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A Val227Ala polymorphism in the peroxisome proliferator activated receptor α (PPARα) gene is associated with variations in serum lipid levels
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1. K Yamakawa-Kobayashi1,
2. H Ishiguro1,
3. T Arinami1,
4. R Miyazaki2,
5. H Hamaguchi1
1. 1Department of Medical Genetics, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan
2. 2Department of Internal Medicine, Kudanzaka Hospital, Kudan-minami, Tokyo 102-0074, Japan
1. Correspondence to:  Dr K Yamakawa-Kobayashi, Department of Medical Genetics Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan;  kkimiko{at}md.tsukuba.ac.jp

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Peroxisome proliferator activated receptors (PPARs) are ligand activated transcription factors that belong to the nuclear receptor superfamily.1, 2 PPARs regulate gene transcription by heterodimerising with retinoic X receptors and binding to DNA sequences, termed PPAR response elements (PPRE), in the promoters of target genes.2, 3

Three different PPAR genes (α, δ/β, and γ), each displaying distinct tissue and developmental expression patterns, have been identified.4, 5PPARα, the first member of the PPAR family to be identified, was cloned as an orphan receptor activated by agents that induce peroxisome proliferation.6 It is expressed primarily in tissues with high levels of fatty acid oxidation, such as those in liver, kidney, heart, and muscle.4, 6 Most target genes of PPARα encode enzymes involved in oxidation of cellular fatty acids, lipid transporters, and apolipoproteins.1, 7 Furthermore, PPARα is known to mediate the actions of fibrates, which are hypolipidaemic drugs that decrease plasma triglycerides and increase high density lipoprotein (HDL) cholesterol concentrations.8, 9 Numerous in vivo and in vitro studies have suggested that PPARα is a key metabolic regulator involved in lipid and glucose homeostasis.1, 10–12 Recently, a Leu162Val polymorphism was identified in the PPARα gene, and this polymorphism was shown to influence plasma lipid concentrations, especially in type II diabetes patients.13, 14 In the present study, we have screened for polymorphisms in the PPARα gene to determine whether genomic variations in this gene influence serum lipid levels, glucose levels, and body mass index (BMI) in healthy subjects.

## SUBJECTS AND METHODS

Taking part in this study were 401 unrelated and apparently healthy subjects (207 men and 194 women) who visited medical clinics in Tokyo for routine medical check ups. They were all Japanese. They ranged in age from 29 to 75 years with a mean age (SD) of 47.7 (SD 9.1) years. The age range of the subjects is broad because we selected them without consideration of their age. The clinical characteristics and serum lipid and fasting glucose levels of the subjects are shown in table 1. People with medical histories that included liver or renal abnormalities or diabetes mellitus and people taking medication known to affect lipid metabolism were excluded from the study. Blood samples were taken after an overnight fast. Informed consent was obtained from all subjects and the study was approved by the Ethics Committee of the University of Tsukuba.

Table 1

Clinical characteristics and serum lipid and glucose levels of the subjects

Serum total cholesterol, HDL-C, triglycerides, and glucose levels were measured by standard enzymatic methods as described elsewhere.15 Serum LDL cholesterol (LDL-C) levels were estimated according to Friedewald's equation.16 Lipid and glucose levels are presented as mg/dl.

The genomic structure of the PPARα gene was deduced through a BLAST search of GenBank (Accession numbers NM_005036 and AL032818). Potential mutations in coding exons and exon-intron boundaries of the PPARα gene were screened in 48 randomly selected subjects by single strand conformation polymorphism (SSCP) analysis with the GenePhor System (Amersham-Pharmacia-Biotec). The genotype of the Val227Ala (T→C, nt 892 in NM_005036) polymorphism was assessed by PCR restriction fragment length polymorphism (RFLP). PCR primers were 5-CCCTCCAAACCCTA GCGATTCGTT-3 and 5-CTGGTTCCATGTTGCCAAGAGAAC-3, and the annealing temperature for amplification was 55°C. The 266 bp PCR product was digested with 2 units of Sau96I (New England BioLabs). The Val227 allele was resistant to digestion, whereas the Ala227 allele was digested into 185 bp and 81 bp fragments. The accuracy of this genotyping method was confirmed by sequence analysis of 10 randomly selected samples. The Leu162Val polymorphism of the PPARα gene was analysed with the method described by Vahl et al.14 To avoid genotyping errors, we repeated the analysis twice.

Differences in lipid levels owing to the genotype of the Val227Ala polymorphism were analysed by multiple linear regression analyses incorporating age and BMI as covariates. Differences in fasting glucose levels and BMI owing to the genotype of the Val227Ala polymorphism were analysed by multiple linear regression analyses incorporating age as a covariate. A p value of <0.05 was considered statistically significant. Statistical analyses were performed with the JMP software package (SAS Institute).

