Association Between the PPARA L162V Polymorphism and Plasma Lipid Levels
The Framingham Offspring Study
Peroxisome proliferator activated receptor (PPAR) alpha is a member of the nuclear receptor superfamily that regulates key proteins involved in fatty acid oxidation, extracellular lipid metabolism, hemostasis, and inflammation. A L162V polymorphism at the PPARA locus has been associated with alterations in lipid and apolipoprotein concentrations. We studied the association among lipids, lipoproteins, and apolipoproteins and the presence of the L162V polymorphism in 2373 participants (1128 men and 1244 women) in the Framingham Offspring Study. The frequency of the less common allele (V162) was 0.069. The V162 allele was associated with increased serum concentrations of total and LDL cholesterol in men (P=0.0012 and P=0.0004, respectively) and apolipoprotein B in men (P=0.009) and women (P=0.03 after adjustment for age, body mass index, smoking, and use of β-blockers, diuretics or estrogens). Apolipoprotein (apo) C-III concentrations were higher in carriers of the V162 allele. The association of the L162V polymorphism on LDL cholesterol concentration was greatest in those who also carried the E2 allele at the APOE locus and the G allele at the APOC3 3238C>G polymorphism. This suggests that alterations in triglyceride-rich lipoprotein metabolism may be involved in the generation of the increase LDL cholesterol observed with the L162V PPARA polymorphism.
The peroxisome proliferator activated receptors (PPARs) are members of the nuclear receptor superfamily. Three subtypes of the receptor (PPARα, PPARβ, and PPARγ) are expressed in humans. PPARα is most commonly expressed in organs and tissues in which fatty acid oxidation is active, such as the liver and muscle, as well as in vascular endothelium and smooth muscle cells. When activated by a ligand, PPARα heterodimerizes with the retinoic X receptor, binds to DNA, and modulates gene transcription.1,2⇓ In addition, ligand-activated PPARα can modulate transcriptional activity through protein-protein interactions with other nuclear transcription factors.3 Our current knowledge indicates that the genes regulated by PPARα participate in the regulation of key proteins involved in extracellular lipid metabolism, fatty acid oxidation, hemostasis, and inflammation.3 As such, PPARα is a candidate gene whose expression or activity may influence cardiovascular disease (CVD) risk through multiple pathways including alterations in lipid concentrations, obesity, insulin resistance, or the inflammatory response.
The gene (PPARA) encoding PPARα is located on the long arm of chromosome 22. Several polymorphisms of the human PPARA gene have been recently described.4– 7⇓⇓⇓ Of these, a C→G transversion at position 484 in exon 5 leads to a substitution of valine for leucine at codon 162 (L162V). The less common allele (V162) has been associated with variations in serum concentrations of total cholesterol,5,7⇓ LDL cholesterol,7 apolipoprotein B (apoB),5,7⇓ high density lipoprotein (HDL) cholesterol4 and apolipoprotein A-I (apoA-I)4 depending on the population studied. In such a situation, where the majority of the association studies are not replicated, we cannot underscore enough the importance of replication studies to define the genetics of complex traits.8 Furthermore, previous studies did not include representatives from all segments of the population, nor did they include significant numbers of women. It was therefore important to study the impact of this polymorphism in a well-characterized cohort representative of the general population.
The aim of our study was to examine the role that the PPARA L162V polymorphism plays in the variability of lipid and apolipoprotein concentrations in a large population sample from the Framingham Offspring Study.
Detailed design and methodology for the Framingham Offspring Study have been previously described.9 In essence, this is a long-term prospective evaluation of risk factors for cardiovascular disease in which participants are the offspring and spouses of the original Framingham Heart Study cohort participants. The analyses carried out in this study included those who attended the 5th examination visit of the study from 1991 to 1995. Coronary heart disease (CHD) included the presence of myocardial infarction, angina pectoris, coronary insufficiency, and coronary death. CVD included CHD plus stroke and peripheral vascular disease. All procedures were approved by the institutional human investigator review boards.
