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  IN THIS Article
 ::  Abstract
 :: Introduction
 ::  Materials and Me...
 :: Results
 :: Discussion
 :: Acknowledgment
 ::  References
 ::  Article Tables

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  Table of Contents     
ORIGINAL ARTICLE
Year : 2012  |  Volume : 58  |  Issue : 3  |  Page : 190-193

Does follistatin gene have any direct role in the manifestation of polycystic ovary syndrome in Indian women?


1 Molecular Anthropology Group, Biological Anthropology Unit, Indian Statistical Institute, Somajiguda, Hyderabad, India
2 Department of Endocrinology, Osmania General Hospital, Somajiguda, Hyderabad, India
3 Anu Test Tube Baby Centre, Somajiguda, Hyderabad, India
4 Department of Human Genetics, Andhra University, Visakhapatnam, Andhra Pradesh, India

Date of Submission08-Jul-2011
Date of Decision01-Nov-2011
Date of Acceptance09-Jun-2012
Date of Web Publication26-Sep-2012

Correspondence Address:
M B Reddy
Molecular Anthropology Group, Biological Anthropology Unit, Indian Statistical Institute, Somajiguda, Hyderabad
India
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Source of Support: Indian Statistical Institute, Kolkata, Conflict of Interest: None


DOI: 10.4103/0022-3859.101386

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 :: Abstract 

Background: Out of a panel of 37 candidate genes tested for linkage with polycystic ovary syndrome (PCOS), the strongest evidence of linkage was reported in the follistatin (FST) gene region. Subsequently, a couple of studies outside India investigated the FST gene for the presence of any mutations and its association with PCOS and the results were found to be largely inconsistent probably due to differences in the ethnic backgrounds and small sample sizes. Aims: To screen the FST gene for mutations and to establish their association pattern with PCOS among a large cohort of South Indian women. Settings and Design: Case-control study. Materials and Methods: PCOS cases were recruited according to the 2003 Rotterdam diagnostic criteria. All the exons of the FST gene were amplified and analyzed in all the cases and controls for the presence of mutations using polymerase chain reaction (PCR) and direct DNA sequencing. Results: A total of 549 women consisting of 250 PCOS cases and 299 controls were recruited for the study. No mutations were found in any of the exons of the FST gene in our Indian sample which is consistent with an earlier finding among the Asian women from Singapore. Although three of the four cohorts of Caucasian background studied earlier reported variants, none of them could establish a strong association with PCOS. Conclusions: The occurrence of the exonic variants of FST gene seems to be dependent on the ethnic background of the subjects under study and its role in the PCOS pathophysiology cannot be established with hitherto available evidence.


Keywords: Follistatin, genetic polymorphism, Indian women, polycystic ovary syndrome


How to cite this article:
Dasgupta S, Pisapati S, Kudugunti N, Kathragadda A, Godi S, Reddy M B. Does follistatin gene have any direct role in the manifestation of polycystic ovary syndrome in Indian women?. J Postgrad Med 2012;58:190-3

How to cite this URL:
Dasgupta S, Pisapati S, Kudugunti N, Kathragadda A, Godi S, Reddy M B. Does follistatin gene have any direct role in the manifestation of polycystic ovary syndrome in Indian women?. J Postgrad Med [serial online] 2012 [cited 2019 Nov 14];58:190-3. Available from: http://www.jpgmonline.com/text.asp?2012/58/3/190/101386



 :: Introduction Top


Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility in women of reproductive age. The pathophysiology of PCOS appears to be multifactorial, with multiple gene and environmental factors contributing to the disease susceptibility. [1],[2] Multiple genetic pathways have been implicated in the pathogenesis of PCOS, including steroid hormone metabolism, gonadotropin action, obesity and energy regulation and insulin action. [3] A large-scale study was conducted among 150 families to test for evidence of linkage or association of 37 candidate genes with PCOS, and the strongest evidence of linkage was reported in the follistatin (FST) gene region. [4] Follistatin is a single-chain glycoprotein expressed in a number of tissues including the ovary and its primary function is to antagonize the activity of activin resulting in some key features of PCOS such as reduced serum follicle stimulating hormone (FSH), impaired ovarian follicle development and increased ovarian androgen production. [5] Due to its functional significance and the strong linkage pattern observed, the FST gene was considered as a suitable candidate for PCOS. [5] There have been a limited number of studies that attempted to identify sequence variants within this gene and their association with PCOS. [3],[5],[6],[7] The results of these studies have been inconsistent. While Jones et al., [3] and Urbanek et al., [5] found a number of mutations albeit only one of those mutations was found to be associated with PCOS in each of these studies, Calvo et al., [6] found only one mutation in Exon 5 which was not significantly associated with PCOS. In contrast to the above findings, among the Caucasians, Liao et al., [7] did not report any mutation in their cohort of Asian women from Singapore. These inconsistent results can be strongly attributed to the ethnic heterogeneity between the samples studied. Despite being an integral part of the larger Asian continent, India with a number of strictly defined endogamous castes and tribes, presents a unique population scenario as compared to the other Asian populations. In this context, it may be noteworthy that earlier studies on Indian populations reflect a totally different pattern of genetic susceptibility to various complex genetic disorders as compared to the populations of other ethnic backgrounds. [8],[9] Therefore, it is imperative to study the association pattern between FST polymorphism and PCOS among the Indian populations to have a more comprehensive understanding of the nature of genetic association in the Asian region. We present here the results of our study on the possible association of FST with PCOS among South Indian women, by far the largest cohort of PCOS women, hitherto studied for FST.


