Polymorphism in the peroxisome proliferator-activated receptor-{gamma} gene in women with polycystic ovary syndrome

S. Korhonen1, S. Heinonen2,8, M. Hiltunen3, S. Helisalmi4, M. Hippeläinen5, R. Koivunen6, J.S. Tapanainen6 and M. Laakso7

1 Department of Obstetrics and Gynaecology, Central Hospital of Mikkeli, 50100 Mikkeli, Departments of 2 Obstetrics and Gynaecology and 3 Neuroscience, University of Kuopio, 70211 Kuopio, Departments of 4 Neurology and 5 Obstetrics and Gynaecology, Kuopio University Hospital, 70211 Kuopio, 6 Department of Obstetrics and Gynaecology, Oulu University Hospital, PO Box 5000, 90014 Oulu and 7 Department of Medicine, Kuopio University Hospital, 70211 Kuopio, Finland

8 To whom correspondence should be addressed. e-mail: seppo.heinonen{at}kuh.fi


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: In view of the strong evidence implicating peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) in adiposity and insulin resistance a study was carried out to investigate PPAR{gamma} genotype frequencies in women with polycystic ovary syndrome (PCOS) and to elucidate its role in the pathogenesis of the syndrome. METHODS: The study involved 135 women with PCOS and 115 healthy control women who were genotyped for a known functional variant of the PPAR{gamma} gene using single strand conformation polymorphism (SSCP) analysis. RESULTS: A significantly different allele distribution of the Pro12 Ala polymorphism of the PPAR{gamma} gene was observed between the two groups, with the frequency of the variant Ala isoform being significantly reduced in the PCOS group (12.6%) when compared with the control group (19.1%) (P = 0.045), at an odds ratio of 0.609 (95% confidence interval: 0.374–0.991). The genotype distributions of the Pro12 Ala polymorphism in the PCOS and control groups were different with borderline significance (P = 0.051). CONCLUSIONS: Our data support a role for PPAR{gamma} gene polymorphism in the pathogenesis of PCOS, the presence of the Ala isoform being protective against the development of PCOS.

Key words: PCOS/polymorphism/PPAR{gamma}


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) gene is located at chromosomal region 3p25 and it is mainly expressed in the adipose tissue where it promotes the differentiation of preadipocytes into adipocytes (Elbrecht et al., 1996Go; Beamer et al., 1997Go). PPARs are transcription factors that belong to the nuclear hormone receptor family. Reduced transcriptional activity of PPAR{gamma} is associated with an increase in insulin sensitivity (Deeb et al., 1998Go; Jacob et al., 2000Go; Stumvoll et al., 2001Go), which is also seen in heterozygous PPAR{gamma} knock-out mice (Miles et al., 2000Go).

The Pro12 Ala polymorphism of the PPAR{gamma} gene has been associated with reduced transcriptional activity of PPAR{gamma} and the presence of the Ala isoform has been linked to both higher insulin sensitivity and lower body mass index (Deeb et al., 1998Go).

Central obesity, insulin resistance, hyperinsulinaemia and hyperandrogenism are typical features in polycystic ovary syndrome (PCOS), and significant number of PCOS patients show impaired glucose tolerance and are in potential risk of developing type 2 diabetes (Dunaif et al., 1989Go; Legro et al., 1999Go; Morin-Papunen et al., 2000Go). It is not clear whether increased serum androgen levels are associated with decreased insulin sensitivity in these patients. The decrease of serum androgen concentrations by means of a GnRH agonist has not been found to alter insulin sensitivity (Geffner et al., 1986Go), but suppression of serum insulin concentrations by means of diazoxide reduces serum testosterone levels in women with PCOS (Nestler et al., 1989Go). This suggests that the direction of causation is from insulin resistance to androgens in the pathogenesis of PCOS. Glucose intolerance has been shown to be ameliorated by thiazolidinediones, synthetic ligands for PPAR{gamma} (Dunaif et al., 1996Go). Pro12Ala polymorphism of the PPAR{gamma} gene attenuates insulin resistance in women with PCOS (Hara et al., 2002Go). In in-vitro studies, the insulin sensitizer troglitazone also inhibits aromatase activity in cultured human ovarian granulosa cells and cancerous granulosa cells (Mu et al., 2000Go, 2001).

