ATHEROTHROMBOTIC DISORDERS: MUTATION IN THE FACTOR XIII GENE

Abstract: The invention relates to a mutation in the Factor XIII A-subunit gene. The Factor XIII A gene product catalyzes the covalent cross linkage of two glutamate amino acid residues with two lysine amino acid residues in the gamma -chain of fibrin followed by the polymerisation of alpha -fibrin to form a blood clot. This application describes how a point mutation in codon 34 of exon 2 which results in a Valine}Leucine substitution (Factor XIII Val34 Leu) is protective against atherothrombotic disorders and more specifically myocardial infarcation. This application describes a method and a kit for use in clinical diagnosis of thrombotic disorders and preventative prophylactics.

Description: ATHEROTHROMBOTIC DISORDERS : MUTATION IN THE FACTOR X I I I GENE The invention relates to a mutation in the Factor XIII A-subunit gene and knowledge thereof for the diagnosis and treatment of associated diseases and particularly but not exclusively atherothrombotic disorders such as myocardial infarction (MI) and deep vein thrombosis. The importance of the coagulation and fibrinolytic cascades in the pathogenesis of myocardial infarction has been demonstrated in prospective studies. These have identified increased concentrations of fibrinogen and suppression of fibrinolysis due to elevated plasminogen activator inhibitor- 1 (PAI-1) concentrations in relation to atherothrombotic disorders. These findings indicate a tendency to increased coagulation with decreased fibrin breakdown in subjects at risk of myocardial infarction and atherothrombotic stroke. Patients with myocardial infarction form abnormal fibrin structures with tight and rigid fibrin networks in association with suppression of fibrinolysis and it has been suggested that factor XIII might have a role in this process (2). The formation of a stable fibrin clot by covalent crosslinking of the a and γ fibrin chains and the formation of platelet aggregate (3-5) requires binding of fibrinogen and von Willebrand factor to the platelet surface (6,7) and involves factor Xllla catalyzing the covalent crosslinking of two glutamyl with two lysyl residues of the γ-chain of fibrin followed by polymerisation of the α-chain (8- 10). Factor XIII, a transamidase found in plasma is a tetramer of two catalytic A-subunits which circulate in plasma in an inactive form bound to two accessory B-subunits (a2b2) (11,12). Factor XIII is distributed intracellularly in platelets, megakaryocytes and macrophages, (13) and both plasma and platelet forms of FXIIIa selectively cross-link fibrin monomers and other substrates including α-2 antiplasmin (14). The A-subunit gene is localised to chromosome 6, spans more than 160kb and contains 15 exons separated by 14 introns (15). Factor XIII is activated by thrombin which cleaves the A-subunit between Arg 37 and Gly 38 releasing the N-terminal 37 residues. This is followed by its separation from the B-subunit in a reaction facilitated by fibrin and calcium ions (16). Crosslinked fibrin shows increased resistance to fibrinolysis and enhanced mechanical strength (17). Several mutations of the factor XIII A-subunit gene have been described, about 20 of which relate to complete absence of the A- subunit and congenital Factor XIII deficiency (18-22). Recently a G-T point mutation in codon 34 of exon 2 which codes for a Valine→Leucine (Factor XIIIVal34Leu) change in the A subunit close to the thrombin activation site has been described (19-21). Although found in some patients with Factor XIII deficiency, the homozygous form has been detected in individuals without a history of a bleeding tendency, indicating that it is unlikely to be involved in the pathogenesis of this rare condition. However, interference with factor XIII activation and crosslinking of fibrin or other substrates may occur as a result of the proximity of Factor XIIIVal34Leu to the thrombin activation site in the A- subunit, thereby contributing to the pathogenesis of thrombotic disorders. We conducted a case control study to investigate the association between the factor XIIIVal34Leu polymorphism and myocardial infarction and to evaluate interactions with fibrinogen and PAI-1 