ORIGINAL ARTICLE: GENETICS
PON1 polymorphisms are associated with polycystic ovary syndrome susceptibility, related traits, and PON1 activity in Indian women with the syndrome Roshan Dadachanji, M.Sc.,a Nuzhat Shaikh, M.Sc.,a Sushma Khavale, B.Sc.,a Anushree Patil, M.D.,b Nalini Shah, M.D.,c and Srabani Mukherjee, Ph.D.a a Department of Molecular Endocrinology and b Department of Clinical Research, National Institute for Research in Reproductive Health (ICMR), and c Department of Endocrinology, Seth GS Medical College, Parel, Mumbai, India
Objective: To investigate the association of paraoxonase 1 (PON1) polymorphisms (L55M and Q192R) with polycystic ovary syndrome (PCOS) susceptibility and its related traits in Indian women. Design: Case-control study. Setting: Academic research institute, infertility, and endocrinology clinics. Patient(s): Controls (n ¼ 326), women with PCOS (n ¼ 482). Intervention(s): None. Main Outcome Measure(s): Genotypic and allelic frequency distribution, genotype-phenotype association, different PON1 activities (lactonase, arylesterase, and paraoxonase). Result(s): The genotypic and allelic frequency distributions of the L55M polymorphism were significantly different between lean controls and lean women with PCOS, and this polymorphism reduced the risk of PCOS development in lean but not in obese Indian women. Furthermore, this polymorphism was significantly associated with decreased 2-hour glucose, apolipoprotein B, free and bioavailable T, and free androgen index concurrent with increased sex hormone-binding globulin (SHBG) and FSH levels only in lean women with PCOS. However, Q192R polymorphism showed comparable genotypic frequency distribution between controls and women with PCOS. PON1 lactonase and arylesterase activities were significantly decreased in women with PCOS compared with controls. PON1 polymorphisms were shown to influence its activities. Conclusion(s): Our study showed that L55M, but not Q192R, polymorphism is significantly associated with reduced PCOS susceptibility only in lean women and also impacts glucose metabolism, lipid parameters, and hyperandrogenemia in them. Our study therefore suggests the possibility of differential genetic Use your smartphone pathophysiology of PCOS between lean and obese women. (Fertil SterilÒ 2015;-:-–-. to scan this QR code Ó2015 by American Society for Reproductive Medicine.) and connect to the Key Words: PCOS, PON1 gene, L55M, Q192R, hyperandrogenemia, PON1 activity Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/dadachanjir-pon1-polymorphisms-pcos/
Received January 21, 2015; revised and accepted March 31, 2015. R.D. has received research fellowships from University Grants Commission, India. N.S. has received research fellowships from Indian Council of Medical Research, and Department of Science and Technology, Government of India. S.K. has nothing to disclose. A.P. has nothing to disclose. N.S. has nothing to disclose. S.M. has received grant from Department of Science and Technology, Government of India. Supported in part by Department of Science and Technology, [SR/SO/HS-64/2010(G)], National Institute for Research in Reproductive Health (NIRRH/ RA/202/11-2014), and Indian Council of Medical Research, Government of India, as well as fellowships from University Grants Commission, India (R.D.), and Department of Science and Technology, Government of India, and Indian Council of Medical Research (N.S.). Reprint requests: Srabani Mukherjee, Ph.D., Department of Molecular Endocrinology, National Institute for Research in Reproductive Health (ICMR), J.M. Street, Parel, Mumbai 400012, India (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2015 0015-0282/$36.00 Copyright ©2015 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2015.03.037 VOL. - NO. - / - 2015
discussion forum for this article now.*
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P
olycystic ovary syndrome (PCOS) is an endocrine enigma, observed in 5%–10% of women of reproductive age (1). It is a heterogeneous condition often accompanied by anovulatory infertility, increased LHto-FSH ratio, hyperandrogenemia, insulin resistance and compensatory hyperinsulinemia, and polycystic ovaries identified on ultrasound (2). Affected women are at risk for 1
