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
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Abstract |
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Key words: PCOS/polymorphism/PPAR
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Introduction |
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The Pro12 Ala polymorphism of the PPAR gene has been associated with reduced transcriptional activity of PPAR
and the presence of the Ala isoform has been linked to both higher insulin sensitivity and lower body mass index (Deeb et al., 1998
).
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., 1989; Legro et al., 1999
; Morin-Papunen et al., 2000
). 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., 1986
), but suppression of serum insulin concentrations by means of diazoxide reduces serum testosterone levels in women with PCOS (Nestler et al., 1989
). 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
(Dunaif et al., 1996
). Pro12Ala polymorphism of the PPAR
gene attenuates insulin resistance in women with PCOS (Hara et al., 2002
). 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., 2000
, 2001).
In view of the strong evidence implicating PPAR in adiposity and insulin resistance, the object of this study was to determine the frequency of the PPAR
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.
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Materials and methods |
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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 38 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, 1961
). 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 PPAR2 gene we used a forward primer, 5'-GACAAAATATCAGTGTGAATTACAGC-3'and a reverse primer, 5'-CCCAATAGCCGTATCTGGAAGG-3' (Elbrecht et al., 1996
). PCRs were carried out in a 6 µl volume containing 50 ng of genomic DNA, 3 pmol of each primer, 10 mmol/l TrisHCl (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 410-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 3739°C). The gel was autoradiographed overnight at 70°C, with intensifying screens.
Statistical analysis for comparing allele frequencies was conducted using Pearsons 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 KruskalWallis 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. HardyWeinberg distribution of genotypes in the PCOS and control groups was assessed by using Genepop web version 3.1c (http://wbiomed.curtin.edu.au/genepop/).
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Results |
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PPAR genotypes were found to be in HardyWeinberg equilibrium in both the study and control groups.
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Discussion |
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How the Pro12 Ala polymorphism of the PPAR 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
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
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., 1999). Although we conclude that polymorphism of the PPAR
gene is likely to be a genetic predisposing factor as regards PCOS in Finnish women, the possibility that polymorphisms of the PPAR
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
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., 1998
; Pihlajamäki et al., 2000
). 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 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
polymorphism and protection against PCOS might be of biological significance, since Pro12 Ala polymorphism of the PPAR
gene attenuates insulin resistance. Although the PPAR
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.
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Acknowledgements |
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References |
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Submitted on August 23, 2002; resubmitted on October 23, 2002; accepted on November 25, 2002.