In view of the strong evidence implicating PPAR{gamma} in adiposity and insulin resistance, the object of this study was to determine the frequency of the PPAR{gamma} polymorphism in a group of women with PCOS. A control group of parous women with no signs of PCOS and a reference population including both men and women, the latter of unknown PCOS status, were also included.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Written approval for the study was obtained from the Ethics Committee of Kuopio University Hospital. Informed consent was obtained from all study subjects and the 115 controls.

Information was collected retrospectively with regard to 135 women with PCOS at endocrinology/infertility clinics in the area of Kuopio and Oulu University Hospitals, Finland, and with regard to 115 non-hirsute, fertile control women with regular cycles and normal ovaries who delivered at Kuopio University Hospital between January 1999 and December 1999. In the study group, the indications for referral were menstrual cycle disturbances, infertility and symptoms of hyperandrogenism. In this study the diagnosis of PCOS was based on observation of anovulation and polycystic ovaries in ultrasonography (eight or more subcapsular follicles of 3–8 mm diameter in one plane in one ovary and increased stroma) and exclusion of other reasons for anovulation and hyperandrogenism. In addition, the study group women had one or more of the following clinical or biochemical disturbances: hirsutism, infertility or laboratory testing revealing androgen excess [serum total testosterone concentration >2.5 nmol/l or free plasma testosterone >40 pmol/l, evaluated by means of assay of sex hormone-binding globulin (SHBG) and total testosterone, and an elevated LH/FSH ratio (>2)]. Hirsutism was defined as the presence of excessive body hair in an androgen-dependent pattern, with a modified Ferriman-Gallwey score of >=8 (Ferriman and Gallwey, 1961Go). Diabetic women were excluded from both study and control groups.

DNA was extracted from peripheral blood lymphocytes using a standard phenol-chloroform extraction method. For amplification of exon B of the PPAR{gamma}2 gene we used a forward primer, 5'-GACAAAATATCAGTGTGAATTACAGC-3'and a reverse primer, 5'-CCCAATAGCCGTATCTGGAAGG-3' (Elbrecht et al., 1996Go). PCRs were carried out in a 6 µl volume containing 50 ng of genomic DNA, 3 pmol of each primer, 10 mmol/l Tris–HCl (pH 8.8), 50 mmol/l KCl, 1.5 mmol/l MgCl2, 0.1% Triton X-100, 100µmol/l dNTP, 0.25 units of DNA polymerase (Dynazyme; DNA polymerase, Finnzymes, Espoo, Finland) and 0.55 µCi of [32P]dCTP. PCR conditions were denaturation at 94°C for 3 s, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 4 min. Variants were detected by single strand conformation polymorphism (SSCP) analysis. PCR products were first diluted 4–10-fold with 0.1% SDS and 10 mmol/l EDTA and then diluted (1:1) with loading mix (95% formamide, 20 mmol/l EDTA, 0.05% bromophenol blue, 0.05% xylene cyanole). After denaturation at 98°C for 3 min, the samples were immediately cooled on ice, and 2 µl of each sample was loaded onto 6% non-denaturing polyacrylamide gel (acrylamide/N,N-methylene-bis-acrylamide ratio 49:1) containing 10% glycerol. Samples were run at two different gel temperatures (29°C and 37–39°C). The gel was autoradiographed overnight at –70°C, with intensifying screens.