ORIGINAL ARTICLE: GENETICS developing future complications, such as impaired glucose tolerance, type 2 diabetes mellitus, dyslipidemia, and hypertension, that may progress to metabolic syndrome and cardiovascular disease (CVD) (3, 4). Manifestation of anatomical and novel markers of subclinical CVD indicates proatherogenic tendencies in these women (5). Nearly 50% of women with PCOS are obese and often present with severe metabolic disturbances and worsened PCOS phenotypes characterized by exacerbated insulin resistant state (6). Obesity and insulin resistance may elevate oxidative stress, subsequently augmenting the progression of this syndrome and its accompanying cardiometabolic complications (7, 8). Women with PCOS exhibit increased oxidative stress with reduced antioxidant capacity and markers of oxidative stress have been correlated with hyperandrogenemia, a hallmark feature of the syndrome (9). Polycystic ovary syndrome is a multigenic disorder and candidate genes involved in steroidogenic and metabolic pathways have been studied to elucidate its genetic predisposition profile. The interactions of multiple genes and environmental signals serve as contributing factors to the complexity of this syndrome (10). However, at present no universal genetic susceptibility markers for PCOS have emerged. The PON1 gene is located on chromosome 7, which encodes the versatile calcium-dependent antioxidant enzyme paraoxonase 1 (PON1) (11). It is predominantly produced in the liver and circulates in blood associated with highdensity lipoprotein (HDL) cholesterol (11). The major cardioprotective roles of PON1 include impeding the oxidation of low-density lipoprotein (LDL) sequestered in the artery wall, preventing oxidative stress-induced damage to cell membranes, promoting macrophage cholesterol efflux, facilitating hydrolysis of homocysteine thiolactone, and as a result reducing the risk of CVD (11). Two widely studied polymorphisms, namely L55M in exon 3 and Q192R in exon 6 of the PON1 gene, have been recognized to modulate the stability and active site of the enzyme, thereby affecting its levels and catalytic efficiency respectively (12). Several studies have documented the association of these polymorphisms with disorders such as cardiovascular disease (13–15), unfavorable lipid profiles (16), and diabetes (17, 18). Reduced PON1 activity has also been reported in obese subjects, which is related to accelerated onset of cardiometabolic abnormalities (19, 20). Obesity and oxidative stress are inter-related and frequently present in women with PCOS, indicating PON1 activity also may be altered in these women. Earlier studies have reported decreased PON1 activity in women with PCOS, which also showed an inverse correlation with hyperandrogenemia (21, 22). A few groups of investigators have also explored the relationship of these polymorphisms with PCOS and its related traits (23–26). Treatment with metformin and hormonal supplements (ethinyloestradiol þ cyproterone acetate) has been shown to increase PON1 activity and subsequently decrease oxidative stress in women with PCOS (27, 28). However, to our knowledge, no conclusive study exploring the association of PON1 polymorphisms and its activity with PCOS risk, and their relationship with cardiovascular and metabolic 2
dysfunction is available. Therefore, we sought to investigate the impact of PON1 polymorphisms on PCOS and its related traits including insulin resistance, hyperandrogenemia, and dyslipidemia, as well as PON1 activity in a case-control study in Indian women.
MATERIALS AND METHODS Subjects The study population consisted of 482 women with PCOS, recruited consecutively from the Infertility Clinic at the National Institute for Research in Reproductive Health and the Endocrinology Clinic of Seth GS Medical College and KEM Hospital, Mumbai, India. Diagnosis of PCOS was defined according to the European Society of Human Reproduction and Embryology (ESHRE)/American Society for Reproductive Medicine (ASRM) Rotterdam 2003 criteria, including the presence of at least two of the following three features: [1] oligomenorrhoea and/or anovulation, [2] clinical and/or biochemical signs of hyperandrogenemia, and [3] polycystic ovaries (PCOs) identified on ultrasound (29). Other related disorders, like nonclassic congenital adrenal hyperplasia, thyroid dysfunction, androgen-secreting tumors, and hyperprolactinemia, were excluded. Study controls were 326 agematched regularly menstruating women with absence of any clinical and/or biochemical signs of hyperandrogenemia and having normal ovaries on ultrasound imaging, enrolled from the local community with the same ethnic background. Participants who had taken any medication that would alter hormonal parameters or lipid and carbohydrate metabolism were excluded from the study. The study was approved by the Human Ethics Committees of both institutions and written informed consent was obtained from each participant. Genotyping was performed for all study participants and comprehensive phenotype characterization was carried out in 285 women with PCOS and 174 controls from whom suitable serum samples were available. Anthropometric data (including height, weight, waist and hip circumferences, body mass index [BMI]) were collected from each participant. To carry out BMI-based analysis, study subjects were categorized as lean (BMI
