelectrophoresis of all concentrates showed a 1.2–3 fold increased antigenic content/unit factor IX clotting activity when compared to plasma. Two dimensional crossed immunoelectrophoresis of standard Factor IX concentrates produced a single sharp peak whether electrophoresed in Ca²⁺ or EDTA containing buffer. “Activated” concentrates produced a dome shaped precipitin arc. The addition of plasma to standard factor IX concentrates yielded a marked shoulder only when the electrophoresis was run in EDTA. This effect could not be reproduced by the addition of antithrombin III (AT-III). The addition of plasma to some “activated” products revealed an even more pronounced heterogeneity whether in Ca²⁺ or EDTA and the addition of AT-III in the same products produced a second precipitin peak. These results indicate that at least three forms of factor IX exist in Factor IX concentrates. The absence of detectable AT-III reacting material in the standard concentrates is $\text{a priori}$ evidence of the absence of major amounts of IXa, whereas the presence of AT-III reacting material in the “activated” concentrates is evidence of biologically active material.