Plasma lipid, lipoprotein, apolipoprotein, and lipoprotein concentrations as well as lipoprotein subclass distributions were assayed as previously described.10
Genomic DNA was isolated from peripheral blood leukocytes by standard methods.11 Genotyping was carried out on an Applied Biosystems 7700 sequence detection system with Taqman probes for allelic discrimination. The method has been used in this laboratory for the detection of polymorphisms in other genes and has been described previously.12,13⇓ We carried out polymerase chain reaction (PCR) amplification of a genomic DNA fragment using the primers 5′-GTG TAT TAC CCT CAC AGG GCT TCT-3′ and 5′-AAA TGT GCA GGG CCA CCT TA-3′. This produced a 181-bp fragment including the polymorphic region. PCR amplification was carried out in the presence of 2 probes with different reporter dyes attached to their 5′ ends (in this case, 6-carbon fluorescein [FAM] and VIC) and a fluorescent quencher (6-carboxy-tetramethylrhodamine [TAMRA] at the 3′ ends. One probe was complementary to the wild-type DNA strand (6FAM-TTT CAC AAG TGC CTT TCT GTC GGG AT-TAMRA) and the other to the DNA strand with the mutation (VIC-TTC ACA AGT GCG TTT CTG TCG GGA T-TAMRA).
PCR was performed in 12-μL reaction mixtures containing 0.4 μmol/L of each primer, 0.05 μmol/L of the L162 probe, and 0.15 μ mol/L of the V162 probe and Taqman universal master mix from Applied Biosystems. After two steps at 50°C for 2 minutes and 95°C for 12 minutes, 47 cycles of 94°C for 20 seconds and 64°C for 1 minute were performed in the ABI Prism 7700 sequence detection system (Applied Biosystems).
During PCR amplification, the complementary strand annealed to the PCR template and Amplitaq Gold DNA polymerase (a component of the universal master mix) cleaved the reporter dye at the 5′ end of probe resulting in increased reporter fluorescence. Thus, homozygotes for the wild-type allele showed fluorescence from only the reporter for the wild-type probe, homozygotes for the mutant allele showed fluorescence only from the reporter for the mutant probe and heterozygotes showed in intermediate fluorescence from both reporters. APOE14 and APOC315 genotypes were determined as previously described.
Statistical analysis was carried out by using Statistical Analysis Software (SAS). The relationship between PPARA genotypes and lipid or apolipoprotein measures was evaluated by using ANCOVA techniques that accounted for the familial relationships among the study participants (by using PROC MIXED in SAS). All statistical data analyses excluded subjects who were on cholesterol-lowering treatments at examination 5. The relationship between each of the lipid or apolipoprotein measurements and PPARA genotype (Table 1) was evaluated both crudely (adjusting for familial relationships only) and after adjusting for familial relationships, age, body mass index (BMI), smoking (cigarettes per day), β -blocker use, diuretic use, and estrogen use (in women). These analyses were performed for men and women separately.
In addition, we evaluated the relationship between PPARA genotype and LDL cholesterol for the different genotypes of APOE and APOC3. This was accomplished by including dummy variables for those in these genotype groups along with the interactions terms with PPARA. Each was evaluated separately in a model that adjusted for familial relationships, age, BMI, smoking, and the treatment variables. The APOE genotypes were grouped as E2/2 and E2/3, E3/3, and E3/4 and E4/4 (40 subjects with E2/4 were excluded from this analysis) and the APOC3 genotypes at position 3238 were grouped as CG and GG or CC. For these analyses, the effects of the L162V polymorphism were in similar directions in men and women. Therefore, the population was analyzed as a whole, with men and women together, after adjusting for gender.
The frequency and phenotypic associations with the L162V polymorphism were analyzed in a total of 2372 subjects (1128 men and 1244 women). The characteristics of the population are shown in Table 1.
PPARA genotype distribution was in Hardy-Weinberg equilibrium. The V162 allele frequency was 0.069, which was similar to that reported in other white populations.
Association of the L162V Polymorphism with CVD Disease and Lipid, Lipoprotein, and Apolipoprotein Concentrations
The associations between lipid and apolipoprotein concentrations and PPARA genotype are shown in Table 2. In men, the less common V162 allele was associated with significantly higher serum concentrations of total cholesterol, LDL cholesterol, apoB, and apoC-III as compared with the L162 (wild-type allele). These differences persisted after adjustment for age, BMI, cigarette smoking, and use of β-blockers/diuretics. In women, a similar trend was seen, but these did not reach statistical significance with the exception of serum apoB (P=0.03) after adjustment for the same covariates.
After adjustment for age, the odds ratio for CVD in men carrying the V162 allele was 0.856 (95% confidence interval, 0.455 to 1.608, P=0.63). The same figure for women was 0.967 (95% confidence interval, 0.465 to 2.013, P=0.93).
