Abstract
BACKGROUND
The importance of apolipoprotein A-V (APOAV) gene variants in the determination of plasma triglyceride levels in humans has been proven in several population studies.
OBJECTIVES
To investigate whether APOAV gene variants are associated with the different plasma cholesterol fractions.
METHODS
The influence of APOAV polymorphisms (T-1131>C, Ser19>Trp and Val153>Met) on plasma cholesterol fractions was evaluated in 1191 men and 1368 women representatively selected from the Czech population. Low-density lipoprotein cholesterol, non-high-density lipoprotein (non-HDL) cholesterol and HDL cholesterol levels were analyzed.
RESULTS
The T-1131>C variation in the APOAV gene was found to affect plasma non-HDL cholesterol, showing significantly higher levels in C-1131 carriers than in T/T-1131 homozygotes. This association was observed in both men (4.61±1.09 mmol/L in C-1131 carriers versus 4.47±1.07 mmol/L in T/T-1131 homozygotes; P<0.01) and women (4.46±1.22 mmol/L in C-1131 carriers versus 4.24±1.17 mmol/L in T/T-1131 homozygotes; P<0.01). Interestingly, when low-density lipoprotein cholesterol (obtained by the Friedewald formula) or HDL cholesterol levels were analyzed, no significant association was detected.
CONCLUSION
The APOAV gene variant T-1131>C may play a role not just in the genetic determination of triglyceride levels but may also influence plasma levels of non-HDL cholesterol.
Keywords: Apolipoprotein A-V, Non-HDL cholesterol, Polymorphism, Triglycerides
Elevated plasma lipid levels have been described as an independent risk factor for cardiovascular disease development (1,2). The final level of plasma lipids results from a combined effect of environmental and genetic predispositions, both of which contribute approximately the same effect.
Genetic predispositions to elevated levels of plasma lipids have been intensively analyzed in the past few years. So far, common variants within the genes for different apolipoproteins, enzymes, receptors and transporters have been associated with plasma lipid levels. In the past few years, apolipoprotein A-V (APOAV) underwent intensive analysis in both animal and human experiments. Transgenic and knock-out mice models of APOAV showed a strong effect on plasma triglyceride (TG) levels, while the total plasma cholesterol levels were not influenced significantly (3).
ApoAV is located on TG rich particles (chylomicrons and very low-density lipoprotein [VLDL]) as well as on high-density lipoprotein (HDL), and enhances the activity of lipoprotein lipase (LPL) (4). In comparison with other apolipoproteins, the plasma concentration of apoAV in humans is low – ranging from approximately 25 μg/L to 400 μg/L (5). When human DNA was screened for APOAV polymorphisms, more than 10 variants were detected (6). The commonly analyzed variants of APOAV are T-1131>C and Ser19>Trp. Strong associations between plasma TG levels and less common alleles, such as T-1131 and Ser19, have been observed in different ethnic groups (6–8). Recently, an association between HDL cholesterol and the Val153>Met variant in women was documented (9). Similar to animal models, total plasma cholesterol in humans is not influenced by APOAV gene variants.
In all large population-based studies, estimations of low-density lipoprotein (LDL) cholesterol have been obtained using the Friedewald formula (10). Because this method is suitable only if plasma TG levels are lower than 4.52 mmol/L, and because in many patients with dyslipidemias a substantial amount of TG could be located in remnant particles or in intermediate-density lipoproteins (IDLs), a growing number of physicians are using non-HDL cholesterol values instead of the classical LDL cholesterol values.
The aim of the present study was to evaluate the association between APOAV gene variants (T-1131>C, Ser19>Trp and Val153>Met) with plasma LDL, HDL and non-HDL cholesterol levels in a large population-based study.
METHODS
Subjects
The 2559 unrelated Caucasians (1191 men and 1368 women, response rate of 84%) included in the present study represented a three-year cohort of a selected sample of 1% of the Czech population. The individuals were recruited between 1997 and 1998, and reinvited between 2000 and 2001 according to the protocol used in the Multinational Monitoring of Trends and Determinants in Cardiovascular Diseases (MONICA) study (11). Analyzed lipid parameters were obtained from the 2000/2001 survey. Written informed consent was obtained from the study participants, and the local ethics committee approved the design of the study. Basic characteristics of the individuals are summarized in Table 1.
TABLE 1.
Basic characteristics of the individuals involved in the study (n=2559)
| Characteristic | Men | Women |
|---|---|---|
| n | 1191 | 1368 |
| Age, years | 49.2±10.8 | 48.8±10.6 |
| Cholesterol, mmol/L | 5.75±1.06 | 5.80±1.15 |
| TG, mmol/L | 1.98±1.28 | 1.46±0.85 |
| HDL cholesterol, mmol/L | 1.26±0.33 | 1.50±0.37 |
| LDL cholesterol, mmol/L | 3.67±0.93 | 3.64±1.04 |
| Non-HDL cholesterol, mmol/L | 4.49±1.04 | 4.29±1.17 |
| Body mass index, kg/m2 | 28.2±4.0 | 27.6±5.5 |
| Diabetes, n (%) | 72 (6.0) | 60 (4.4) |
| Hypertension, n (%) | 489 (41.1) | 457 (33.4) |
| Smoking prevalence, n (%) | 389 (32.7) | 348 (25.4) |
Data are presented as the mean ± SD unless otherwise indicated. HDL High-density lipoprotein; LDL Low-density lipoprotein; TG Triglyceride
DNA and biochemical analysis
DNA was isolated by a standard method (12). Individual APOAV variants were analyzed using the procedure described previously (13–15).