 :: Materials and Methods Top


Study design

PCOS cases and controls were enrolled for the study during July 2008 to April 2009. We were able to recruit a larger number of samples, which far exceeds the estimated number of samples (N=221) required for obtaining a high statistical power (≥90%) {using Naings online calculator}. Patients were recruited from the gynecology clinic of a public hospital as well as from an infertility clinic, as per the Rotterdam criteria, 2003, [10] according to which any two of the following three conditions need to be fulfilled for the inclusion: (i) presence of clinical and/or biochemical signs of hyperandrogenism, (ii) infrequent periods with intermenstrual interval of more than 35 days, and (iii) polycystic ovaries; an ovary with the ultrasound appearance of more than 10 subcapsular follicles (<10 mm in diameter) in the presence of prominent ovarian stroma was considered polycystic. The adolescent cases in this study were evaluated for clinical/biochemical hyperandrogenism as well as polycystic pattern of the ovaries and not just their menstrual irregularities before recruiting them as PCOS cases. Apart from the clinical evaluation by the gynecologists and endocrinologists, the basic biochemical tests were conducted for all the patients before diagnosing them as PCOS and to exclude other related disorders. Patients with hyperprolactinemia, thyroid and adrenal diseases, 21-hydroxylase deficiency, and androgen-secreting tumors were excluded. The weight and height of the subjects were recorded. Hirsutism was defined as a Ferriman-Gallwey score of more than 5. [11],[12] All other biochemical tests including luteinizing hormone (LH), FSH, lipid profile were done additionally for the patients since they were not the routine tests prescribed by the clinicians at the time of PCOS diagnosis. Once the diagnosis was made, these additional tests were prescribed to the patients and subsequently those hormonal tests could be done in most cases, not in all. We present here the relevant hormonal data which pertain to the physiological pathway of the gene under study, in this case, follistatin.

Normal controls with no history of treatment for fertility, with normal menstrual cycles every 25-32 days and with no signs of clinical hyperandrogenism (hirsutism, acne, alopecia) were recruited from the family planning centre of the public hospital and from the general population. Intravenous blood samples (~5 ml) were collected from both the patients and controls. Informed written consent was obtained from each subject prior to enrollment in the study. The study protocol was approved by the institutional review committee.

DNA extraction, amplification and sequencing

DNA was extracted from the peripheral blood samples of the patients and control using the phenol-chloroform method. [13] We carried out PCR amplification and sequencing to screen the FST exonic regions using the forward and reverse primers. Each PCR was optimized with respect to the concentration of Mg 2+ ions. The PCR-mix consisted of 10xPCRBuffer, 10 μM dNTP-mix, 1 μM of each primer, 1 U Taq-polymerase and 40 ng template DNA in a reaction volume of 10 μl. Reactions were carried out in an ABI GeneAmp9700 thermal cycler (Applied Biosystems, Foster City, CA). Forward and reverse primers and annealing temperature are given in [Table 1].
Table 1: Primers used for PCR and direct DNA sequencing of the human follistatin gene

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Cycle sequencing of PCR products were carried out with either the forward or the reverse primers using the Big-Dye Terminator ready reaction kit (Applied Biosystems, Foster City, CA). Extended products were purified by ethanol precipitation and analyzed on an ABI 3730 automated DNA Analyzer (Applied Biosystems, Foster City, CA).

