Association of the T45G polymorphism in exon 2 of the adiponectin gene with polycystic ovary syndrome: role of {Delta}4-androstenedione

Dimitrios Panidis1,4, Anargyros Kourtis1, Asterios Kukuvitis1, Dimitrios Farmakiotis1, Nectaria Xita2, Ioannis Georgiou3 and Agathocles Tsatsoulis2

1 Division of Endocrinology and Human Reproduction, Second Department of Obstetrics and Gynecology, Aristotle University of Thessaloniki, Thessaloniki, 2 Division of Endocrinology, Department of Medicine and 3 Genetics Unit, Department of Obstetrics and Gynecology, University of Ioannina, Ioannina, Greece

4 To whom correspondence should be addressed at: 119 Mitropoleos Street, 54622, Thessaloniki, Greece. Email: argic{at}med.auth.gr


    Abstract
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
BACKGROUND: Insulin resistance is a prominent feature of polycystic ovary syndrome (PCOS), independent of obesity. It is possible that insulin resistance in PCOS is genetically determined. Adiponectin is a protein that modulates insulin action and is regarded as a possible link between adiposity and insulin resistance. The objective of this study was to examine the role of the adiponectin gene T45G polymorphism, located in exon 2, in PCOS, since this polymorphism has been shown to be associated with obesity and insulin resistance. SUBJECTS AND METHODS: Two hundred and thirty-two women were studied, and were classified as follows: 132 women with PCOS [62 with body mass index (BMI) >25 kg/m2 and 70 with BMI <25 kg/m2] and 100 ovulating women without hyperandrogenemia (controls: 19 with BMI >25 kg/m2 and 81 with BMI <25 kg/m2). From all subjects a whole-blood sample was taken and was used for isolation of peripheral blood leukocytes. The adiponectin T45G polymorphism, located in exon 2, was genotyped by amplification of genomic DNA. In all subjects, serum gonadotropin, androgen, 17-OH-progesterone, fasting glucose, insulin and adiponectin levels were measured between the third and sixth day of the menstrual cycle. RESULTS: A statistically significant difference was observed in the frequency of GG and TG genotypes between women with PCOS (40/132; 30.3%) and controls (19/100; 19.0%). In a subgroup of PCOS women with high {Delta}4-androstenedione levels ({Delta}4A >3.11 ng/ml), a statistically significant difference between the frequencies of the genotypes was also noticed compared with the control group, in contrast to the subgroup with relatively low {Delta}4-androstenedione levels ({Delta}4A <3.11 ng/ml). No significant associations were found between this adiponectin polymorphism and BMI, testosterone level, adiponectin levels and glucose-to-insulin ratio. CONCLUSIONS: Our study suggests that adiponectin polymorphisms are not causatively involved in the metabolic disturbances of PCOS, but that an interaction between adiponectin and steroid synthesis or action might exist.

Key words: adiponectin/polycystic ovary syndrome/gene polymorphism


    Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Polycystic ovary syndrome (PCOS) is characterized by chronic anovulation and hyperandrogenism, being the most frequent cause of anovulatory infertility (Lobo, 2003Go). Furthermore, insulin resistance with compensatory hyperinsulinemia are prominent features of the syndrome, possibly genetically determined (Goodarzi and Korenman, 2003Go; Lord et al., 2003Go). It is also possible that the molecular mechanisms of these disorders in PCOS are different from those observed in obesity and diabetes mellitus type 2 (DM2) when the latter are not associated with reproductive morbidity.

The infamous ‘metabolic syndrome’ or ‘X-syndrome’, i.e. the combination of insulin resistance with hypertension, dyslipidemia and central obesity, is nowadays regarded as a major risk factor for the development of DM2 and cardiovascular disease (Reusch, 2002Go). Furthermore, reproductive disorders are often observed in pre-menopausal women with metabolic syndrome. Therefore, the combined metabolic and reproductive morbidity of PCOS has recently led to the introduction of the term ‘XX-syndrome’ (Sam and Dunaif, 2003Go).

Once regarded as an inert depot of triglycerides, adipose tissue has recently emerged as an active endocrine organ that regulates energy metabolism by secreting a number of substances, so-called ‘adipo(cyto)kines’ (Ahima and Flier, 2000Go). Apparently, these peptides influence peripheral insulin sensitivity, playing an important role in the pathogenesis of metabolic syndrome (Mantzuzawa et al., 1999Go).

One relatively well-studied molecule of this kind is adiponectin, identified in 1995 by Scherer et al. (1995)Go. Adiponectin is the only known adipocytokine, the levels of which are reduced in obese, compared with normal, individuals (Arita et al., 1999Go). Adiponectin has been shown to have anti-atherogenic effects (Ouchi et al., 1999Go; 2001Go; Okamoto et al., 2000Go), and a number of studies also suggest a potent insulin-sensitizing action of this hormone (Lindsay et al., 2002Go; Daimon et al., 2003Go; Spranger et al., 2003Go; Tschritter et al., 2003Go).