Lipoprotein parameters were measured enzymatically by the World Health Organization Regional Lipid Reference Centre, IKEM (Prague, Czech Republic), on a Roche COBAS MIRA autoanalyzer (Roche Diagnostics, Switzerland), using conventional enzymatic methods with reagents from Boehringer Mannheim Diagnostics and Hoffmann-La Roche. Non-HDL cholesterol was calculated by subtracting HDL cholesterol from total cholesterol. LDL cholesterol was calculated using the Friedewald formula (10). Body mass index was calculated as weight in kilograms divided by the square of height in metres.
Statistical analysis
Statistical analysis was performed using ANOVA to analyze the differences among genotypes. Individuals with TG levels greater than 4.52 mmol/L (11 women and nine men) and individuals who did not have all APOAV variants genotyped were excluded from the analysis.
RESULTS
T-1131>C polymorphism
Distributions of the APOAV T-1131>C polymorphism genotypes are summarized in Table 2. The frequencies of the alleles and genotypes were found to be similar to the frequencies described previously in Caucasian populations (7,15).
TABLE 2.
Distribution of the apolipoprotein A-V variants in the Czech population
| Polymorphism | Men, n (%) | Women, n (%) |
|---|---|---|
| T-1131>C | ||
| T/T | 957 (85.5) | 1086 (83.7) |
| T/C | 141 (12.6) | 197 (15.2) |
| C/C | 21 (1.9) | 15 (1.2) |
| Ser19>Trp | ||
| Ser/Ser | 982 (87.4) | 1092 (84.8) |
| Ser/Trp | 141 (12.5) | 188 (14.6) |
| Trp/Trp | 1 (0.1) | 7 (0.5) |
| Val153>Met | ||
| Val/Val | 1047 (93.6) | 1219 (93.7) |
| Val/Met | 71 (6.4) | 82 (6.3) |
| Met/Met | 0 (0) | 0 (0) |
Both male and female carriers of the APOAV allele C-1131 had higher plasma TG levels than T/T-1311 homozygotes (P<0.001) (13).
Also, non-HDL cholesterol was influenced significantly by this variant. The male carriers of the C-1131 allele had significantly higher plasma levels of non-HDL cholesterol (4.61±1.09 mmol/L) than the male T/T-1131 homozygotes (4.47±1.07 mmol/L) (P<0.01). A similar association was observed in women (4.46±1.22 mmol/L in C-1131 allele carriers versus 4.24±1.17 mmol/L in T/T-1131 homozygotes; P<0.01).
In contrast, the LDL cholesterol levels (obtained using the Friedewald formula) and the HDL cholesterol levels were not influenced significantly by this variant (Table 3).
TABLE 3.
Effect of the T-1131>C polymorphism in the apolipoprotein A-V gene on plasma cholesterol levels
| Men
|
Women
|
|||||
|---|---|---|---|---|---|---|
| T/T-1131 | C-1131 | P | T/T-1131 | C-1131 | P | |
| n | 957 | 162 | 1086 | 212 | ||
| TC | 5.72±1.04 | 5.77±1.00 | NS | 5.78±1.14 | 5.94±1.18 | NS |
| LDL-C | 3.65±0.95 | 3.68±0.98 | NS | 3.61±1.04 | 3.78±1.12 | NS |
| HDL-C | 1.25±0.34 | 1.21±0.37 | NS | 1.51±0.37 | 1.47±0.37 | NS |
| Non-HDL-C | 4.47±1.07 | 4.61±1.09 | 0.01 | 4.24±1.17 | 4.46±1.22 | 0.01 |
Data are presented as the mean ± SD unless otherwise indicated. The values for cholesterol levels are in mmol/L. HDL-C High-density lipoprotein cholesterol; LDL-C Low-density lipoprotein cholesterol; NS Not significant; TC Total cholesterol
Ser19>Trp polymorphism
The frequencies of the APOAV Ser19>Trp genotypes are summarized in Table 2. These values were found to be similar to the frequencies described previously (6,16). In both men and women, this polymorphism significantly influenced plasma levels of TGs – Trp19 carriers had higher TG levels than Ser/Ser19 homozygotes (P<0.001) (13).
In contrast to the T-1131>C variant, non-HDL cholesterol levels were not influenced by this variant for either men or women (data not shown).
Val153>Met polymorphism
The frequencies of the APOAV Val153>Met genotypes are summarized in Table 2. This variant was found to have no effect on plasma levels of TGs, but a sex-specific effect on plasma levels of HDL cholesterol was detected (9).