Statistical analysis

All the statistical analyses were performed using SPSS statistical software (Version 19.0, SPSS Inc., Chicago, IL, USA). A χ2 test was carried out to test for differences between the case and control groups in terms of the anthropometric characteristics. Independent sample t-test was used to compare differences in the mean values of biochemical parameters for the PCOS subjects categorized according to body mass index (BMI). Significance level was set at 5% for all the tests.


 :: Results Top


A total of 549 women consisting of 250 PCOS cases (aged 14-40 years) and 299 controls (aged 14-47 years) were enrolled for the present study. The anthropometric/clinical characteristics of PCOS cases and controls are presented in [Table 2]. PCOS subjects had a significantly higher mean value for BMI and waist-hip ratio (WHR). The mean age of menarche, however, was significantly lower in the PCOS group compared to the controls. The proportion of obese women (BMI≥25) within the PCOS group was significantly higher than in the control group (55.1% vs. 15.1%, respectively; P<0.001). Comparison of the biochemical parameters between the lean and obese PCOS cohort revealed that although the mean levels of LH and FSH are not significantly different between the lean and obese PCOS cases (P>0.1), a higher LH: FSH ratio (characteristic feature of PCOS) is evident among the obese group. Moreover, obese PCOS cases had a significantly higher mean level of cholesterol and triglycerides than the lean PCOS cases [Table 3]. All the six exons of the follistatin gene were successfully amplified by PCR in the entire cohort. No mutations/ polymorphisms were detected in any of the exons.
Table 2: Anthropometric/clinical characteristics of PCOS subjects and controls

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Table 3: Hormonal profile of PCOS cases

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 :: Discussion Top


We screened the follistatin gene in the largest cohort of PCOS subjects studied so far in a case-control setup, the first study of its kind in India. Consistent with the earlier findings among Chinese PCOS women, [7] no mutations could be identified in the exonic regions of the FST gene among the Indian women. This overlapping nature of findings could well be due to the similarity in the ethnic backgrounds representing the Asian phenotype in general. The other three studies conducted on Caucasian cohorts [3],[5],[6] depicted a different perspective on the possible role of FST in PCOS. Though they could identify certain mutations/polymorphisms, their association pattern with the disease phenotype could not be established leading to an overall conclusion that the contribution of FST towards PCOS pathology is uncertain.

Follistatin is a single-chain glycoprotein that is expressed in many tissues including the ovary, adrenal cortex, pituitary and pancreas. This polypeptide specifically binds activin, neutralizing its biological activity. Activin causes ovarian follicular development, inhibits theca cell androgen production, increases pituitary FSH secretion and pancreatic insulin secretion. Since follistatin inhibits the activity of activin, its altered activity due to over-expression would therefore be expected to affect follicular development, ovarian androgen production, pituitary FSH secretion, and insulin release which turn out to be characteristic features of PCOS. [5],[6] Based on its functional relevance and evidence of strong linkage with PCOS, the follistatin gene emerged as the strongest candidate. [4]

Urbanek et al., [5] screened the gene for sequence variants and for assessing their associative pattern with PCOS among the Caucasian cohort. Extensive sequencing revealed variants at 17 sites, but none of them showed any association with PCOS except the one variant in Exon 6 that showed a slightly elevated transmission disequilibrium test TDT score with marginal statistical significance. Additionally, they compared mRNA levels of follistatin in cultured fibroblast cells from PCOS and control women, the results of which also did not yield any significant differences. Therefore, the etiological basis of this gene could not be established.

Subsequently, Liao et al., [7] screened 64 Chinese patients with PCOS for mutations in the entire coding region of the FST gene using PCR-based single strand conformation polymorphism (SSCP) and DNA sequencing and could not detect any variants. In another study, 34 PCOS patients from Spain were screened [6] and only a single nucleotide change of a G to A at cDNA position 951 in exon 5 was observed, resulting in a silent mutation in 2 of 34 patients and 1 of 15 controls, negating the role of follistatin in PCOS etiology. Thus, the abnormalities in the coding regions of the follistatin gene could not be related to PCOS in white women from Spain. On the other hand, in a relatively recent study, Jones et al., [3] performed a case-control analysis among western Australian women comprising 173 PCOS cases and 107 controls, taking into account seven known single nucleotide polymorphisms (SNPs) from the promoter region, intron 1, exon 4 and exon 5 of the follistatin gene. They found an association between the intronic SNP and two androgenic parameters of free androgen index (FAI) and sex hormone binding globulin (SHBG) levels; subjects carrying the mutant allele represented a relatively severe form of hyperandrogenemia. However, they did not find any difference in the allele frequencies of other polymorphic loci between cases and controls thereby suggesting that FST is not the main susceptibility locus in most of the patients. Nevertheless, it may provide a clue to a possible role of this gene in the etiology of the disease in a subset of PCOS subjects. The lack of consistency in the results of the above mentioned studies underscores the importance of [1] ethnic differences in the frequency of susceptible genetic variants resulting in different association patterns, [2] small sample sizes and [3] relatively modest contribution of FST towards the PCOS pathophysiology only in certain ethnic groups. Alternatively, it may also be representing linkage with another polymorphic locus in the chromosomal region of the FST gene. [3]