The adiponectin gene consists of three exons and two introns spanning a 17-kb region, and has been located in chromosome 3q27 (Scherer et al., 1995Go; Hu et al., 1996Go). One common and two rare genetic polymorphisms in the adiponectin gene have been identified in non-diabetic populations (Schaffler et al., 2000Go; Takahashi et al., 2000Go). The silent T/G polymorphism in exon 2 of the human adiponectin gene (T45G->Gly15Gly) could somehow affect plasma adiponectin levels (Takahashi et al., 2000Go; Yang et al., 2003aGo; bGo). This highly prevalent polymorphism has recently been reported to be associated with the risk of obesity, insulin resistance, DM2 and high levels of low-density lipoprotein cholesterol (Hara et al., 2002Go; Menzaghi et al., 2002Go; Stumvoll et al., 2002Go; Ukkola et al., 2003Go; Yang et al., 2003aGo; bGo). In contrast, others have found no association of this particular locus with obesity or DM2 (Schaffler et al., 2000Go; Takahashi et al., 2000Go; Zietz et al., 2001Go; Filippi et al., 2004Go).

Despite the numerous reports studying the association between adiponectin gene polymorphisms and insulin resistance or obesity, until now, no studies have examined adiponectin gene polymorphisms in women with PCOS. In this study, we investigated the possible association of the T45G adiponectin gene polymorphism with PCOS.


    Subjects and methods
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 Introduction
 Subjects and methods
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 Discussion
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Subjects
One hundred and thirty-two women with PCOS were selected. They had all presented at our gynecological endocrinology infirmary with at least one of the following signs: oligomenorrhea, fertility problem, hirsuitism, acne or male-pattern alopecia. None of the women had galactorrhea, nor any systemic disease that could possibly affect their reproductive physiology. Furthermore, no woman reported use of any medication that could interfere with the normal function of the hypothalamic–pituitary–gonadal axis. One hundred healthy women with regular menses and without hyperandrogenemia volunteered as controls. All women in the study were genetically unrelated.

All 232 women were divided into two groups, based on the diagnosis of PCOS: group I, 132 women with PCOS [62 with body mass index (BMI) >25 kg/m2 and 70 with BMI <25 kg/m2]; and group II, 100 ovulating women (19 with BMI >25 kg/m2 and 81 with BMI <25 kg/m2) without hyperandrogenemia (controls). Diagnosis of PCOS in women from group I was based on the presence of chronic anovulation (less than six cycles in 12 months) and hyperandrogenemia. Furthermore, other common causes of hyperandrogenism (prolactinoma, congenital adrenal hyperplasia, Cushing syndrome and virilizing ovarian or adrenal tumours) were excluded, in accordance with the criteria proposed in 1990 by the NIH and revised in 2003 (Zawadski and Dunaif, 1992Go; The Rotterdam ESHRE/ASRM Sponsored PCOS Consensus Workshop Group, 2004Go). All women in group II had normal ovulating cycles (28±2 days, mean±SD) with blood progesterone levels >10 ng/ml in two consecutive cycles, and no signs of hyperandrogenism.

In all women, the basal serum levels of FSH, LH, testosterone, {Delta}4-androstenedione ({Delta}4A) and dehydroepiandrosterone sulfate (DHEA-S) were measured. Serum levels of prolactin (PRL), 17{alpha}-OH-progesterone, sex-hormone-binding globulin (SHBG), glucose and insulin were also measured. In 61 women with PCOS and 25 controls, serum adiponectin levels were also available. All measurements were performed between the third and sixth day of the menstrual cycle. Free androgen index (FAI) was calculated according to the equation: testosterone (nmol/l)x100/SHBG (nmol/l). Glucose-to-insulin ratio was also calculated.

Blood samples were collected between the third and sixth day of a spontaneous menstrual cycle, at 09:00, after an overnight fast. Informed consent was obtained from all 232 women and the study was approved by the Institutional Review Board. All women with PCOS were outpatients attending the Endocrine Unit of the 2nd Department of Obstetrics and Gynecology of the Aristotle University of Thessaloniki at the Hippocration Hospital of Thessaloniki. Control women were volunteers. Genotype analysis was performed at the Genetics Unit of the Department of Obstetrics and Gynecology at the University of Ioannina.

Assay methods
Hormonal measurements. The basal serum levels of FSH, LH, testosterone, {Delta}4A, DHEA-S, PRL, 17{alpha}-OH-progesterone, SHBG, adiponectin, insulin and glucose were measured, as described previously (Panidis et al., 2003Go).

