Coronary artery disease (CAD) is a multifactorial disease caused by genetic and environmental factors. Apolipoprotein genes involved in lipoprotein synthesis and metabolism are considered excellent candidates for studying the susceptibility to CAD and myocardial infarction (MI) 1. Functional variants of genes encoding lipoproteins are responsible in part for between-individual variation in the plasma levels of lipoproteins and, therefore, they are related with the risk for atherosclerosis 2.

Among apolipoproteins, apolipoprotein (a) is a highly glycosylated protein component of lipoprotein (a) 3. It is a highly polymorphic protein whose size varies depending on the number of Kringle IV type 2 (KIV) repeats 4. Several polymorphisms of the APO (a) gene have been described in the 5'-untranslated region 5. A pentanucleotide repeat (PNR) polymorphism (TTTTA)n located 1.3 kb upstream of the first exon in the APO (a) gene (chromosome 6, 6q26-q27) may explain a part of the between-individual variability of lipoprotein Lp(a) 6. However, inconsistent results have been published concerning association of this polymorphism with CAD and/or MI 78.

APO E gene, the most extensively studied polymorphism among apolipoproteins, has three common alleles designated E2, E3 and E4 coding for three protein isoforms (apo E2, apo E3 and apo E4, respectively) 9 that serve as a ligand for members of the low-density lipoprotein receptor (LDLR) family. This function is extremely important for the catabolism of triglyceride rich particles. It has been demonstrated that the E4 allele is associated with increased levels of plasma cholesterol, LDL-C, and increased risk for CAD 1011.

Apolipoproteins CI and CII are constituents of very low-density and high-density lipoproteins. The genes coding for these apolipoproteins are close to the APO E gene on the long arm of chromosome 19 (19 q_13,2). The apo CI protein inhibits the bindings of beta very low-density and intermediary density lipoproteins to lipoproteins' receptors. Apo CII is required as a cofactor in the hydrolysis of triacylglycerides of chylomicrons and VLDL by lipoprotein lipase. Two restriction polymorphisms, the HincII site in the promoter region of the APO CI gene and the AvaII site in the intron 3 of the APO CII gene 1213, have been described in these proteins. The restriction site of APO CI has been associated with a significant increase of APO CI gene transcription 14. Since these three genes are located in the same chromosome, the effect of the APO E, CI and CII gene cluster on serum lipid levels has been studied, particularly in Chinese Han samples of CAD patients 1516. The results suggest that the linkage disequilibrium between APO E and APO CI should be, in part, responsible for the development of CAD.

In the present study we analyze the polymorphism of the four apolipoprotein genes [APO (a) PNR, APO E, APO CI and APO CII] in the Tunisian population and we check the relation of these polymorphisms and homocysteine, lipid and apolipoprotein levels to CAD.

Allele and genotype frequencies of the APO (a) PNR, APO E, APO CI and APO CII polymorphisms analyzed in controls and patients are presented in Table 1. In all cases, genotype distributions were in Hardy-Weinberg equilibrium. APO E-APO CI-APO CII haplotypes are indicated in Table 2.

In this study we have analyzed for the first time the polymorphisms of APO (a) PNR, APO E, APO CI and APO CII genes together with several lipid and apolipoprotein levels in Tunisian CAD patients and in controls. This constitutes an approach to the genetic characterization of the lipid profiles of Tunisian cardiovascular patients and supplies anthropological data on the Tunisian population.

Although APO (a) PNR, APO CI and APO CII gene polymorphisms have been analyzed in only few populations from South Europe (Table 2), they show frequency distribution between Mediterranean populations similar to that found according to APO E gene and other widely analyzed genetic markers [for review see 20262728]. In fact, we have found modest significant differences among our Tunisian sample and those of South Europe only for the APO (a) PNR distribution. Thus, populations of the two Mediterranean shores share a relative genetic homogeneity that probably reflects a common origin and/or remarkable levels of gene flow.