Interaction of Other Genetic Polymorphisms on the Association Between LDL Cholesterol and the L162V Polymorphism
In an attempt to understand the basis by which the PPARA L162V polymorphism influences LDL cholesterol concentrations, we assessed potential interactions with other loci involved in triglyceride-rich lipoprotein (TRL) metabolism. Specifically, we examined interactions with the APOE locus (E2, E3, or E4 alleles) and the 3238C>G polymorphism at the APOC3 locus. Figure 1 shows that the association of high LDL cholesterol with the V162 allele is most pronounced in carriers of the E2 allele at the APOE locus and the G3238 allele at the APOC3 locus.
Several polymorphisms at the PPARA locus have been described. Most of them being rare or not encoding any amino acid change.5 We have focused our study on the L162V polymorphism for the following reasons. First, the frequency of the V allele (≈0.07 in whites) is the highest among the single-nucleotide polymorphisms reported at this locus. This allele frequency gave us sufficient statistical power to address the primary aims of our study. Second, this polymorphism has been previously associated with alterations in lipoprotein concentrations in both diabetic and nondiabetic subjects without drug treatment.4,5,7⇓⇓ In contrast, the R131Q polymorphism has been found to be associated with apo A-I concentrations only in diabetic men.4 As can be seen in Table 1, diabetes mellitus was uncommon in our study population. Finally, it has been shown that the V162 allele encodes a protein with altered ability to activate transcription in vitro.4,6⇓ These two studies showed increased transcriptional activation associated with the V162 allele, although only one6 showed that the effect was dependent on the concentration of agonist to which the cells were exposed. The R131Q polymorphism, however, was activated by a PPARα agonist in a manner that was similar to the wild-type receptor.6 As such, it seems likely that the L162V polymorphism is a functional polymorphism. Nevertheless, we cannot reject the hypothesis that this polymorphism is in linkage disequilibrium with another polymorphism, which in turn, could be causal for the reported associations.
As previously described by Vohl et al7 and Lacquemant et al,5 we found that the V162 allele was associated with higher serum concentrations of LDL cholesterol (136 versus 127 mg/dL in men and 128 versus 124 mg/dL in women) and apoB (120 versus 114 mg/dL in men and 112 versus 108 mg/dL in women). However, the magnitude of the difference was smaller in women, reaching statistical significance for apoB only after adjustment for age, BMI, and β-blocker, estrogen, and diuretic use (Table 2). To put this data in context, with the same population, the presence of the E4 allele was associated with increased LDL cholesterol compared with those homozygous for the E3 allele from 130 to 133 mg/dL in men and from 126 to 132 mg/dL in women. Likewise, the E4 allele was similarly associated with higher apoB compared with E3 homozygotes (120 versus 115 in men and 114 versus 110g/L in E3 women). Therefore, the PPARA locus appears to have a similar impact on LDL cholesterol variability in the population as the one previously demonstrated for the APOE locus.
Our data shows a nonsignificant trend to decreased risk of CVD in male and female carriers of the V162 allele despite the increase in LDL cholesterol associated with this allele. However, the number of CVD cases was small (Table 1) and the odds ratio far from statistically significant. Thus, it is still uncertain whether the presence of this polymorphism alters the risk of CVD. In fact, given the frequency of the V162 allele in this population and assuming a baseline proportion of CHD of 5.06% in women and 8.28% in men, we estimate that we have less than 80% power to detect any odds ratio greater than 0.4 in women and 0.44 in men with an alpha of 0.05.
The V162 allele was also associated with increased concentrations of plasma apoC-III. ApoC-III has an inhibitory effect on lipoprotein and hepatic lipase16 activities, and its plasma concentrations are an important determinant of the catabolism of TRL.17 Transcriptional repression of the APOC3 gene is one of the major pathways through which fibrates, known agonists for PPARα, lower serum triglycerides and influence lipid metabolism by binding to a DR-1 site in the APOC3 promoter.18 In addition, within the apoC3 promoter, there is an insulin response element (IRE). Polymorphisms in the IRE render the gene unresponsive to the normal transcriptional repression by insulin.19 These polymorphisms are in linkage disequilibrium with the 3238C>G polymorphism at the APOC3 locus.20 Because both the DR-1 site and the IRE are found in the APOC3 promoter, it is rational to hypothesize that a polymorphism at the PPARA locus could interact with the 3238C>G polymorphism in APOC3.
