Mol. Hum. Reprod. Advance Access originally published online on February 6, 2007
Molecular Human Reproduction 2007 13(4):237-241; doi:10.1093/molehr/gal120
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Polymorphism of the follistatin gene in polycystic ovary syndrome
1 Department of Endocrinology and Diabetes 2 Western Australian Institute of Medical Research 3 Keogh Institute for Medical Research, Sir Charles Gairdner Hospital, Perth, Australia 4 School of Medicine and Pharmacology, University of Western Australia, Perth, Australia 5 School of Biological Sciences, Murdoch University, Australia
6 To whom correspondence should be addressed at: Keogh Institute for Medical Research, Sir Charles Gairdner Hospital, Nedlands, WA 6009, Australia. Tel: +61 8 9346 2466; Fax: +61 8 9346 3221; E-mail: bstuckey{at}cyllene.uwa.edu.au
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Abstract |
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Follistatin has been reported as a candidate gene for polycystic ovary syndrome (PCOS) from linkage and association studies. Acting to regulate the development of ovarian follicles and as an antagonist to aromatase activity, alterations in follistatin function or expression may result in key features of PCOS such as reduced serum FSH, impaired ovarian follicle development and augmented ovarian androgen production. We investigated polymorphisms in the FST gene to determine if genetic variation is associated with susceptibility to PCOS or key phenotypic features of PCOS patients in a case–control association study. One hundred and seventy-three PCOS patients of Caucasian descent (mean age 30.0 ± 4.8 years), conforming to the NIH diagnostic criteria, were recruited from a clinical practice database and 107 normal ovulating women (mean age 38.8 ± 13.4 years) were recruited from the general community as control subjects. Morphometric data, biochemistry and genomic DNA were collected from study subjects and genotyping was performed on seven Single nucleotide polymorphisms (SNPs) in the FST gene region. Allele frequencies of the SNPs were rs1423560 G/C (0.99/0.01), rs3797297 C/A (0.80/0.20), rs11745088 C/G (0.98/0.02), rs3203788 A/T (0.98/0.02) and rs1062809 G/C (1.00/—), rs1127760 A/T (0.98/0.02) and rs1127761 A/T (0.98/0.02), and these were not significantly different between the PCOS and control groups (P < 0.05). Statistical analysis revealed significant associations between the SNP rs3797297 and sex hormone-binding globulin (P = 0.04) and free androgen index (FAI) (P < 0.01). We conclude that FST is not a susceptibility locus for PCOS; however, the SNP rs3797297 from FST gene was associated with androgenic markers for PCOS and may be of importance in the hyperandrogenaemia of the disease.
Key words: androgen/follistatin/FST/polycystic ovary syndrome/polymorphism
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Introduction |
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Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility in women of reproductive age (Urbanek et al., 2003) and may affect 6.5–8% of women in the developed world (Goodarzi and Azziz, 2006). The pathophysiology of PCOS appears to be multifactorial, with multiple gene and environmental factors contributing to disease susceptibility and expression (Balen, 2004). There is strong evidence for a genetic component in PCOS, as familial clustering of PCOS and its symptoms are seen frequently in the family members of PCOS patients. To date, no study has convincingly established a mode of inheritance for the disorder (Amato and Simpson, 2004). The majority of studies suggest a dominantly inherited trait of varied expression with low penetrance. The diversity of PCOS within families and between PCOS patients suggests that inheritance of the disorder could be both multifactorial and polygenic (Legro and Strauss, 2002).
Multiple genetic pathways have been implicated in the pathogenesis of PCOS including steroid hormone metabolism, gonadotropin action, obesity and energy regulation and insulin action (Dunaif et al., 1988; Acien et al., 1999; Moran et al., 2003; Moran and Norman 2004). In 39 affected sister pairs tested for evidence of linkage or association between PCOS and hyperandrogenaemia, evidence of linkage was reported in the follistatin gene region (Urbanek et al., 1999). Follistatin is a single-chain glycoprotein (Calvo et al., 2001) expressed in a number of tissues including ovary, adrenal cortex, pituitary and pancreas (Eldar-Geva et al., 2001) and its primary function is to fine-tune the activity of activin (Schneyer et al., 2004). Activin stimulates FSH production by the pituitary and augments its action on granulosa cells. Conversely, follistatin neutralizes activin and thereby antagonizes folliculogenesis and aromatization. (Phillips and Krester, 1998). Up-regulation of follistatin function or expression could result in some key features of PCOS such as reduced serum FSH, impaired ovarian follicle development and augmented ovarian androgen production. In animal models, overexpression of follistatin has been shown to result in a PCOS-like phenotype (Guo et al., 1998).