## RESULTS AND DISCUSSION

A missense polymorphism, Val227Ala (GTC→GCC), was identified in the PPARα gene by SSCP analysis followed by sequence analysis. Of 401 subjects, 362 were homozygous for the Val227 allele, 37 were heterozygous for the Val227 and Ala227 alleles, and two were homozygous for the Ala227 allele. The distribution of these genotypes did not deviate from Hardy-Weinberg equilibrium.

Table 2 shows the mean values for serum lipid levels, fasting glucose levels, and BMI according to the PPARα genotype for both men and women and total subjects. Because only two subjects were homozygous for the Ala227, we classified the subjects into two groups, carriers of the Ala227 allele and non-carriers.

Table 2

Effects of the Val227Ala polymorphism of the PPARα gene on the serum lipid and glucose levels and BMI

Sex, age, and BMI affect serum lipid levels; therefore, we examined the relationship between the Val227Ala genotype and lipid levels separately in men and women, and p values were calculated after adjustments for age and BMI by multiple linear regression analysis. It is also known that dietary habits, exercise, smoking, and alcohol intake influence serum lipid levels, but we could not obtain information regarding such factors for our subjects.

Among women, the mean serum total cholesterol and triglyceride levels in carriers of the Ala227 allele were significantly lower than those in non-carriers (p=0.046 and p=0.038, respectively). The mean LDL-C level in carriers of the Ala227 allele was lower than that in non-carriers; however, the difference was not significant (p=0.066). In men, a similar trend was observed, but the difference was not significant. Mean HDL-C, fasting glucose levels, and BMI did not differ significantly between the genotype groups in either women or men. In the total subjects, the mean serum total cholesterol level in carriers of the Ala227 allele was significantly lower than that in non-carriers (p=0.033). The lipid profiles of the carriers with the Ala227 allele appear favourable compared with those of non-carriers.

The Val227Ala site is located in the region between the DNA binding and ligand binding domains of the PPARα gene, which is also thought to contain the dimerisation domain. It is possible that the substitution of Val to Ala at codon 227 causes a functional change in PPARα and that the Ala227 isoform has higher activity than the Val227 isoform. However, we have no experimental evidence to support this hypothesis. It will be necessary to examine differences in the transactivation activities in vitro as a result of this amino acid substitution.

It was reported that phenotypes related to lipid and glucose metabolism are strongly influenced by sex in PPARα deficient mice.17, 18 It is assumed that oestrogen signalling plays a role in lipid and glucose homeostasis via regulation of fatty acid utilisation pathways.18, 19 In our present study, the findings suggest that PPARα gene polymorphisms affect serum lipid levels, especially in women. Menopause has a significant effect on serum oestrogen and lipid levels in women; however, we could not obtain information regarding the menopausal status of our subjects. For convenience, we selected 95 women under 45 years from our subjects, assuming that most women under 45 years are premenopausal. A stronger association between the genotypes of the Val227Ala polymorphism and total cholesterol levels was observed in women under 45 years than in the total population of 194 women (p=0.023 v p=0.046) (table 2). Such data appear to support the above assumption for a relationship between oestrogen signalling and lipid homeostasis.

Recently, a Leu162Val polymorphism was identified in white populations.13, 14, 20 Vohl et al14 found an association between the Leu162Val polymorphism and hyperapobetalipoproteinaemia in non-diabetic subjects. Flavell et al13 reported associations between the Leu162Val polymorphism and total cholesterol, HDL-C, and apolipoprotein AI levels in type II diabetic subjects. Furthermore, it was reported that the Val162 isoform increases PPRE dependent transcriptional activity compared with the Leu162 isoform in in vitro transient transfection assays.13, 20 However, we did not find this polymorphism in our Japanese subjects. In contrast, the Val227Ala polymorphism has not been detected in white populations,13, 14, 20 suggesting that there is variability in the frequencies of PPARα gene polymorphisms between ethnic groups.

Many association studies of polymorphic markers in candidate genes related to lipoprotein metabolism and interpersonal variations in serum total and LDL cholesterol levels have been reported.21–23 However, many of the relationships appear to differ between populations with the exception of the apolipoprotein E polymorphism.21–24 Further epidemiological and genetic studies are needed to understand the relationship between the Val227Ala polymorphism in the PPARα gene and serum lipid levels. It will also be interesting to examine the relationship between responses to fibrates and the Val227Ala genotype of the PPARα gene.

## Acknowledgments

We are grateful to the subjects for participating in this study. This study was supported by a Grant-in-Aid for Scientific Research from Ministry of Education, Science, Sports and Culture.

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