concentrations and polymorphisms in the genes coding for these proteins. We have suφrisingly found that the Factor XIIIVal34Leu polymorphism is protective against atherothrombotic disorders and more specifically myocardial infarction. It therefore follows that knowledge of this particular genotype can be used to advantage in clinical diagnosis of thrombotic disorders. It is therefore an object of the invention to provide a means of diagnosing the susceptibility to or likely existence of a thrombotic disorder comprising a determination of the point mutation Val34Leu in exon 2 of the A-subunit gene Factor XIII. According to a first aspect of the invention there is therefore provided a method of diagnosing the susceptibility to and/or the likely existence of a thrombotic disorder comprising determining whether the presence of a point mutation in codon 34 of exon 2 of the A-subunit gene of Factor XIII resulting in a valine to leucine substitution and where such a mutation exists concluding that an individual is likely to have at least a degree of protection against a thrombotic disorder; and where such a mutation does not exist concluding that an individual is less likely to have a degree of protection against a thrombotic disorder. It will be apparent to those skill in the art that once the above diagnosis has been made conventional courses of therapy can be adapted accordingly. Thus, in the instance when an individual does not have the Factor XIIIVal34Leu polymorphism then the number, nature and regime of the anti-coagulants may be adapted accordingly. In a preferred method of the invention the diagnosis is undertaken using a polymerase chain reaction. According to a yet further aspect of the invention there is provided a kit for undertaking the diagnosis of the invention which kit includes DNA primers suitable for use in PCR reaction to amplify a region of exon 2 where said mutation is located. Ideally said DNA primers are represented by the following primers; ACCCAGAGTGGTGGGGAAG as a 5' primer; and GACCTTGTAAAGTCAAAAATGTC as a 3' primer; and suitable reaction mixture, SSCP loading buffer; and optionally, an acrylamide/bis, preferably 10%, gel; and optionally, a suitable stain such as silver nitrate. In the preferred kit of the invention controls may be provided such as examples of gels stained to show a wild type, heterozygote or homozygote mutant condition. In addition, or alternatively, said kit may further include test DNA corresponding to a wild type heterozygote or homozygote condition. In this latter instance it is preferred that the test DNA provided with the kit is processed in parallel with test DNA from an individual whose genotype is to be determined. The nature of the invention will now be described by way of example only with reference to the following information wherein: Figure 1 shows the principal structural characteristics of Factor XIII A-subunit polypeptide showing Factor XIIIVal34Leu in relation to thrombin activation site. The lower panel shows the genotype pattern obtained by SSCP; Figure 2 shows cadaverin incoφoration into fibrin in wild-type subjects, subjects heterozygous for the FXIHVal34Leu polymoφhism and those subjects homozygous for FXIIIVal34Leu. Table 1 shows clinical and biochemical characteristics of all patients investigated for the presence of coronary artery disease by angiography (n=398) and normal controls recruited from family practitioners (n=196); Table 2 shows clinical and biochemical characteristics of all patients investigated for the presence of coronary artery disease according to a past history of myocardial infarction (n=197) compared to those without a history of myocardial infarction (n=201); Table 3 shows prevalence of Factor XIII genotypes in subjects investigated for coronary atheroma with and without a past history of myocardial infarction; Table 4 shows haemostatic and other selected cardiovascular risk factors in subjects possessing the Factor XIIIVal34Leu polymoφhism with a history of MI (n=27) and without (n=83) and in homozygous wild type with (n=76) and without (n=89) a history of MI; Table 5 shows prevalence of PAI-1 genotype in patients with and without myocardial infarction and further characterised by Factor XIII genotype; Table 6 shows odds ratios for past history of myocardial infarction in relation to possession of Factor XIIIVal34Leu and other selected risk factors in subjects characterised for presence of atheroma by angiography (n=398); and Table 7 shows the prevalence of Factor XIII genotypes in subjects of different ethnic origins. METHODS Selection of Case Patients and Control Subjects 398 consecutive Caucasian patients admitted for routine angiography for investigation of chest pain and suspected coronary artery disease (CAD) were recruited to the study. 