The role of follistatin in ovarian follicular development, secretion of FSH and production of androgens makes it a credible candidate for PCOS (Urbanek et al., 2000). The discovery that follistatin levels are significantly higher in PCOS patients, independent of obesity (Eldar-Geva et al., 2001), suggests that alteration in follistatin function may contribute to the PCOS phenotype. Previous investigations of polymorphisms in this region and their role in PCOS have focused on those in the coding regions of the gene (Calvo et al., 2001; Liao et al., 2000; Urbanek et al., 2000). Furthermore, the data available on allele frequencies of single nucleotide polymorphisms (SNPs) in the FST region and the linkage disequilibrium (LD) between these remain relatively sparse, making it difficult to select haplotype tag SNPs for studies of PCOS. On the basis of previously reported linkage and association between the FST gene region and knowledge of its ovarian function, we hypothesized that both coding and non-coding polymorphisms in the gene region may be associated with susceptibility to PCOS and key clinical and phenotypic features of PCOS. In this study, we report the analysis of seven SNPs in the FST gene region, in a well-characterized cohort of PCOS and healthy control women.
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Materials and methods |
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Study design
A cohort of 173 Caucasian women conforming to NIH diagnostic criteria for PCOS (Zawadski, 1992) was recruited retrospectively from an endocrinology clinical practice. Other causes of hyperandrogenism and anovulation were excluded. A control cohort of 107 healthy Caucasian women were recruited from the general population of Perth, Western Australia by advertisement. A questionnaire was administered to controls to establish menstrual cycle, weight, height, prevalence of PCOS related symptoms (hirsutism, acne, impaired glucose tolerance), ethnic background, fertility history, family incidence of PCOS and PCOS-related symptoms and activity levels. These women had a history of regular menstrual cycles and no history or evidence of PCOS related symptoms such as oligoanovulation and hirsutism. Western Australia has a cosmopolitan population, the majority of whom are of British or Western European descent. Approval for the study was obtained from the Human Ethics Committee of Sir Charles Gairdner Hospital and written informed consent was obtained from each study participant.
Clinical and biochemical measurements
General medical, ethnicity and reproductive history data were collected from PCOS patients and control participants via questionnaire. PCOS patients underwent a thorough clinical and biochemical examination. A family history of PCOS was an exclusion criterion for control subjects. Weight and height were measured and body mass index (BMI) was derived by the computation: weight (kg)/height (m2).
Total cholesterol, triglycerides, high-density lipoprotein (HDL)-cholesterol, glucose and insulin were measured after an overnight fast of 10 h with water allowed. Oral glucose tolerance test was performed using standard 75 g glucose load and blood glucose and insulin collected at 0, 60 and 120 min. Total cholesterol, triglycerides and HDL were measured by LX20 Timed-end-point method (Beckman-Coulter, Fullerton, USA). Low-density lipoprotein (LDL)-cholesterol was calculated by the Friedewald formula: LDL-C = total cholesterol – [(0.46 x TG) + HDL-C]. Glucose was measured by LX20 Oxygen rate method employing a Beckman oxygen electrode (Beckman-Coulter) and insulin by Tosah AIA 600 two-site immunoenzymometric assay (Tosoh Corporation, Tokyo, Japan). LH was measured by Immulite 2000 immunometric assay (DPC, Los Angeles, CA, USA), FSH, estradiol and dehydroepiandrosterone sulphate (DHEA-S) by Immulite 2000 solid-phase, two-site chemiluminescent enzyme immunometric assay (DPC, Los Angeles, USA). Testosterone was measured by solid-phase radioimmunoassay (DPC) and sex hormone-binding globulin (SHBG) by Immulite 2000 immunometric assay (DPC). Androstenedione was measured by radioimmunoassay (Dia Sorin Inc., Stillwater, USA) and 17-hydroxyprogesterone by Coat-a-Count solid phase, radioimmunoassay (DPC).