Also, for this variant, no associations with plasma LDL cholesterol or non-HDL cholesterol levels were found in either men or women (data not shown).
DISCUSSION
The present study was conducted to investigate the role of common gene variants of APOAV in the determination of plasma non-HDL and LDL cholesterol levels.
Although the absolute impact varied, the T-1131>C variant in the APOAV gene had a significant effect on plasma levels of non-HDL cholesterol, but not on LDL cholesterol levels (obtained using Friedewald formula) in the same individuals. This effect was detectable in both men and women.
The present results, which are from a large population study cohort, represent the first such data to show a positive association between the common polymorphism T-1131>C in the APOAV gene and plasma non-HDL cholesterol levels in the general Caucasian population. Ser19>Trp and Val153>Met variants have no effect on plasma non-HDL cholesterol in either men or women.
The results of most of the genetic associations’ studies undertaken are not consistent and reproducible each time. The causes of these differences are insufficient number of individuals and differences in sex, ethnicity, age, body mass index, diets, etc. To minimize the chance of false-positive or false-negative results, we used the protocol from the MONICA study. This protocol was prepared for the World Health Organization’s monitoring of cardiovascular risks and is considered to be one of the best possible selective criteria for preparation of a representative population sample.
Approximately 2500 unrelated Caucasian individuals (equally distributed between sexes, wide age range) were included in the present study. Although, according to the selection criteria used, one would not expect any bias in sample selection, the T-1131>C genotype frequencies were not in Hardy-Weinberg equilibrium – there were more C/C-1131 homozygotes than expected. This discrepancy is surprising, especially because more than 20 other polymorphisms (two mentioned variants in the APOAV; insertion/deletion in the angiotensin-converting enzyme gene; apolipoprotein E polymorphism; insertion/deletion variant in the apolipoprotein C1 gene; T-159>C in the CD14 gene; G-174C variant in interleukin-6; variants in the preproghrelin, lipopolysaccharide-binding protein and MTHFR genes; etc [Hubacek, partially unpublished results]) were analyzed so far in this population and no such discrepancy was observed previously.
A growing number of physicians are using non-HDL cholesterol instead of calculated LDL cholesterol for estimating cardiovascular risk.
The reasons for using non-HDL cholesterol instead of calculated LDL cholesterol for estimating lipoprotein-related risk for complications of atherosclerotic vascular disease are well accepted, and were reviewed approximately 10 years previously (17). Non-HDL cholesterol is an estimate of not only LDL particles but also all atherogenic lipid particles, because it encompasses IDL and VLDL particles. Measuring non-HDL cholesterol has several practical advantages. First, it can be calculated in the nonfasting state and the calculation is very simple. It can also be calculated in the setting of elevated TG levels, when estimation of LDL cholesterol with the Friedewald formula is not accurate. Additionally, a recent study (18) found that the calculated and directly measured LDL cholesterol concentrations are significantly different – more than 90% of direct measurements of LDL cholesterol exceeded calculated LDL cholesterol. Finally, the Friedewald formula is also less accurate in patients with diabetes (18).
Recent studies (19–22) have concentrated on non-HDL cholesterol as a predictor of coronary artery disease (CAD) or myocardial infarction in differently designed studies.
Rallidis et al (19) have found that in young individuals, non-HDL cholesterol was the strongest predictor for myocardial infarction among all studied conventional risk factors. Similarly, in The Bogalusa Heart Study (20), non-HDL cholesterol was as good as or better than other widely recommended lipoprotein measures in the identification of subclinical atherosclerosis in young adults. Non-HDL cholesterol is also the best predictor of CAD among nondiabetics (21). Finally, the largest study published so far (18,255 participants from the Health Professionals Follow-up Study [22]) supported the concept that both non-HDL cholesterol and apolipoprotein B are more predictive in the development of CAD than LDL cholesterol.
Despite these facts, no results have been published on the associations between genetic variants and plasma levels of non-HDL cholesterol.
It is known that apoAV is important for the activation of plasma LPL. This enzyme metabolizes TGs in VLDL particles (and in chylomicrones as well) and plays a key role in the conversion of these particles to IDL and further to LDL particles. Although the mechanism by which the APOAV variant influences non-HDL cholesterol level is not completely clear, we speculate that slightly lower activation of LPL could lead to the delayed metabolism of TG-rich particles and elevated levels of intermediate metabolites (remnants and IDL particles) in plasma. These individual effects do not necessarily need to be significant (23), but, if analyzed together, with other fractions creating a non-HDL cholesterol pool, the effect could be significant.
Our results suggest that the T-1131>C variant in the APOAV gene could be a significant genetic determinant not only for plasma levels of TGs, but also for plasma non-HDL cholesterol, thus affecting two independent risk factors for CAD development.
ACKNOWLEDGEMENTS:
ACKNOWLEDGEMENTS: This work was supported by project number 1M0510 (Ministry of Education, Youth and Sports of the Czech Republic) and by Institute for Clinical and Experimental Medicine, Prague, Czech Republic, project number 00023001.
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