Our study describes for the first time the simultaneous analysis of the genetic variants HindIII, S447X, D9N and N291S (LPL), S19W and -1131T/C (APOA5) and the APOE polymorphism and their association with TG levels in a large, well-characterized Spanish Mediterranean population. The results clearly show an independent effect of the polymorphisms studied, TG-lowering in the case of HindIII and S447X and with a clear TG-raising effect in the case of the D9N, N291S, S19W and -1131T/C variants and the APOε4 allele. Thus, combinations of lowering and/or raising polymorphisms showed a TG gradient below or above the TG levels of the reference (no variant) group. Moreover, the variant combination consisting of two lowering polymorphisms was protective against HTG, whereas having single or combined TG-raising variants was independently associated with HTG.
The frequency of carriers of LPL
-D9N and N291S polymorphisms was around 3%, i.e., between the 1% and 7% described for healthy Caucasian persons [26
], though no studies have, as far as we are aware, been carried out in Mediterranean populations of this size. We have only been able to find one Mediterranean study, that of Choumeriannou et al
], which showed a similar frequency (3.6%) for the LPL
-N291S variant in a group of 84 Greek patients with familial hypercholesterolaemia. We have found no population studies in our area with which to compare the frequency of the D9N polymorphism and the allele frequency data presented is therefore original for this variant. The minor allele frequencies of APOA5
- S19W and -1131T/C variants (6.2% and 6.8%, respectively) were similar to those of Caucasians elsewhere [17
] and similar to those recently described in the Spanish EPIGEM population [30
]. The protective LPL
variants HindIII and S447X, and the APOE
polymorphism have been studied in our country [14
] and were found to be within the range reported for other populations from southern Europe [32
Our work confirms the associations found in previous studies between the gene variants studied and TG levels [12
], though it is the first time they have been analyzed simultaneously and in a large population, thereby showing their individual independent effect. Assuming a modest role of single polymorphisms as markers of complex diseases the study of variant combinations provides complementary information that could be clinically meaningful. Recent studies on genetic predisposition to CVD reinforce this idea as they show that certain polymorphism combinations, including variants in genes analyzed in our work, can predict CVD [4
The results of our study show that the most favourable variant combination consisted of the presence of just TG-lowering variants of LPL
and the least favourable was the presence, with no protective alleles, of at least one TG-raising variant. These results are in agreement with those of previous studies analyzing combinations of LPL
] or combinations of LPL
]. Additionally, our data suggest that the protective effect of HindIII and S447X is retained, not only versus the variants of LPL
] but also versus those of APOA5
, which is a novel aspect of our study.
A recent report dealing with a Czech cohort forming part of the MONICA study [35
] found that the raising effect of the APOA5
variants (-1131T/C or S19W) was significant in females but in males a non-significant trend was observed. Moreover, APOA5
did not affect plasma TG levels if the APOE
-ε4 allele was present. Our results differ, as we clearly found an additive and independent effect of these variants of APOA5
and the other polymorphisms studied, with no heterogeneity according to gender. The lack of effect described by Hubacek et al
is controversial, as previous results from the same group showed an increased frequency of the APOA5
-ε4 combination in persons with severe HTG as compared with a healthy population [36
]. Sousa et al
] have also described an interaction for the risk of developing severe HTG associated with the combination APOA5
-ε2. In our study, however, we did not find a main TG-raising effect associated with the APOE
-ε2 allele; conversely, this lack of association with TG levels has been described in other populations for the APOE
-ε4 allele [38
]. On other hand, the higher TG levels seen by us in APOA5*APOE
and other variant combinations were not significantly associated with an interaction effect. It is important to note the differences between these studies regarding their design (case-control, population-based,...) and in terms of the statistical approach (univariate or regression analyses, different covariates used,...), and there may also be situations of limited statistical power. Finally, a major consideration is that the differences in the environmental and genetic context of the populations studied may strongly influence the results obtained and thus explain the lack of concordance among these studies.
As expected, the presence of a TG-raising polymorphism, and even more so that of a combination of these variants, was associated with a greater likelihood of HTG, as Figure clearly shows. Prior analyses by another group [38
] have shown an increase in the OR with effect from two TG-raising variants, depending on the waist circumference; indeed, the effect was amplified when the number of TG-raising polymorphisms rose together with the presence of abdominal obesity. This contrasts with data from the present study, since the association with HTG with effect from one TG-raising variant was independent of waist circumference. The combined analysis of the TG-lowering and TG-raising variants, which is original to this study, partly explains these differences, as the TG-lowering polymorphisms were not analysed in the study by Brisson et al
. As can be seen in Figure , the association of the TG-raising variants with HTG lost its significance, even with just one protective variant, i.e. with the LPL
- HindIII intronic polymorphism. Therefore, our results reinforce the concept that this polymorphism has a modestly advantageous effect independent of the S447X variant (in linkage disequilibrium with it and classically considered functional) and highlight the importance of analyzing both protective variants.
Finally, the association of the APOA5
polymorphisms S19W and -1131T/C, and the APOE
non-ε3 genotype with severe HTG has been recently reported [40
]. This report also highlights the multiplicative effect in the value of the Odds Ratios for severe HTG of combinations of six TG-raising polymorphisms, always with one of the APOA5
variants. Unfortunately, despite the fact that our study population was large, the relatively small number of persons with TG-raising combinations and with HTG limits the statistical power to detect certain potential interactions between variants.
Although our study supports previous results from other groups and provides new findings with consistent results, certain limitations are involved. Firstly, it is obvious that the characteristics inherent to the study population (geographical area, age, state of health) condition the results obtained. Secondly, analysis of more variants in more genes as well as other environmental variables [41
], such as diet and physical activity, not assessed in our study might likely better explain variability in TG levels and the manifestation of HTG.