The homeostasis model assessment (HOMA) for insulin resistance was calculated using the formula: (glucose x insulin)/22.5. Free androgen index was calculated as: (testosterone/SHBG) x 100.
SNP selection and genotyping
Genomic DNA was extracted and purified from EDTA whole blood obtained from each subject. SNPs were selected using frequency and validation data available in public databases such as dbSNP with the aim of providing substantial coverage of the gene region by exploiting LD (i.e. haplotype tagging). Six SNPs were selected for full analysis based on the success of amplification in multiplex reactions and position within the gene region. SNP from promoter and coding regions were favoured for genotyping.
The FST gene maps to chromosome 5q11.2 and spans 5329 bases from 52,812,352 to 52,817,659 (NCBI Genome Build 36.1). Genotyping was completed on 280 subjects for six SNPs in the FST gene: rs1423560, rs3797297, rs11745088, rs3203788, rs1127760 and rs1127761 (Figure 1). The SNP rs1062809 was also screened, but was found to be non-polymorphic in our series and was dropped from further consideration. Analysis of SNPs was by single nucleotide extension reaction using MALDI-ToF mass spectrometry on a Voyager DE Pro 6066 (Applied Biosystems, Foster City, USA) with reactions performed as previously described (Wise et al., 2003). Large-scale typing was preceded by a screening assay on a panel of 92 DNA samples to validate the allele frequencies of the SNPs selected for study. Random duplicate genotyping was routinely undertaken throughout the study and indicated a genotyping error rate of < 1.5%.
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Statistics
Statistical analysis was performed using Statistica for Windows Version 5.1 (Statsoft Inc., Tulsa, USA). A chib square test was used to confirm that the genotype data did not deviate from Hardy–Weinberg equilibrium and to test for differences in allele frequency between PCOS and control groups. Analysis of variance was used to detect significant (P < 0.05) difference in biochemical parameters for subjects grouped by genotype, and post hoc analysis of means utilized Scheffe's test. Kruskal–Wallis test was used in cases of unequal variance. Correction for multiple testing using a permutation approach simulated 10 000 random re-assignments of phenotype data with genotypes held constant. Forward stepwise multiple linear regression was used to further define the possible association between independent variables and PCOS (with FAI used as a surrogate end-point). In this model, FAI was regressed against BMI, HOMA and genotype. COCAPHASE and QTPHASE programs in UNPHASED (Dudbridge, 2003) were used to perform haplotype analysis and JLIN (Carter et al., 2006) was used to plot LD.
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Results |
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Clinical and biochemical features of the cohort
The anthropometric and clinical features of the PCOS and control groups are shown in Table I. The mean age of the control group was slightly older than that of the PCOS women, but this is not relevant when comparing genotype frequencies as done in this study. PCOS subjects had a higher BMI than the control women. Age at menarche was younger in the PCOS group compared with the control group, but this did not achieve statistical significance. The biochemical characteristics of the PCOS group are shown in Table II.
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Locus specific analyses: effects of individual SNP genotypes on phenotype data
The position, function and observed allele distribution for each SNP is given in Table III. Genotype data was tested for deviation from Hardy–Weinberg equilibrium both separately, as case and control groups, and combined, and all SNPs were in Hardy–Weinberg equilibrium. There were no differences in allele frequencies between case and control groups for the SNPs we studied in the FST gene.