As is the case for the L162V polymorphism at the PPARA locus, the data concerning the effects of the 3238C>G polymorphism on lipid and apolipoprotein concentrations are not consistent between study populations. The rare G3238 allele has been associated with elevated serum triglyceride21,22⇓or apoC-III23 concentrations in many, but not all, 15,24,25⇓⇓ studies. A previous study using the same study population as our study failed to demonstrate any association between the genotype and either of these two variables in men or women.15 The authors suggested that significant gene-gene or gene-environment interactions might exist that mask the effect of this polymorphism in this study population. This hypothesis is now supported by the finding that inclusion of the L162V polymorphism in PPARA in the model resulted in a significant association between apoC-III concentration and the 3238C>G polymorphism that was absent previously (data not shown).15 We found that the effect of the L162V polymorphism in PPARA on apoC-III concentration was seen most prominently among carriers of the G3238 allele (Figure 1). The interaction term PPARA×APOC3 genotype was of borderline statistical significance (P=0.07 data not shown) when included in the model.
We also found that genetic variation at the APOE locus modulated the impact of the L162V polymorphism on LDL cholesterol concentrations. Polymorphisms at the APOE locus are some of the most important genetic determinants of LDL cholesterol concentrations in the general population. The E2 allele is associated with low concentrations of LDL cholesterol in this14 and other populations.26 The E2 allele is also known to demonstrate impaired TRL binding by the LDL receptor27 and more recently, has been shown to be associated with impaired lipoprotein lipase–mediated lipolysis of TRLs.28 The greatest increase in LDL cholesterol associated with the L162V polymorphism (Figure 1) was seen in carriers of the E2 allele and the smallest in those with the E4 allele.
We believe these data provide some insight into the pathways that could be involved in generating the association between LDL cholesterol concentration and the L162V polymorphism at the PPARA locus. Both the 3238C>G polymorphism in APOC3 and the E2 allele in APOE are associated with decreased TRL clearance. The predominant effect of the L162V polymorphism in PPARA thus occurs in those who are already genetically predisposed to have an impaired TRL catabolism. These findings, along with the increased apoC-III concentrations observed, suggest that alterations in TRL metabolism may be involved in the generation of higher LDL concentrations observed in this and previous studies. The in vitro evidence that the V162 allele is associated with altered transcriptional activation and the knowledge that several of the genes involved in TRL metabolism are regulated by PPARα (eg, hepatic lipase, lipoprotein lipase, and apoC-III) would be in line with this hypothesis.
The absence of any association between the L162V polymorphism at the PPARA locus and TRL or triglyceride concentrations may appear to negate this hypothesis. Several possible reasons exist for this discrepancy. Intra-individual variation in the plasma VLDL cholesterol concentration is large, whereas that for LDL cholesterol is relatively small. This phenomenon will reduce the chance of finding an association between a genetic variant and plasma TRL concentrations, especially when the changes are small, as in this case. Alternatively, additional gene-gene or gene-diet interactions may exist that modulate the effect of the L162V polymorphism, and this will need to be addressed in future studies. Potential gene-environment interactions may be particularly relevant to this polymorphism. It has been found that the mutation shows opposing effects on gene transcription depending on the concentration of exposure to a PPARα ligand.6 At low concentrations of an agonist, it shows reduced ability, whereas at high concentrations, it shows increased ability to activate gene transcription. The level of exposure to known endogenous (such as nonesterified fatty acids) or exogenous ligands (such as dietary polyunsaturated fatty acids) may therefore be important in determining the biochemical phenotype observed with this polymorphism.
In conclusion, in a largely normolipemic population, the L162V polymorphism was associated with increased plasma concentrations of total cholesterol, LDL cholesterol, and apoB. Therefore, our data, in combination with previous reports, underscore the relevance of this locus as a modulator of cholesterol levels in the population. In addition, increased concentrations of apoC-III and increased effect in those subjects who also carried the G3238 allele at the APOC3 locus and the E2 allele at the APOE locus, suggest that alterations in TRL metabolism may be involved in the generation of the biochemical phenotype associated with this genotype.
Supported by NIH/NHLBI grant no. HL54776, NIH/NHLBI contract no. 1-38038, and contracts 53-K06-5-10 and 58-1950-9-001 from the US Department of Agriculture Research Service.
Received November 19, 2001; revision accepted January 23, 2002.
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