PON1 polymorphisms are associated with polycystic ovary syndrome susceptibility, related traits, and PON1 activity in Indian women with the syndrome Roshan Dadachanji, M.Sc.,a Nuzhat Shaikh, M.Sc.,a Sushma Khavale, B.Sc.,a Anushree Patil, M.D.,b Nalini Shah, M.D.,c and Srabani Mukherjee, Ph.D.a a Department of Molecular Endocrinology and b Department of Clinical Research, National Institute for Research in Reproductive Health (ICMR), and c Department of Endocrinology, Seth GS Medical College, Parel, Mumbai, India
Objective: To investigate the association of paraoxonase 1 (PON1) polymorphisms (L55M and Q192R) with polycystic ovary syndrome (PCOS) susceptibility and its related traits in Indian women. Design: Case-control study. Setting: Academic research institute, infertility, and endocrinology clinics. Patient(s): Controls (n ¼ 326), women with PCOS (n ¼ 482). Intervention(s): None. Main Outcome Measure(s): Genotypic and allelic frequency distribution, genotype-phenotype association, different PON1 activities (lactonase, arylesterase, and paraoxonase). Result(s): The genotypic and allelic frequency distributions of the L55M polymorphism were significantly different between lean controls and lean women with PCOS, and this polymorphism reduced the risk of PCOS development in lean but not in obese Indian women. Furthermore, this polymorphism was significantly associated with decreased 2-hour glucose, apolipoprotein B, free and bioavailable T, and free androgen index concurrent with increased sex hormone-binding globulin (SHBG) and FSH levels only in lean women with PCOS. However, Q192R polymorphism showed comparable genotypic frequency distribution between controls and women with PCOS. PON1 lactonase and arylesterase activities were significantly decreased in women with PCOS compared with controls. PON1 polymorphisms were shown to influence its activities. Conclusion(s): Our study showed that L55M, but not Q192R, polymorphism is significantly associated with reduced PCOS susceptibility only in lean women and also impacts glucose metabolism, lipid parameters, and hyperandrogenemia in them. Our study therefore suggests the possibility of differential genetic Use your smartphone pathophysiology of PCOS between lean and obese women. (Fertil SterilÒ 2015;-:-–-. to scan this QR code Ó2015 by American Society for Reproductive Medicine.) and connect to the Key Words: PCOS, PON1 gene, L55M, Q192R, hyperandrogenemia, PON1 activity Discuss: You can discuss this article with its authors and with other ASRM members at http:// fertstertforum.com/dadachanjir-pon1-polymorphisms-pcos/
Received January 21, 2015; revised and accepted March 31, 2015. R.D. has received research fellowships from University Grants Commission, India. N.S. has received research fellowships from Indian Council of Medical Research, and Department of Science and Technology, Government of India. S.K. has nothing to disclose. A.P. has nothing to disclose. N.S. has nothing to disclose. S.M. has received grant from Department of Science and Technology, Government of India. Supported in part by Department of Science and Technology, [SR/SO/HS-64/2010(G)], National Institute for Research in Reproductive Health (NIRRH/ RA/202/11-2014), and Indian Council of Medical Research, Government of India, as well as fellowships from University Grants Commission, India (R.D.), and Department of Science and Technology, Government of India, and Indian Council of Medical Research (N.S.). Reprint requests: Srabani Mukherjee, Ph.D., Department of Molecular Endocrinology, National Institute for Research in Reproductive Health (ICMR), J.M. Street, Parel, Mumbai 400012, India (E-mail: [email protected]). Fertility and Sterility® Vol. -, No. -, - 2015 0015-0282/$36.00 Copyright ©2015 American Society for Reproductive Medicine, Published by Elsevier Inc. http://dx.doi.org/10.1016/j.fertnstert.2015.03.037 VOL. - NO. - / - 2015
discussion forum for this article now.*
* Download a free QR code scanner by searching for “QR scanner” in your smartphone’s app store or app marketplace.