Statistical analysis for comparing allele frequencies was conducted using Pearson’s {chi}2-test (asymptotic P-values; SPSS 9.0 software) and the level of statistical significance was defined as P < 0.05. Odds ratios (ORs) as estimates of relative risk of the disease were calculated on the basis of 95% confidence intervals (CI). A Kruskal–Wallis test was done on the genotypes since it allows the exploiting of the fact that the genotypes are ordinal (i.e. Pro/Pro, Pro/Ala, Ala/Ala) and to look for trends. Hardy–Weinberg distribution of genotypes in the PCOS and control groups was assessed by using Genepop web version 3.1c (http://wbiomed.curtin.edu.au/genepop/).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The clinical characteristics of the study group women are shown in Table I. As dictated by our inclusion criteria, diabetic women were excluded from this study. The 135 women with PCOS were genotyped for Pro12 Ala substitution in the gene encoding PPAR{gamma}. Table II presents the PPAR{gamma} genotype and allele frequencies. Population frequencies were derived from the same geographical area and they were based on 649 tested subjects in the general population (Pihlajamäki et al., 2000Go). The frequency of the Ala substitution allele was 12.6% among PCOS cases and 19.1% among the control women (P = 0.045), which in turn corresponded well to the population frequency (16.0%), as expected. The Ala substitution allele was significantly less frequent among women with PCOS when compared with the control group, at an OR of 0.609 (95% CI: 0.374–0.991).


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Table I. Clinical characteristics of the women with PCOS
 

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Table II. Peroxisome proliferator-activated receptor-{gamma} genotype frequencies among women with PCOS and healthy controls, and in the general population
 
The distribution of genotypes was different in the PCOS group and in the controls with borderline significance (P = 0.051, Kruskal-Wallis). Affected women were then divided into subgroups according to the number of Ala substitution alleles: no Ala substitution allele (genotype Pro/Pro), or one or two Ala substitution alleles (genotypes Pro/Ala and Ala/Ala). Using the control group as a reference, pooled Pro/Ala and Ala/Ala genotypes were associated with protection against PCOS at a borderline level of significance (P = 0.055), and an odds ratio of 0.581 (95% CI: 0.333–1.014). In the PCOS group, the mean body mass indices were not statistically different between the three genotypes.

PPAR{gamma} genotypes were found to be in Hardy–Weinberg equilibrium in both the study and control groups.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this study, we investigated the possible association between PPAR{gamma} polymorphism and the occurrence of PCOS. Given the familial tendency of PCOS and the likely pathogenetic role of insulin resistance in the disease, we investigated the role of a functional variant in the gene encoding PPAR{gamma} in women with PCOS (Franks et al., 2001Go). Based on a priori information we hypothesized that the common Pro allele would increase the risk of PCOS, whereas the Ala variant would be protective against the disease. We found that the functional variant leading to reduced transcriptional activity of the PPAR{gamma} gene appeared to exert a protective effect against PCOS, as expected. The common PPAR{gamma} Pro12 Ala polymorphism is also associated with decreased risk of type 2 diabetes (Altshuler et al., 2000Go) which is in accordance with the present results in view of the overlap between PCOS and type 2 diabetes.

How the Pro12 Ala polymorphism of the PPAR{gamma} gene produces or contributes to the clinical syndrome remains unclear, but the results of previous studies linking insulin resistance to PCOS and the likely pathogenetic role of PPAR{gamma} in insulin resistance are in accordance with the results of our study. Clearly, relating genotype to phenotype in an appropriate manner will be one of the future challenges in diseases that result from varying susceptibility to a wide range of environmental factors, mediated by many different genes. Family studies have demonstrated the importance of genetic factors in PCOS, but the precise mode of inheritance and the molecular basis of the disorder remain uncertain. The study of PCOS is also hampered by the lack of a male phenotype. However, the Ala allele of the PPAR{gamma} gene may be advantageous against the development of PCOS when gene-environment interactions are considered. On the other hand, these results imply that the Pro allele may be a susceptibility factor for PCOS without being necessary for it.

The Finnish population is considered to be a genetic isolate, and therefore it is ideal for genetic association studies (Peltonen et al., 1999Go). Although we conclude that polymorphism of the PPAR{gamma} gene is likely to be a genetic predisposing factor as regards PCOS in Finnish women, the possibility that polymorphisms of the PPAR{gamma} gene segregate with PCOS in other populations with a different ethnic background should be confirmed across populations. Furthermore, our study size did not permit us to analyse data separately for PCOS women with varying degrees of insulin resistance. Attempts to relate the PPAR{gamma} genotype to a quantitative trait such as body mass index (BMI) showed no difference between genotypes in the present study, although recent studies have shown that the Ala 12 allele is associated with high BMI and with dyslipidaemia in elderly subjects (Beamer et al., 1998Go; Pihlajamäki et al., 2000Go). This may indicate that the functional role of Pro12 Ala substitution differs depending on the degree of obesity.

