In this study, we anticipated an interaction between total cholesterol levels and the MTTP -164T > C polymorphism with regard to the CVD risk. The presence of a statistically significant interaction confirmed our hypothesis and indicated carriers of the C allele of the MTTP -164T > C polymorphism with plasma total cholesterol levels lower than 200 mg/dL had an increased risk of CVD. The association seemed to be stronger for IS than for MI, but differences in the associations were not supported by competing risk analysis. Conversely, the MTTP -164 C-allele showed a lower CVD, and MI, risk in participants with cholesterol levels higher than 200 mg/dL. Similar relationships were observed considering LDL-cholesterol with levels lower and higher than 130 mg/dl suggesting that LDL is the driving cholesterol component. However, the value of LDL levels seems to be limited as they had to be estimated based on the Friedewald formula . In fact further studies are needed to replicate these findings.
The association between MTTP –I128T polymorphisms and CVD risk observed in the replication cohort showed a similar trend within the strata of cholesterol levels higher than 200 mg/dL. However, considering that the number of cases in the Heinz Nixdorf Recall study is small further replication studies are needed. To our knowledge, this is the first prospective study showing such an effect of MTTP on risk of IS.
With regard to the association between the MTTP -164T > C polymorphism and cholesterol levels, previous studies observed inconsistent results. Few studies reported a slight cholesterol lowering effect of the rare alleles of the MTTP promoter polymorphisms [7, 10]. Ledmyr et al. investigated the association between the MTTP -493 G/T polymorphism and cholesterol in both healthy and hyper-cholesterolemic individuals, and observed decreased levels of total cholesterol in carriers of the -493 T variant [7, 10]. Furthermore, Phillips et al. in a small study including 82 patients with type 2 diabetes mellitus (T2DM) of a Caucasian population found that the subjects heterozygous for the -493 G/T had lower LDL-cholesterol and, in the postprandial phase, higher apoB48 levels in the VLDL fraction. The authors suggested that the -493 G/T polymorphism seemed to confer protection against atherosclerosis in T2DM patients . In contrast, Jou et al. observed that total cholesterol, LDL-, and non HDL-cholesterol levels were higher according to the rare allele of the MTTP -493 G/T polymorphism when disease free young African Americans were investigated [19, 20]. Further, Lundahl et al. observed lower serum triglyceride levels in subjects affected by familial hypercholesterolemia and homozygous for the rare allele of the MTTP -493 G/T genotype .
Overall, these studies seem to suggest that MTTP regulates lipids differently in the presence or absence of disease, although the occurrence of a possible interaction between the LDL receptor and the MTTP genes is not excluded [7, 14]. Our results seem to be in line with these hypotheses. On one hand we observed slightly higher total and LDL-cholesterol levels in subjects free of CVD and homozygous for the rare -164 C allele, and on the other, lower total cholesterol and triglyceride levels according to the rare allele of the MTTP -164T > C polymorphism in the group of future CVD cases.
It has been shown that the C–allele of the MTTP-164 T > C polymorphism is homologous to a putative sterol response element (SRE) binding site and as such confers a reduced MTTP expression [26–28]. These findings come from an experimental study in which Hagan et al. demonstrated that human MTTP promoter activity is up-regulated by cholesterol . The mechanism based on which cholesterol regulates MTTP gene expression is linked to the presence of a modified SRE in the MTTP promoter . When cholesterol levels are low, the sterol regulatory element binding protein (SREBP) acts as transcription factor, binds to the SRE thereby inhibiting MTTP gene expression . In contrast, in presence of cholesterol the modified SRE likely binds a new SREBP family member thus up-regulating MTTP expression [27, 28]. These observations suggest that MTTP gene expression is differently regulated by high and low cholesterol levels.
Despite the lack of significant associations between the -493G > T or -164T > C single nucleotide polymorphisms (SNPs), coronary heart disease and blood lipids observed in two previous studies [24, 25], recently Aminoff et al. put forward that carriers of the rare -164C allele are at increased risk of IHD . They substantiated their findings by showing in vivo that the presence of the rare alleles of the -493G > T and -164T > C SNPs confer lower MTTP transcription in the heart, liver and macrophage. This mechanism, in turn, by causing the lipid accumulation in the heart would provoke an increased IHD risk. Indeed, our findings are in line with those of Aminoff et al. though they concluded that the increased IHD risk observed according to the -164C variant was independent of plasma lipids. As mentioned above, because human MTTP promoter activity is positively regulated by cholesterol , it is reasonable to assume that subjects with low cholesterol levels have, in general, a lower MTTP gene expression. Thus, in this low risk group carriers of the -164C variant, compared to carriers of the common allele, might be at increased CVD because of their lower MTTP gene expression. At the same time, if one would consider the observed associations as those mimicking MTTP inhibitors, then these findings could further highlight the concerns expressed by Aminoff et al. regarding the long term side effects MTTP inhibition may generate [26, 47].
In contrast, in subjects with higher cholesterol levels we observed a reduced, though borderline significant, CVD risk accordingly to the MTTP gene -164 C variant. Our findings suggest that there could be an antagonistic (qualitative) interaction between cholesterol levels and MTTP -164 T > C polymorphism. These observations warrant further investigation.
The main limitation of this study is that the plasma lipoprotein and apolipoprotein levels, which are important in the effect of MTTP in cardiovascular disease, were not measured; our analyses on triglyceride levels were based on both fasting and non-fasting subjects; we estimated the LDL-cholesterol levels based on Friedewald equation. Strength of our study includes its prospective design. Furthermore, all cases of MI and IS were validated by medical records and were derived from a cohort population with a very high follow-up coverage.