196 healthy Caucasian control subjects were recruited from local Family Health Services Authority general practice registers. Control subjects with no documented history of either stable or unstable angina or myocardial infarction were matched with the case patients for age, and race, (Table 1). All subjects gave informed consent according to a protocol approved by the United Leeds Teaching Hospitals NHS Trust and the Pinderfields Health Trust Ethics Committees. A clinical history was recorded for each patient and normal volunteer. A study of the incidence of MI and the prevalence of the G— T point mutation in Asian Indians and Pima Indians as compared to the 196 healthy Caucasian control population was undertaken using 160 Asian Indian subjects (high incidence of MI) and 304 Pima Indians (low incidence of MI). Samples were collected from Pima Indian subjects as part of a long term epidemiological study of diabetes and its complications in this population (34). Samples were collected from healthy Asian control subjects living in the Leeds and Bradford area as part of a study of thrombotic risk factors in this population. The ethnic origins of the 160 Asian subjects studied were 9 Punjabi, 9 Gujarati, 2 Hindu, 86 Muslim, 5 Bengali and 49 Seikh. There was no significant difference between the genotype frequencies in subjects originating from these different areas of Asia (data not shown). Additionally, the correlation between the incidence of MI and the prevalence of the G— >T point mutation was extended to patients having a history of deep thrombosis. This study used 217 patients known to have this condition compared to 250 age-matched controls. Identification of Coronary Artery Disease and myocardial infarction Results of angiography were reported by cardiologists blind to patient status. Angiographic results were graded as normal, single, double or triple vessel disease based on the presence of ≥ 50% stenosis in a major coronary artery or one of their branches. Myocardial infarction was ascertained by reference to patients' hospital case notes using criteria defined by the World Health Organisation (WHO). At least two of the three following criteria were required to make a diagnosis of myocardial infarction: ST elevation of 1mm in two or more successive leads; typical chest pain longer than 20 minutes duration and a rise in creatinine kinase of more than twice the baseline level. Analysis of circulating factors Venous blood was taken before 9.00am following an overnight fast using a 19G butterfly needle. In the patients, blood for DNA extraction and lipids was collected from all 398 subjects, whilst plasma samples for tests of haemostasis were collected from a subset of 275 subjects who attended one centre. DNA and plasma for tests of haemostasis were available in all 196 control subjects. Samples for measurement of circulating fibrinogen were taken into 0.1M trisodium citrate at room temperature and centrifuged at 2,500g for 15 min at room temperature. Samples for PAI-1 Ag were taken into 0.1M trisodium citrate on an ice/water mixture and separated within 30 min by centrifugation at 2500g for 30 min at 4°C. Plasma samples were snap frozen in liquid nitrogen and stored at -40°C. Fibrinogen levels were determined by the method of Clauss, using a KC10 coagulometer (Amelung) (23). ELISA techniques were used for the estimation of PAI-1 Ag (Biopool, Sweden). Intra-assay and inter- assay coefficients of variation were 2.0% and 3.5% for fibrinogen and 5.0% and 8.0% for PAI-1 Ag respectively. Extraction of DNA and Determination of Genotypes Extraction of DNA: Genomic DNA had been extracted from 10ml of whole blood as