In conclusion, a significant association was found between the single polymorphism located in the coding region of the PPAR{gamma} gene and PCOS, which gave us evidence to accept the original hypothesis. Various candidate genes have been proposed as important contributors to PCOS but none has yet been accepted as a major cause of the condition. The association between Pro12 Ala PPAR{gamma} polymorphism and protection against PCOS might be of biological significance, since Pro12 Ala polymorphism of the PPAR{gamma} gene attenuates insulin resistance. Although the PPAR{gamma} gene has to be considered as a strong candidate associated with the disease, there may be differences across populations and in affected individuals due to the multifaceted nature of PCOS.


    Acknowledgements
 
This study was supported by grants from the Sigrid Juselius Foundation and the Academy of Finland.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Altshuler, D., Hirschorn, J.N., Klannemark, M., Lindgren, C.M., Vohl, M-C., Nemesh, J., Lane, R.L., Schaffner S.F., Bolk, S., Brewer, C. et al. (2000). The common PPAR{gamma}Pro12 Ala polymorphism is associated with decreased risk of type 2 diabetes. Nature Genet., 26, 76–80.[CrossRef][ISI][Medline]

Beamer, B.A., Negri, C., Yen, C.J., Gavrilova, O., Rumberger, J.M., Durcan, M.J., Yarnall, D.P., Hawkins, A.L., Griffin, C.A., Burns, D.K. et al. (1997). Chromosomal localization and partial genomic structure of the human peroxisome proliferator activated receptor-gamma (hPPAR gamma) gene. Biochem. Biophys. Res. Commun., 233, 756–759.[CrossRef][ISI][Medline]

Beamer, B., Yen, C., Andersen, R., Muller, D., Elahi, D., Cheskin, L., Andres, R., Roth, J. and Shuldiner, A.R. (1998) Association of the Pro12Ala variant in the peroxisome proliferator-activated receptor-gamma2 gene with obesity in two Caucasian populations. Diabetes, 47, 1806–1808.[Free Full Text]

Deeb, S.S., Fajas, L., Nemoto, M., Pihlajamäki, J., Mykkänen, L., Kuusisto, J., Laakso, M., Fujimoto, W. and Auwerx, J.A. (1998) Pro12Ala substitution in PPAR gamma 2 is associated with decreased receptor activity, lower body mass index and improved insulin sensitivity. Nature Genet., 20, 284–287.[CrossRef][ISI][Medline]

Dunaif, A., Segal, K.R., Futterweit, W. and Dobrjansky, A. (1989) Profound peripheral insulin resistance, independent of obesity, in polycystic ovary syndrome. Diabetes, 38, 1165–1174.[Abstract]

Dunaif, A., Scott, D., Finegood, D., Quintana, B. and Whitcomb, R. (1996) The insulin-sensitizing agent troglitazone improves metabolic and reproductive abnormalities in the polycystic ovary syndrome. J. Clin. Endocrinol. Metab., 81, 3299–3306.[Abstract]

Elbrecht, A., Chen, Y., Cullinan, C.A., Hayes, N., Leibowitz, M., Moller, D.E. and Berger, J. (1996) Molecular cloning, expression and characterization of human peroxisome proliferator activated receptors gamma 1 and gamma 2. Biochem. Biophys. Res. Commun., 224, 431–437.[CrossRef][ISI][Medline]

Ferriman, D. and Gallwey, J.D. (1961) Clinical assessment of body hair growth in women. J. Clin. Endocrinol. Metab., 21, 1440–1447.[ISI]

Franks, S., Gharani, N. and McCarthy, M. (2001) Candidate genes in polycystic ovary syndrome. Hum. Reprod. Update, 7, 405–410.[Abstract/Free Full Text]

Geffner, M.E., Kaplan, S.A., Berch, N., Golde, D.W., Landaw, E.M. and Chang, R.J. (1986) Persistence of insulin resistance in polycystic ovarian disease after inhibition of ovarian steroid secretion. Fertil. Steril. 45, 327–333.[ISI][Medline]