Genotype analysis. Genomic DNA was isolated from peripheral blood leukocytes of women with PCOS and the controls. The adiponectin T45G polymorphism, located in exon 2, was genotyped by amplification of genomic DNA using the following primers: F5'-GAATGAGACTCTGCTGAGATGG and R5'- TATCATGTGAGGAGTGCTTGGATG. PCR products were obtained using 25 µl reactions [0.3 µg genomic DNA, 2 pmol/µl primers, 2 mmol/l each of deoxy-ATP, TGP, CTP and TTP, 0.5 U TaqDNA polymerase (Invitrogen, Life Technologies, USA), 1 mmol/l MgCl2] in a thermal cycler (PTC-100; MJ Research, Inc., USA). The amplification conditions were as follows: 94°C for 5 min, followed by 30 cycles of 30 s at 94°C, 30 s at 60°C and 90 s at 72°C, and ending with a single 10 min extension step at 72°C. The resulting fragment was 372 bp in length. The polymorphism was typed with enzyme Smal (BioLabs Inc., New England). Digestion of the G allele produced two fragments with lengths 216 and 156 bp. The digestion products were resolved after electrophoresis in 2% agarose gel (Figure 1).



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Figure 1. Comparison of BMI, FAI and glucose-to-insulin ratio between the three genotypes, in women with PCOS (mean±SD).

 
Statistical analysis
The distributions of genotypes were compared between study groups by {chi}2-tests. Differences in biochemical parameters and BMI between the three genotypes were assessed by the t-test for independent samples, after log transformation, in order to achieve a more normal distribution. All results are reported as the mean±SD. In both studied groups the genotype frequency distributions of T45G polymorphism were in Hardy–Weinberg equilibrium. All analyses were performed with the SPSS statistical package, version 11.5 (SPSS Inc., Chicago, IL, USA). Statistical significance was set at 5%


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 Subjects and methods
 Results
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Endocrine and anthropometric data of women with PCOS and controls are shown in Table I. BMI values, FAI, testosterone, {Delta}4A and DHEA-S levels were higher in the PCOS group compared with controls, whereas glucose-to-insulin ratio and adiponectin levels were lower (P<0.05 for BMI, P<0.005 for adiponectin, P<0.001 in all other comparisons).


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Table I. Endocrine and anthropometric data of women with PCOS and controls (mean±SD statistical significance)

 
The frequency of the TT compared with the TG, GG and TG + GG genotypes in women with PCOS and in controls is shown in Table II. Although no difference was observed when {chi}2-test was performed on the distributions of the three genotypes separately, a statistically significant difference ({chi}2=3.833, P<0.05) was observed in the frequency of GG and TG genotypes between women with PCOS (40/132; 30.3%) and controls (19/100; 19.0%). In both groups studied, the genotype frequency distributions of this polymorphism were in Hardy–Weinberg equilibrium ({chi}2=2.77, {chi}2=1.01, P>0.05). There was no difference in the frequency of the TT compared with the TG, GG and TG + GG genotypes between normal-weight and obese or overweight women.


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Table II. The frequency of the TT compared with the TG+GG genotypes in women with PCOS and in controls, and statistical significance

 
Women with PCOS were divided into two subgroups, based on mean serum concentration of {Delta}4A (3.11 ng/ml). Again, no difference was observed when {chi}2-test was performed on the distributions of the three genotypes separately. However, in the subgroup of PCOS women with high {Delta}4A levels (>3.11 ng/ml), a statistically significant difference ({chi}2=3.95, P<0.05) between the frequencies of the TG + GG genotypes was noticed compared with the control group. However, no significant difference was observed between the subgroup with relatively low {Delta}4A (<3.11 ng/ml) and the control group (Table III). No difference was observed when women with PCOS were again divided into two subgroups, based on mean values of testosterone and DHEA-S levels, FAI or the glucose-to-insulin ratio.


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Table III. The frequency of the TT compared with the (TG+GG) genotypes in women with PCOS with high or low {Delta}4A levels and in controls, and statistical significance

 
No difference in testosterone, DHEA-S or {Delta}4A levels, BMI, FAI or the glucose-to-insulin ratio was observed between the three genotypes. The above comparisons did not yield any significant differences whether performed in all women or separate groups (women with PCOS and controls) (Figure 1). Subjects with PCOS carrying the G allele had lower adiponectin levels, but the difference was not statistically significant (Figure 2).



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Figure 2. Comparison of serum adiponectin levels between the three genotypes, in women with PCOS and controls (mean±SD).