described previously (24). Identification of Factor XIII genotype: A 183-bp fragment of exon2/intron B of factor XIII was amplified by the polymerase chain reaction (PCR), with 5'-ACCCAGAGTGGTGGGGAAG as a 5'primer and 5'- GACCTTGTAAAGTCAAAAATGTC as a 3'primer. PCR was carried out using a 25μl reaction mix containing 25pmol of each primer, lOOng DNA, 200μM of each dNTP, lOmM Tris HCl (pH 8.8), l.OmM MgC12, 50mM KC1, 0.1% Triton X-100 and 1U Dynazyme II DNA polymerase (Flowgen, Sittingbourne, Kent) with 30 cycles of 94°C for 1 min, 56°C for 1 min, 72°C for 1 min followed by a final 5 min extension step at 72°C. To detect the substitution of Guanine to Thymine (Val34— >Leu) we used a single stranded conformational polymoφhism (SSCP) technique (Fig 1). Following PCR the amplicons were mixed with SSCP loading buffer, denatured, cooled on ice and loaded onto a vertical gel of 10% acrylamide/bis solution 19:1 (Giro BRL, Life Technologies Ltd., Paisley, England), and electrophoresed at room temperature for 16 hours at 8 W constant power in 0.5 x TBE running buffer. Gels were stained with silver nitrate. Genotype was classified by two independent observers as wild type 1/1 (Val, Val), heterozygote 1/2 (Val, Leu) or homozygote mutant 2/2 (Leu, Leu). Confirmation of genotypes was carried out by sequencing a random selection of samples using dye-labelled terminators (Abi PrismTM Dye Terminator Cycle Sequencing Ready Reaction Kit, Perkin Elemer, Warrington, England) (26) Sequence analyses were performed using an ABI 373A sequencer. There were no discrepancies between genotypes determined by the two methods. Identification of fibrinogen and PAI-1 genotypes: Genotypes at the fibrinogen -455 G/A polymoφhism and the PAI-1 gene 4G/5G promoter polymoφhism were detected as previously described (27,28). Statistical analysis BMI and circulating concentrations of fibrinogen, PAI-1 antigen and triglycerides were log transformed to normalise the distribution and allow analysis by parametric tests. Log transformed results are expressed as geometric mean and anti-logged 95% confidence intervals. All other values are expressed as mean (95% confidence intervals). Data on smoking, hypertension, diabetes and sex were compared by the chi-squared test. Differences in levels between groups were compared using an unpaired Student t-test. Allele frequencies were compared in patients with and without, MI and to controls by gene counting and analysed by the chi-squared test. Logistic regression models, including age, sex, BMI, triglycerides, smoking, hypertension and FXIII genotype as co- variants, were used to identify determinants of myocardial infarction in patients with and without MI, and odds ratios are presented with 95% confidence intervals. Another logistic regression model including BMI, triglycerides, PAI-1 antigen, sex, cholesterol, hypertension and FXIII genotype as covariants, was used to identify determinants of MI in patients with MI and in healthy controls and odds ratios are presented with 95% confidence intervals. A p- value of 0.05 or less was considered to indicate statistical significance. All statistical analyses were performed using the SPSS for windows statistical package (SPSS Inc., Chicago, USA). RESULTS Characteristics of the Study Population All patients and controls: Table 1 shows the prevalence of selected risk factors for coronary heart disease among the age matched 398 patients and 196 controls. Cholesterol, BMI, PAI-1 antigen, and triglycerides levels in all patients were significantly higher than in the controls (p<0.0001). There were no differences in fibrinogen concentrations between the groups. Patients characterised for a past history of myocardial infarction: In the 398 patients who underwent angiography, 25% had normal coronary arteries, and 19%, 21% and 35% had single, double and triple vessel disease, respectively. By WHO criteria 197 subjects had a confirmed history of myocardial infarction and 201 subjects had no history of myocardial infarction. In the group with a history of myocardial infarction, there were significantly more males (p=0.0003) and subjects had increased BMI, (mean, 95%CI) 27.4 (26.8-28.0) v's 26.5 (26.0-27.0), p=0.02 (Table 2). Possession of