Hara, M., Alcoser, S.Y., Qaadir, A., Beiswenger, K.K., Cox, N.J. and Ehrmann, D.A. (2002) Insulin resistance is attenuated in women with polycystic ovary syndrome with the Pro(12)Ala polymorphism in the PPARgamma gene. J. Clin. Endocrinol. Metab., 87, 772–775.[Abstract/Free Full Text]

Jacob, S., Stumvoll, M., Becker, R., Koch, M., Nielsen, M., Loblein, K., Maerker, E., Volk, A., Renn, W., Balletshofer, B. et al. (2000) The PPAR gamma 2 polymorphism pro12Ala is associated with better insulin sensitivity in the offspring of type 2 diabetic patients. Horm. Metab. Res., 32, 413–416.[ISI][Medline]

Legro, R.S., Kunselman, A.R., Dodson, W.C. and Dunaif, A. (1999) Prevalence and predictors of risk for type 2 diabetes mellitus and impaired glucose tolerance in polycystic ovary syndrome: a prospective, controlled study in 254 affected women. J. Clin. Endocrinol. Metab., 84, 165–169.[Abstract/Free Full Text]

Miles, P.D., Barak, Y., He, W., Evans, R.M. and Olefsky, J.M. (2000) Improved insulin-sensitivity in mice heterozygous for PPAR-gamma deficiency. J. Clin. Invest., 105, 287–292.[Abstract/Free Full Text]

Morin-Papunen, L.C., Vauhkonen, I., Koivunen, R.M., Ruokonen, A. and Tapanainen, J.S. (2000) Insulin sensitivity, insulin secretion, and metabolic and hormonal parameters in healthy women and women with polycystic ovarian syndrome. Hum.Reprod., 15, 1266–1274.[Abstract/Free Full Text]

Mu, Y.M., Yanase, T., Nishi, Y., Waseda, N., Oda, T., Tanaka, A., Takayanagi, R. and Nawata, H. (2000) Insulin sensitizer, troglitazone, directly inhibits aromatase activity in human ovarian granulosa cells. Biochem. Biophys. Res. Commun., 271, 710–713.[CrossRef][ISI][Medline]

Mu, Y.M., Yanase, T., Nishi, Y., Takayanagi, R., Goto, K. and Nawata, H. (2001) Combined treatment with specific ligands for PPARgamma: RXR nuclear receptor system markedly inhibits the expression of cytochrome P450arom in human granulosa cancer cells. Mol. Cell. Endocrinol., 181, 239–248.[CrossRef][ISI][Medline]

Nestler, J.E., Barlascini, C.O., Matt, D.W., Steingold, K.A., Plymate, S.R., Clore, J.N. and Blackard, W.G. (1989) Suppression of serum insulin by diazoxide reduces serum testosterone levels in obese women with polycystic ovary syndrome. J. Clin. Endocrinol. Metab., 68, 1027–1032.[Abstract]

Peltonen, L., Jalanko, A. and Varilo, T. (1999) Molecular genetics of the Finnish disease heritage. Hum. Mol. Genet., 8, 1913–1923.[Abstract/Free Full Text]

Pihlajamäki, J., Miettinen, R., Valve, R., Karjalainen, L., Mykkänen, L., Kuusisto, J., Deeb, S., Auwerx, J. and Laakso, M. (2000) The Pro12A1a substitution in the peroxisome proliferator activated receptor gamma 2 is associated with an insulin-sensitive phenotype in families with familial combined hyperlipidemia and in nondiabetic elderly subjects with dyslipidemia. Atherosclerosis, 151, 567–574.[CrossRef][ISI][Medline]

Stumvoll, M., Wahl, H.G., Loblein, K., Becker, R., Machicao, F., Jacob, S. and Haring, H. (2001) Pro12Ala polymorphism in the peroxisome proliferator-activated receptor-gamma 2 gene is associated with increased antilipolytic insulin sensitivity. Diabetes, 50, 876–881.[Abstract/Free Full Text]

Submitted on August 23, 2002; resubmitted on October 23, 2002; accepted on November 25, 2002.