 

    Discussion
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 Subjects and methods
 Results
 Discussion
 References
 
The novel ‘adipokine’ adiponectin has been proposed as a possible mediator of target tissue sensitivity to insulin (Chandran et al., 2003Go; Diez and Iglesias, 2003Go; Goldfine and Kahn, 2003Go). The strong linkage disequilibrium with DM2, adiposity and insulin levels, found in the region where the adiponectin gene is located (Kissebah, 2000; Vionett, 2000), suggests that somewhere on this locus a common genetic variant must have an influence on obesity and/or glucose homeostasis. Exactly what genetic alterations are in linkage disequilibrium with adiponectin gene polymorphisms is not clear at present.

The purpose of our study was to examine for the first time: (i) the frequency of the common T45G polymorphism in exon 2 of the adiponectin gene in women with PCOS, compared with normal controls; and (ii) the effect of this polymorphism on anthropometric, hormonal and metabolic parameters of the syndrome. In all groups, no association of T45G polymorphism with BMI and glucose-to-insulin ratio was observed.

This finding is in concordance with recent studies, where no association was found between obesity or insulin resistance and the T45G polymorphism (Schaffler et al., 2000Go; Takahashi et al., 2000Go; Zietz et al., 2001Go; Filippi et al., 2004Go). Nevertheless, significant associations between this highly prevalent polymorphism and obesity or insulin resistance have also been reported (Hara et al., 2002Go; Menzaghi et al., 2002Go; Stumvoll et al., 2002Go; Ukkola et al., 2003Go; Yang et al., 2003aGo; bGo). Our findings support the hypothesis that the T45G polymorphism in exon 2 of the adiponectin gene is not directly linked to obesity and the metabolic disturbances of the ‘X-syndrome’, as demonstrated in PCOS, at least in the population studied.

It is interesting, though, that different frequencies of the TG+GG genotypes were found in women with PCOS compared with controls. Furthermore, the frequency of GG+TG genotypes in a subgroup of PCOS women with relatively higher {Delta}4A levels was significantly higher compared with the control group, although no difference was noticed between the subgroup of PCOS women with relatively lower {Delta}4A levels and the control group. Testosterone levels and FAI were not correlated with genotype frequencies in a similar way. In a recently published study (Panidis et al., 2003Go) it was demonstrated that {Delta}4A, but not testosterone, levels were negatively correlated with serum adiponectin levels. Moreover, this correlation was BMI-independent.

Subjects with PCOS carrying the G allele had lower adiponectin levels, but without statistical significance (Figure 2). This finding is in agreement with previous data (Takahashi et al., 2000Go). Furthermore, a significant correlation of another polymorphism (G276T) with lower circulating adiponectin has been observed (Hara et al., 2002Go). The two polymorphisms might be in linkage disequilibrium, and the G/G haplotype has been strongly associated with the metabolic disturbances of PCOS, as well as lower plasma adiponectin concentration (Menzaghi et al., 2002Go).

Therefore, although the number of women homozygous for the G allele in our study was relatively small to draw firm conclusions, we postulate that there is a complex relationship, possibly a negative feedback loop, between adiponectin and the hypothalamic–pituitary–gonadal axis, specifically steroid synthesis or action. Indeed, in vitro studies have shown that both glucocorticoids and androgens (Nishizawa et al., 2002Go) down-regulate the expression of adiponectin, and there is substantial evidence suggestive of a complex interaction between this hormone and gonadal function (Combs et al., 2003Go).

However, in a previous study (Panidis et al., 2003Go), no difference was found in plasma adiponectin levels between women with PCOS and BMI-matched controls, which is in agreement with another recently published report (Orio et al., 2003Go). It was concluded that circulating adiponectin is not actively involved in the metabolic aspects of the syndrome. Nevertheless, it is possible that adipokines also act in a paracrine manner (Panidis et al., 2004aGo). For instance, a very recent study suggests that resistin could be involved in the pathogenesis of the syndrome as a local factor (Seow et al., 2004Go), although no difference in plasma levels was observed, in concordance with our own report on resistin levels in PCOS (Panidis et al., 2004bGo).

In conclusion, our findings suggest an interaction between adiponectin and steroid synthesis or action in PCOS, although the physiological significance of such a relationship remains obscure at present. No statistically significant link between the T45G adiponectin gene polymorphism and circulating adiponectin, obesity or the metabolic aspects of PCOS was observed, although women with the syndrome carrying the G allele had a tendency for lower serum adiponectin levels. Since the T45G polymorphism is a synonymous mutation, the exact molecular mechanisms responsible for the biological effects of this variation are not known at present. It is plausible that this polymorphism is in linkage disequilibrium with some other functional genetic alterations. More data are needed to specify the systemic and local function of the newly identified ‘adipocytokines’ in the pathophysiology of PCOS.


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Submitted on January 26, 2004; accepted on April 30, 2004.