the PAI-1 4G/4G genotype was significantly associated with myocardial infarction (p<0.01) There were no differences in fibrinogen genotype or levels of PAI-1, cholesterol or triglyceride between groups. Prevalence of Factor XIII Mutation in Patients and Controls Genotype frequencies for patients and controls are shown in Table 3. The factor XIII mutation was found in 258 out of all 594 samples (43%) representing an allele frequency of 24.6%. The distribution of factor XIII genotype was not significantly different between all patients and controls (p=0.3) and there was no relationship between possession of the Factor XIIIVal34Leu polymoφhism and extent of coronary atheroma (data not shown). Prevalence of Factor XIII Mutation in Patients with and without MI There was a significant difference in the genotype distribution of factor XIII comparing patients with and without MI with an increased prevalence of the homozygous wild type in subjects with MI (68% v's 50%) and a lower prevalence of the heterozygous state (27% v's 45%), p=0.0009. (Table 3). Comparing patients with MI to healthy control subjects, there was a significant difference in the genotype distribution (p=0.005). Interactions between Factor XIIIVal34Leu and selected Haemostatic risk factors Table 4 shows selected cardiovascular risk factors in patients subdivided according to possession of Factor XIIIVal34Leu (grouped as homozygous and heterozygous), compared to homozygous wild type and with and without a history of myocardial infarction. There were no differences in any of the risk factors in subjects homozygous for the wild-type, either with or without a history of myocardial infarction. In subjects possessing the Factor XIIIVal34Leu there were significant increases in PAI-1 in subjects with a history of myocardial infarction compared to those without a history, 27.9 (21.1-36.9) v's 16.7 (13.7-20.3) ng/ml, respectively, p=0.004 and there were weakly significant differences in BMI (Table 4). In patients with the Factor XIIIVal34Leu polymoφhism who had sustained a myocardial infarction there were significantly increased numbers of subjects possessing the PAI-1 4G/4G genotype 46% v's 24%, p=0.005, (Table 5). In logistic regression models, construction of an interaction term confirmed this relationship between Factor XIIIVal34Leu and PAI-1 4G/4G genotype. There were no other genotypic associations in any group for PAI-1, or fibrinogen (data not shown). Odds ratios for selected risk factors and myocardial infarction Patients in relation to a history of myocardial infarction: In a logistic regression model comparing patients with MI to those without MI, including age, BMI, triglycerides, smoking, hypertension, sex and FXIII genotype as covariances, age, BMI, sex and FXIII were the only factors significantly associated with MI. Odds ratios (95% confidence intervals) for an increase in 10 years of age was 1.42 (1.39-1.46), p=0.008, for an increase of BMI of lkg/m2: 1.06 (1.00-1.13), p=0.05, for males compared to females: 1.49 (1.2-1.9), p=0.001, and for the possessing the FXIII mutation compared to wild type: 0.67 (0.85-0.54), p=0.0006, (Table 6). Patients with myocardial infarction and healthy controls: In a logistic regression model comparing patients with MI to healthy controls including BMI, triglycerides, PAI-1 antigen, sex, cholesterol, hypertension and FXIII genotype as covariants, sex, PAI-1 antigen, hypertension, cholesterol and FXIII genotype were factors significantly associated with MI. The odds ratios (95% confidence intervals) were for males compared to females 1.52 (1.11-2.10), ρ=0.01, for an increase of lng/ml PAI-1 antigen: 1.04 (1.01-1.06), p=0.0004, for hypertension compared to non-hypertensives: 2.40 (1.07-5.40), p=0.03, for an increase in cholesterol of 1 mmol/1: 1.41 (1.07-1.87), p=0.01 and for those possessing the FXIII mutation compared to wild type: 0.63 (0.87-0.46), p=0.004. There were no differences in levels of any of the circulating factors by factor XIII genotypes. Deep Vein Thrombosis An investigation of the protective properties of the G-→T point mutation has been extended to other thrombotic disorders. In a study of 217 subjects with a history of deep vein thrombosis compared to 215 