Comparative analyses between control and CAD groups from Tunisia have shown important observations, particularly in comparison with those found in other populations. Concerning the analyses of plasma lipid mean concentrations, the significant high values of TC, LDL-C, apo B and TG, and the significant low values of HDL-C, apo A-I, and apo E shown by Tunisian patients with CAD have been also observed in other populations 2930. The homocysteine concentration, that shows mean values around 28.4% higher in CAD Tunisian patients than in controls, presents a linear relationship with the severity of CAD (14.67 μmol/L, 17.46 μmol/L, and 18.7 μmol/L for 1, 2 and 3 affected vessels respectively, p < 0.05). This relationship suggests that high homocysteine levels are associated with CAD and with its severity. Similar observations have been found in Iranian and Greek populations 3132 but not demonstrated in a sample of American population 33. It will be interesting to check this association in other populations and to add the quantification of homocysteine to the classic parameters analyzed in the routine practice.

In the case of the APO CI and APO CII polymorphisms, no association was noted with CAD. In fact, only few studies have analyzed the association of these polymorphisms with the disease. In a study on the Chinese population 16 the authors found that only APO CI polymorphism was associated with CAD.

Regarding the APO (a) PNR polymorphism, we ascertained a global negative association between the number of TTTTA repeats and CAD. This agrees with conclusions relating to Russian, French and Irish populations 834. However, general associations between "short" TTTTA repeats (less than 10 repeats) and MI or CAD have been reported in other populations 7233536. In addition, in this study we have noted that when the allele distribution is considered in relation to the severity of the disease (Table 6) a trend, although not significant, appears towards an increase of larger (≥ 10 repeats) alleles in the subgroup with major severity. While, in a previous study on the Japanese population, the authors showed that the homozygous genotype for the "short" 8 repeats allele was related to the number of diseased vessels 7.

The differences in results on CAD risk noted between populations probably reflect the complexity of the involvement of many genetic and environmental factors in CAD appearance. In fact, these risk factors are probably candidate factors and the appearance of the CAD does not need the presence of all of them. Namely, in a given population the presence of only some (effecter factor group) of these candidate factors, are enough for favoring the CAD appearance without or with slight secondary influence of the other factors. The effecter factor group is very likely the result of a complex gene-environmental interaction, which can vary from a population to another. Our observations relating to APO E polymorphism may illustrate this proposal.

In some populations (Italian, Turk and Iranian) a significantly higher frequency of APO E4 allele was observed in CAD patients compared with healthy subjects 10113037. Thus, APO E4 allele can be considered among candidate risk factors for CAD. However, in our Tunisian population as in Kuwaiti, Polish and Finnish populations 383940 no significantly higher frequency of APO E4 allele has been found in CAD patients. Namely, in these populations APO E4 allele is not among effecter factor group and it is without or with slight secondary influence on the appearance of CAD. In fact, this slight secondary influence is shown both in the Tunisian and the Finnish 40 populations by an association between the APO E4 allele and the CAD severity.

The study of the impact of APO E genotype variations on serum lipid parameters shows that CAD patients and healthy subjects with APO E4 allele have higher levels of atherogenic parameters (TC, LDL-C and apo B), whereas subjects with APO E2 allele have low levels of these parameters. Such APO E allele effects have been also noted in some other populations 1130. The impact of APO E allelic variation in the TC, LDL-C and apo B levels is mainly due to up and down regulation of LDL receptors by the E2 and E4 alleles respectively 41.

The effect of APO E4 on CAD severity could be the consequence of its association to significant high levels of atherogenic parameters and its association to low levels of apo E. In fact, the significant low mean concentration of apo E in CAD patients, observed also in the Italian population 1030 suggests that apo E plays an anti-atherogenic role.

The effect of APO E genotypes on HDL-C concentration has not demonstrated the same consistency across populations. In the present study, no association has been found between APO E polymorphism and HDL-C concentrations in both patient and control groups. The relation of APO E polymorphism and HDL-C has been reported in some populations 113742 but not in others 3043. These differences between populations can be explained by gene-environmental interaction. Indeed, HDL-C levels are affected by exercise and alcohol consumption 4445.

The authors declare that they have no competing interests.

RB performed all the experiments and data analyses and contributed in the interpretation of results and in the editing of the manuscript, EE performed a direct help and supervision of all DNA analyses and contributed in data analyses and in the review of the manuscript, PM performed a general supervision of all DNA analyses and data analyses and participated in the review of the manuscript, MH participated in control's group collection, KBH participated in coronary artery disease patient collection, HC supervised all the work and participated in the result interpretations and in the editing and the review of the manuscript. All authors read and approved the final manuscript.