P
olycystic ovary syndrome (PCOS) is an endocrine enigma, observed in 5%–10% of women of reproductive age (1). It is a heterogeneous condition often accompanied by anovulatory infertility, increased LHto-FSH ratio, hyperandrogenemia, insulin resistance and compensatory hyperinsulinemia, and polycystic ovaries identified on ultrasound (2). Affected women are at risk for 1
ORIGINAL ARTICLE: GENETICS developing future complications, such as impaired glucose tolerance, type 2 diabetes mellitus, dyslipidemia, and hypertension, that may progress to metabolic syndrome and cardiovascular disease (CVD) (3, 4). Manifestation of anatomical and novel markers of subclinical CVD indicates proatherogenic tendencies in these women (5). Nearly 50% of women with PCOS are obese and often present with severe metabolic disturbances and worsened PCOS phenotypes characterized by exacerbated insulin resistant state (6). Obesity and insulin resistance may elevate oxidative stress, subsequently augmenting the progression of this syndrome and its accompanying cardiometabolic complications (7, 8). Women with PCOS exhibit increased oxidative stress with reduced antioxidant capacity and markers of oxidative stress have been correlated with hyperandrogenemia, a hallmark feature of the syndrome (9). Polycystic ovary syndrome is a multigenic disorder and candidate genes involved in steroidogenic and metabolic pathways have been studied to elucidate its genetic predisposition profile. The interactions of multiple genes and environmental signals serve as contributing factors to the complexity of this syndrome (10). However, at present no universal genetic susceptibility markers for PCOS have emerged. The PON1 gene is located on chromosome 7, which encodes the versatile calcium-dependent antioxidant enzyme paraoxonase 1 (PON1) (11). It is predominantly produced in the liver and circulates in blood associated with highdensity lipoprotein (HDL) cholesterol (11). The major cardioprotective roles of PON1 include impeding the oxidation of low-density lipoprotein (LDL) sequestered in the artery wall, preventing oxidative stress-induced damage to cell membranes, promoting macrophage cholesterol efflux, facilitating hydrolysis of homocysteine thiolactone, and as a result reducing the risk of CVD (11). Two widely studied polymorphisms, namely L55M in exon 3 and Q192R in exon 6 of the PON1 gene, have been recognized to modulate the stability and active site of the enzyme, thereby affecting its levels and catalytic efficiency respectively (12). Several studies have documented the association of these polymorphisms with disorders such as cardiovascular disease (13–15), unfavorable lipid profiles (16), and diabetes (17, 18). Reduced PON1 activity has also been reported in obese subjects, which is related to accelerated onset of cardiometabolic abnormalities (19, 20). Obesity and oxidative stress are inter-related and frequently present in women with PCOS, indicating PON1 activity also may be altered in these women. Earlier studies have reported decreased PON1 activity in women with PCOS, which also showed an inverse correlation with hyperandrogenemia (21, 22). A few groups of investigators have also explored the relationship of these polymorphisms with PCOS and its related traits (23–26). Treatment with metformin and hormonal supplements (ethinyloestradiol þ cyproterone acetate) has been shown to increase PON1 activity and subsequently decrease oxidative stress in women with PCOS (27, 28). However, to our knowledge, no conclusive study exploring the association of PON1 polymorphisms and its activity with PCOS risk, and their relationship with cardiovascular and metabolic 2
dysfunction is available. Therefore, we sought to investigate the impact of PON1 polymorphisms on PCOS and its related traits including insulin resistance, hyperandrogenemia, and dyslipidemia, as well as PON1 activity in a case-control study in Indian women.
MATERIALS AND METHODS Subjects The study population consisted of 482 women with PCOS, recruited consecutively from the Infertility Clinic at the National Institute for Research in Reproductive Health and the Endocrinology Clinic of Seth GS Medical College and KEM Hospital, Mumbai, India. Diagnosis of PCOS was defined according to the European Society of Human Reproduction and Embryology (ESHRE)/American Society for Reproductive Medicine (ASRM) Rotterdam 2003 criteria, including the presence of at least two of the following three features: [1] oligomenorrhoea and/or anovulation, [2] clinical and/or biochemical signs of hyperandrogenemia, and [3] polycystic ovaries (PCOs) identified on ultrasound (29). Other related disorders, like nonclassic congenital adrenal hyperplasia, thyroid dysfunction, androgen-secreting tumors, and hyperprolactinemia, were excluded. Study controls were 326 agematched regularly menstruating women with absence of any clinical and/or biochemical signs of hyperandrogenemia and having normal ovaries on ultrasound imaging, enrolled from the local community with the same ethnic background. Participants who had taken any medication that would alter hormonal parameters or lipid and carbohydrate metabolism were excluded from the study. The study was approved by the Human Ethics Committees of both institutions and written informed consent was obtained from each participant. Genotyping was performed for all study participants and comprehensive phenotype characterization was carried out in 285 women with PCOS and 174 controls from whom suitable serum samples were available. Anthropometric data (including height, weight, waist and hip circumferences, body mass index [BMI]) were collected from each participant. To carry out BMI-based analysis, study subjects were categorized as lean (BMI