age-matched controls the FXIIIVal34Leu polymoφhism is protective against deep vein thrombosis with a lower frequency in patients than in controls (p<0.004 unpublished data). Ethnic Differences in Susceptibility to MI The prevalence of the G— >T point mutation in exon 2 of the Factor XIII A- subunit gene in 196 healthy Caucasian control subjects was 41% heterozygotes and 7% homozygotes. This study found a higher prevalence of the protective mutation (48% heterozygotes, 16% homozygotes for the mutation, p<0.00001) in a group of 304 Pima Indians and a lower incidence of the mutation (17.5% heterozygotes, 3.75%, homozygotes for the mutation p<0.00001) in 160 Asian Indian subjects. These results show that the trend in the prevalence of heart disease in these populations is the reverse of the trend in the frequency of this mutation as would be expected for a mutation offering protection from MI. Although the observed changes may reflect random ethnic differences this study does support the hypothesis that the observed differences in Factor XIII genotype frequencies could explain the different incidence of coronary heart disease and myocardial infarcation in these populations. Estimate of Fibrin Crosslinking by Cadaverin Incorporation The degree of fibrin crosslinking was estimated in the three Factor XIII genotypes by measuring cadaverin incoφoration according to ( ). There is a significant correlation between the presence of the FXIIIVal34Leu mutation and the incoφoration of cadaverin into fibrin indicating the suprising observation that the presence of FXIIIVal34Leu may lead to a more stable thrombus once formed. DISCUSSION Our data demonstrate an inverse association between possession of the Val34Leu mutation in exon 2 of the gene encoding the A-subunit of factor XIII and the occurrence of myocardial infarction. These results are consistent with the hypothesis that possession of the Factor XIIIVal34Leu polymoφhism confers a degree of protection against progression to myocardial infarction in subjects with coronary artery atheroma. In those patients with both Factor XIIIVal34Leu and myocardial infarction, there were higher levels of PAI-1 and an increased frequency of the PAI-1 4G/4G genotype to suggest that impaired fibrinolysis negates the postulated protective effects of Factor XIIIVal34Leu. In logistic regression models possession of Factor XIIIVal34Leu was associated with a marked risk reduction giving an odds ratio of 0.67, whilst conventional risk factors including increasing age, male sex, increased cholesterol and hypertension were associated with an increased risk of myocardial infarction. The Factor XIIIVal34Leu polymoφhism was originally described in a population of Finnish subjects in whom the allele frequency was 23% of 600 normal controls (20), similar to that in our study population. In both studies ratification of genotype was confirmed by sequencing to indicate that these results represent genuine population frequencies. Subsequently two further publications have reported this polymoφhism in relation to factor XIII deficiency (19) and in small groups of Finnish, Russian, German and Japanese subjects (21) in whom population frequencies were not described. The correlation between the incidence of the Factor XIIIVal34Leu polymoφhism and protection against MI is supported by the results of our analysis of the frequency of the mutation in Asian Indians and Pima Indians. Asian Indians have a high incidence of MI when compared to Caucasian ( intermediate incidence of MI) and Pima Indians ( low incidence of MI). Genotype comparisons of Pima Indians ( 48% heterozygote, 16% homozygote for the mutation) with Caucasian ( 41% heterozygote , 7% homozygote) and Asian Indians ( 17.5% heterozygote, 3.7% homozygote) shows a marked correlation between the incidence of cardiovascular disease and the prevalence of the protective substitution of valine to leucine , please see table7. The formation of crosslinked fibrin from fibrin monomer is pivotal to the development of a stable thrombus. This process involves the action of thrombin on fibrinogen to produce soluble fibrin and the activation of factor XIII which crosslinks fibrin rendering it more resistant to fibrinolysis. In