SISTEMA DE ENERGÍA

6.5 ECTIM study

The ECTIM study (Etude Cas Témoin sur l ’Infarctus du Myocarde - Case-Control Study on Myocardial Infarction ) is a large multi-centre study including samples from Belfast, Lille, Strasbourg and Toulouse, centres at differing risk of CHD. The ratio of CHD incidence between Belfast and Toulouse is approximately 2.5 to 1. The aims of this study were both to compare the frequency of DNA polymorphisms in patients who have had a myocardial infarction and controls to examine possible association of the Arg/Gln353 polymorphism with risk of IHD, and to study the association between these

polymorphisms and measured levels of risk factors for MI i.e. plasma lipoprotein levels and coagulation factor levels. All the individuals in the study were men, the control sample being drawn at random from GP practices in the four areas and the patients being aged between 25 and 64 who had had a myocardial infarction according to MONICA category I (Lowel 1990).

There were no observed differences in the frequency of the Arg353 allele between patient

and control groups and so it appears that the Arg/Gln353 allele does not affect an

individuals risk of IHD. However, if the polymorphism proves to be causative in determining plasma level of FVIIc, itself a risk factor, it follows that the polymorphism must have some effect on risk, albeit perhaps small.

The association between FVIIc and genotype and the genotype-specific correlation between FVIIc and triglycerides was examined in the four centres included in the study.

1 5 8

The results show that the association was present in all the centres and in both patient and control groups, apart from the Lille controls, although the number of samples in this group was by far the smallest. There appeared to be no evidence of the genotype- specific correlation between FVIIc and triglycerides observed in the other studies, which were done on much smaller numbers of individuals. One reason for this may be because the ECTIM study was the only study presented in this thesis in which the blood samples on which measurement of the variables was carried out were fasting samples. Therefore, postprandial hypertriglyceridaemia would not influence FVIIc and a genotype-specific difference in correlation between FVIIc and triglycerides would not be expected. However, another difference between this study and all the previous studies is that the FVIIc assays differed. The BCMU (MRC Epidemiology and Medical Care Unit at Northwick Park Hospital (now at St.Bartholomew’s Hospital Medical School) assay, which was used in all except the ECTIM study, was developed before the discovery o f the vitamin K-dependent coagulation factor Protein C. The importance of Protein C in the haemostatic process was discovered when a deficiency of this protein was found to cause thromboembolic disorders (Griffin et al 1981). Protein C is activated by thrombin in conjunction with the endothelial cell thrombin receptor thrombomodulin. Thrombomodulin acts to enhance thrombin activation of Protein C approximately 30,(XX)-fold. Activated Protein C, together with its cofactor Protein S, inactivates FV and FVIII and increases levels of plasminogen activator by inhibiting the activation of its inhibitor PAI-1. Presence of Protein C in plasma prolongs the clotting time by inactivating FV and FVIII and enhancing fibrinolysis (for review see Bouma 1988). The ECMU assay uses normal bovine plasma, which has had all the vitamin K- dependent clotting factors removed by barium sulphate precipitation. Factor II, factor IX and factor X are added back to the plasma and FVII coagulant activity is then

1 5 9

measured in sample plasma using this mixture. The assay used in the ECTIM study however uses human congenitally FVII deficient plasma, which contains all of the vitamin K-dependent clotting factors including Protein C ( except for FVII). Because o f the inhibitory effects of Protein C and Protein S, this makes the assay far less sensitive to circulating activated FVII than the ECMU assay. The congenitally FVII deficient plasma will support the coagulant activity of activated FVII but, because of the presence of Protein C, dampens the autoactivation of FVII. This means that the range of FVIIc which can be detected with this assay is narrower than for the ECMU assay, a property which probably accounts for the lack of genotype-specific differences in the correlation between plasma FVIIc and triglycerides in the ECTIM study.

6.6 Conclusions

The Arg/Gln353 polymorphism is associated with differences in both FVII antigen levels

and FVII coagulant activity. FVIIc was strongly associated with the Arg/Gln353

polymorphism in all the different population samples studied, including those from different ethnic groups, from both men and women, and from both MI patients and healthy people. In addition, this association has recently been observed in a study of healthy women in the USA (Elaine Meilahn and colleagues, personal commmunication). Two of the studies described in this thesis also showed a genotype-specific correlation between both FVIIag and FVIIc and triglycerides, and in the case of FVIIag this was also evident with serum cholesterol levels. Together these results strongly suggest that the Arg/Gln353 amino acid substitution is affecting production of the FVII molecule and

its activity in the presence of lipids. The diversity of population samples and the consistency of the results, together with the fact that an amino acid substitution occurs.

1 6 0

make it highly unlikely that the Arg/Gl%g3 polymorphism is not causing the observed

differences and is merely a marker for another change elsewhere in the gene, although this can still not be ruled out entirely.

161

7 Final conclusions

Both fibrinogen and FVII levels are strong risk factors for IHD. The risk associated with elevated levels of these plasma proteins is greater than that for serum cholesterol level, and the difference in risk is even more pronounced for fatal events, as shown in the NPHS (Meade et al 1986). The studies presented in this thesis are primarily concerned with gene-environment interaction, with IL- 6 in the case of the fibrinogen

work and triglycerides in the case of the FVII studies. If the polymorphisms in these genes are proved to be predictors of interaction with environmental factors, which may to be the case, they would be more useful in predicting risk than a single measure of the risk factor in question, i.e. plasma fibrinogen or plasma FVIIc, because of the intra­ individual variation in levels of these factors.

It may be useful to know an individuals Arg/Glnjjj genotype in order to identify those individuals who would benefit from low fat diets. Table 1 shows the results from the Ethnic study (5.2) presented in a different way, that is, with the genotype groups divided according to whether the plasma triglyceride level was above or below the mean. These data show that it is only the Arg^^g homozygotes with a plasma triglyceride level above the mean who have appreciably raised plasma FVIIc. Therefore, if there proves to be a causal relationship between triglycerides and FVII activation, it is this group who would derive the greatest benefit from advice to adopt a lower dietary fat intake. However, since most people will be Arg^^g homozygotes, a low fat diet would be beneficial in the majority of cases.