Overall, we conclude that the occurrence of the exonic variants of FST seems to be dependent on the ethnic background of the subjects under study and that the abnormalities in the coding region of this gene do not appear to play a key role in PCOS. Nonetheless, future studies are warranted to replicate these findings in relatively larger cohorts encompassing the ethnic heterogeneity from different global regions before reaching an unequivocal conclusion. Further, it is necessary to identify variants in the promoter region and other regulatory sites of the FST gene that would better explain the function of follistatin and its effect on the PCOS pathophysiology.


 :: Acknowledgment Top


The authors thank Director, Indian Statistical Institute for logistic support and the Director, Centre for Cellular and Molecular biology, Hyderabad, for providing us access to the DNA sequencing facility to run the plates processed at ISI.

 
 :: References Top

1.Homburg R. What is polycystic ovarian syndrome? A proposal for a consensus on the definition and diagnosis of polycystic ovarian syndrome. Hum Reprod 2002;17:2495-9.  Back to cited text no. 1
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2.Azziz R, Woods KS, Reyna R, Key TJ, Knochenhauer ES, Yildiz BO. The prevalence and features of the polycystic ovary syndrome in an unselected population. J Clin Endocrinol Metab 2004;89:2745-9.  Back to cited text no. 2
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3.Jones MR, Wilson SG, Mullin BH, Mead R, Watts GF, Stuckey BG. Polymorphism of the follistatin gene in polycystic ovary syndrome. Mol Hum Reprod 2007;13:237-41.  Back to cited text no. 3
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4.Urbanek M, Legro RS, Driscoll DA, Azziz R, Ehrmann DA, Norman RJ, et al. Thirty-seven candidate genes for polycystic ovary syndrome: Strongest evidence for linkage is with follistatin. Proc Natl Acad Sci U S A 1999;96:8573-8.  Back to cited text no. 4
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5.Urbanek M, Wu X, Vickery KR, Kao LC, Christenson LK, Schneyer A, et al. Allelic variants of the follistatin gene in polycystic ovary syndrome. J Clin Endocrinol Metab 2000;85:4455-61.  Back to cited text no. 5
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6.Calvo RM, Villuendas G, Sancho J, San Millán JL, Escobar-Morreale HF. Role of the follistatin gene in women with polycystic ovary syndrome. Fertil Steril 2001;75:1020-3.  Back to cited text no. 6
    
7.Liao WX, Roy AC, Ng SC. Preliminary investigation of follistatin gene mutations in women with polycystic ovary syndrome. Mol Hum Reprod 2000;6:587-90.  Back to cited text no. 7
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8.Aruna M, Nagaraja T, Andal S, Tarakeswari S, Sirisha PV, Reddy AG, et al. Role of progesterone receptor polymorphisms in the recurrent spontaneous abortions: Indian case. PLoS One 2010;5: E8712.  Back to cited text no. 8
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9.Dhandapany PS, Sadayappan S, Xue Y, Powell GT, Rani DS, Nallari P, et al. A common MYBPC3 (cardiac myosin binding protein C) variant associated with cardiomyopathies in South Asia. Nat Genet 2009;41:187-91.  Back to cited text no. 9
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10.Rotterdam ESHRE/ASRM-Sponsored PCOS Consensus Workshop Group. Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril 2004;81:19-25.  Back to cited text no. 10
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11.Ferriman D, Gallwey JD. Clinical assessment of body hair growth in women. J Clin Endocrinol Metab 1961;21:1440-7.  Back to cited text no. 11
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12.Mifsud A, Ramirez S, Yong EL. Androgen receptor gene CAG trinucleotide repeats in anovulatory infertility and polycystic ovaries. J Clin Endocrinol Metab 2000;85:3484-8.  Back to cited text no. 12
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13.Sambrook J, Fritschi EF, Maniatis T. Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory Press; 1989.  Back to cited text no. 13
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3]

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