addition, a number of proteins in the coagulation and fibrinolytic cascades are substrates for Factor XIII including α2-antiplasmin which is crosslinked to the fibrin a chain whilst retaining its full inhibitory capacity. Lysis of fibrin is dependent on binding of tissue plasminogen activator and plasminogen to fibrin with local plasmin production in a milieu that is protected from circulating inhibitors such as PAI- 1. Under these circumstances local inhibition of fibrinolysis is largely dependent on effective crosslinking of α2-antiplasmin to the fibrin clot. The results from our study indicate that the Factor XIIIVal34Leu affects thrombin activation of the Factor XIII A subunit to produce fibrin that is less resistant to fibrinolysis due to alteration in rates of crosslinking of both fibrin monomer and α2- antiplasmin. The importance of the fibrinolytic system in the maintenance of vascular patency and the development of myocardial infarction has been demonstrated in prospective clinical studies (29,30). However, levels of PAI-1 are strongly related to the features of insulin resistance, most notably triglyceride and insulin concentrations (28) and a recent publication from the ECAT study group reported that PAI-1 is not a risk factor independent from the other features of this syndrome (31). Circulating levels of PAI-1 relate to a 4G/5G polymoφhism 675bp from the transcription start site with levels some 25% higher in those with 4G/4G genotype compared to 5G/5G (32) and possession of the 4G/4G genotype has been related to risk of myocardial infarction (33). In our study, the PAI-1 4G/4G genotype was associated with myocardial infarction in all patients and in regression models, PAI-1 levels were associated with increased risk . Investigation of those subjects with Factor XIIIVal34Leu who had a myocardial infarction demonstrated increased PAI-1 concentrations and a higher prevalence of the 4G/4G genotype. Although there were more subjects possessing wild-type with myocardial infarction, the association between the PAI-1 4G/4G genotype and myocardial infarction was not apparent in this group. This indicates that this relationship is principally mediated in subjects possessing Factor XIIIVal34Leu. If this proves to be the case, the findings in this study could provide one possible explanation for the increased vascular risk in insulin resistant states, most notably non-insulin dependent diabetes mellitus (NIDDM). In NIDDM subjects, the ubiquitous increase in PAI-1 levels would increase vascular risk in those possessing Factor XIIIVal34Leu above that of the non-insulin resistant population. Finally the Factor XIIIVal34Leu mutation has also been shown to have protective properties in other thrombotic disorders. In a study of 217 subjects with a history of deep vein thrombosis compared to 250 age-matched controls the Factor XIIIVal34Leu mutation was shown to be protective against this condition. This supports a contention that the mutation may have additional protective properties against a range of thrombotic disorders. With regard to the function of the FXIIIVal34Leu mutation, thus far we have shown that alteration in the mutated molecule paradoxically leads to increased crosslinking of fibrin. The data presented in Figure2 shows the incoφoration of cadaverin into fibrin in the three genotypes. It is apparent that cadaverin incoφoration, as an indirect measure of fibrin crosslinking, is higher in the mutant genotypes than in wild-type. The results from this study indicate a major gene effect conferring protection against myocardial infarction and deep vein thrombosis associated with Factor XIIIVal34Leu. The increased PAI-1 concentrations and PAI-1 4G/4G genotype in subjects with Factor XIIIVal34Leu and myocardial infarction indicates interaction with the insulin resistant syndrome to increase vascular risk. These findings need to be confirmed in a separate population, and preferably in a prospective study. REFERENCES 1. Grant PJ, Prentice CRM. 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Table 1 PATIENT CONTROL p value (n=398) (n=196) AGE (years) 58.5 (57.6-59.5) 58.1 (56.1-60.1) 0.65 SEX (m : f) 267 : 131 98 : 98 <0.0001 CHOLESTEROL (mmol/l) 6.3 (6.2-6.4) 5.8 (5.6-6.0) <0.0001 BMI (kg/m 2 ) 26.9 (26.6-27.3) 25.4 (24.8-26.0) <0.0001 "FIBRINOGEN (g/l) 3.19 (3.09-3.29) 3.14 (3.03-3.26) 0.56 *PAI-1 ANTIGEN (ng/ml) 19.7 (17.8-21.9) 8.3 (7.2-9.6) <0.0001 TRIGLYCERIDES (mmol/l) 1.9 (1.8-2.0) 1.5 (1.4-1.6) <0.0001 PLATELET COUNT (10 9 /l) 252 (245-260) 229 (207-251) 0.6 Table 2 PATIENTS WITHOUT Ml PATIENTS WITH Ml p value (n=201) (n=197) AGE (years) 57.8 (56.5-59.0) 59.4 (58.0-60.8) 0.09 CHOLESTEROL (mmol/l) 6.2 (6.0-6.4) 6.3 (6.2-6.5) 0.4 BMI (kg/m 2 ) 26.5 (26.0-27.0) 27.4 (26.8-28.0) 0.02 *FIBRINOGEN (g/l) 3.2 (3.09-3.32) 3.16 (2.95-3.38) 0.7 FIBRINOGEN -455 G/A 27% 30% 0.5 *PAI-1 ANTIGEN (ng/ml) 18.7 (16.3-21.4) 21.9 (18.5-25.9) 0.2 PAI-1 4G/4G 27% 36% <0.01 TRIGLYCERIDES (mmol/l) 1.8 (1.7-2.0) 2.0 (1.9-2.2) 0.09 PLATELET COUNT (10 9 / I) 257 (246-269) 248 (238-258) 0.2 CURRENT SMOKER (n : y) 157 : 44 163 : 34 0.2 HYPERTENSION (n : y) 130: 71 140: 57 0.2 SEX (m : f) 118 : 83 149: 48 0.0003 Table 3 Myocardial infarct No Myocardial Infarct Healthy Controls (n=197) (n=201) (n=196) Factor XIII G/G 68% 50% 52% (n=134) (n=100) (n=102) G/T 27% 45% 41% (n=54) (n=90) (n=80) T/T 5% 5% 7% (n=9) (n=11) (n=14) Table 4 WILD TYPE Factor XIIIVal34Leu NO HISTORY p value NO HISTORY p value HISTORY OF Ml HISTORY OF Ml OF Ml OF Ml n= 89 76 83 27 AGE (years) 57.6 58.7 0.4 58.2 61.4 0.100 (55.7-60) (56.6-60.8) (56.4-60.1) (58-65) CHOLESTEROL 6.4 6.4 1.0 6.1 6.2 0.600 (mmol/l) (6.1-6.6) (6.1-6.7) (5.9-6.4) (5.9-6.6) TRIGLYCERIDE 1.8 1.9 0.5 2.0 2.2 0.300 (mmol/l) (1.5-2.0) (1.7-2.1) (1.7-2.2) (1.9-2.6) FIBRINOGEN 3.3 3.3 0.8 3.2 3.4 0.200 (g/l) (3.1-3.4) (3.1-3.5) (3.1-3.4) (3.2-3.8) PAI-1 (ng/ml) 20.9 21.0 1.0 16.7 27.9 0.004 (17.4-25) (17.6-25.3) (13.7-20.3) (21.1-36.9) BMI (Kg/m 2 ) 26.0 26.7 0.2 26.6 28.3 0.040 (25.3-26.7) (25.8-27.8) (26-27.3) (27-30.0) Table 5 MYOCARDIAL INFARCT No MYOCARDIAL INFARCT FXIII Wild-type 4G/4G 42 (33%) 31 (32%) PAI-1 genotype 4G/5Q 66 (52%) 44 (45%) 5G/5G 20 (15%) 23 (23%) p=0.3 FXIIIVal34Leu 4G/4G 24 (43%) 23 (26%) PAI-1 genotype 4G/5G 25 (45%) 42 (47%) 5G/5G 7 (12%) 25 (27%) p=0.03 Table 6 FACTOR ASSESSMENT ODDS RATIO p value (95% CI) FXIIIVal34Leu Possession of FXIIIVal34Leu 0.67 0.0006 polymorphism (0.54-0.85) AGE 10 year increments 1.42 0.008 (1.39-1.46) SEX Male sex 1.49 0.001 (1.2-1.9) PAI-1 Ag Increase of 1 ng/ml 1.04 0.0004 (1.01-1.06) CHOLESTEROL Increase of 1 mmol/l 1.41 0.01 (1.07-1.87) HYPERTENSION History of hypertension 2.40 0.03 Hypertensive therapy (1.07-5.4) Recorded BP> 140 / 90 Table 7

Claims: CLAIMS 1. A method of diagnosing the susceptibility to and/or likely existence of a thrombotic disorder comprising determining the presence of a point mutation in exon 2 of the Factor XIII A gene wherein said point mutation results in the conversion of a valine amino acid to a leucine amino acid at codon 34 and where such a mutation exists concluding that an individual is likely to have at least a degree of protection against a thrombotic disorder; and where such a mutation does not exist concluding that an individual is less likely to have a degree of protection against a thrombotic disorder. 2. A method of diagnosis according to Claim 1 wherein said method involves a polymerase chain reaction. 3. A kit for use in diagnosing the susceptibility to and/or likely existence of a thrombotic disorder involving the determination of the existence of a point mutation in exon 2 of the Factor XIII A gene wherein said point mutation results in the conversion of a valine amino acid to a leucine amino acid at codon 34 comprising: DNA primers suitable for undertaking a PCR reaction and amplfying a region of exon 2 where said point mutation is located. 4. A kit according to Claim 3 wherein said DNA primers are represented by the sequences; ACCCAGAGTGGTGGGGAAG as a 5' primer; and GACCTTGTAAAGTCAAAAATGTC as a 3' primer; and 5. A kit according to Claim 3 or 4 wherein said kit contains control nucleic acid sequences to facilitate the identification of wild-type, and the heterozygote or homozygote mutant condition.

Inventors: GRANT PETER JOHN, GRANT, PETER, JOHN
Applicants: GRANT PETER JOHN, UNIV LEEDS, GRANT, PETER, JOHN, UNIVERSITY OF LEEDS
Application year: 1997
Main classification: C12Q1
IPC codes: A61K48, C12N15, C12Q1