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However, ANOVA revealed significant associations between SNP in FST and a number of phenotypic markers of PCOS. rs3797297 was significantly associated with FAI (P < 0.01; Table IV) and was also significantly associated with SHBG level (P = 0.04). Correction for multiple testing utilizing 10 000 re-assignments using a MANOVA approach produced a P-value of 0.04 for both FAI and SHBG. The SNP rs11745088 showed evidence of association with DHEA-S, but there was a low number of subjects with the polymorphic allele (homozygous GG, 5.1 ± 2.56 µM l–1, n = 77, versus heterozygote GC, 9.1 ± 1.8 µM l–1, n = 2; P = 0.033) and there were no homozygous CC individuals seen in our cohort, so this result must be viewed with caution. There were no other associations between any of the SNPs and PCOS phenotypes we studied. In multiple linear regression, HOMA and genotype for rs3797297 were predictors of FAI (P = 0.011 and P = 0.031, respectively) (Table V).
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Haplotype analysis
The LD between the SNPs we studied is shown in Figure 2, and it suggests that haplotype analysis might be informative. Analysis showed that four common haplotypes comprising rs1423560, rs3797297, rs11745088, rs3203788, rs1062809, rs1127760 and rs1127761 account for 94.5% of the population (G-C-C-A-G-A-A, 74.5%; G-A-C-A-G-A-A, 17%; G-C-G-A-G-A-A, 2%; G-C-G-A- G-A-T, 2%). In a test of association between all four haplotypes and phenotypic variables of PCOS, we found no association with haplotype (P > 0.05), suggesting that the individual SNPs or other polymorphisms in strong LD with rs3797297 are responsible for the observed effects and these are not enhanced by including haplotypes across the gene, in the analysis.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Our data suggest that polymorphism in the follistatin gene is associated with key androgenic phenotypes of PCOS. In this study, the SNP rs3797297, located in intron 1 of the FST gene, was significantly associated with both FAI and SHBG. In both instances, post hoc analysis revealed that the main effect was due to subjects who were homozygous for rare A-allele of the polymorphism. The data for rs3797297 do not appear to be consistent with a co-dominant mode of expression at this locus. In subjects with two copies of the polymorphic allele at this locus, the FAI is well above the reference range, representing more severe hyperandrogenaemia than in those women with a copy of the ancestral allele. Thus, a recessive effect appears more likely. In the case of the SNP rs11745088, which was significantly associated with DHEA-S level, only two heterozygotes were identified for whom DHEA-S was available, with both these individuals having elevated DHEA-S. Although our study is larger than many molecular genetics studies of PCOS published to date, the power of the study remains relatively modest. Calculations show that this sample has a power of 0.85–0.918 with an
of 0.05 to detect changes in SHBG and 0.761–0.92 to detect changes in FAI for an SNP such as rs3797297 depending on the model used. Therefore, replication of these preliminary data in a larger well-characterized cohort will be needed to provide confidence in this result. To further study a potential role for the FST gene in PCOS, we used multiple linear regression as a means of clarifying the relative contribution of other variables to variation in FAI in our PCOS cohort. In addition to genotype, we included other primary markers of PCOS, such as HOMA and BMI, in the regression model. This analysis indicated that both HOMA and genotype for rs3797297 are significant predictors of FAI potentially explaining 23 and 6% of the variance in FAI, respectively.
Follistatin is a single-chain glycoprotein that primarily acts to regulate the activity of activin, which is responsible for ovarian follicular development, inhibition of theca cell androgen production and increases in both pituitary FSH secretion and pancreatic insulin secretion (Calvo et al., 2001). Primarily synthesized in the granulosa cells of the ovarian antral follicle, follistatin mRNA increases within the dominant follicle during development and declines during the atretic process (Lin et al., 2003). Follistatin acts to suppress aromatase activity in the granulosa cell and also LH-stimulated progesterone release from thecal cells (Phillips and Krester, 1998). The overexpression of follistatin in mice has been shown to result in arrested ovarian follicular development and reduced levels of FSH, both key phenotypes of PCOS (Urbanek et al., 2000).
The initial identification of FST as a candidate locus for the hyperandrogenaemia associated with PCOS resulted from a study of both linkage and association of 37 candidate genes by Urbanek et al. (1999). The reported association in this study was with the marker D5S623, located 
500 kb from the FST gene. Additional studies of the FST locus have been focused on either mutations or SNP in the coding region of the gene and have not been conducted in a case–control study (Calvo et al., 2001, Urbanek et al., 2000). Urbanek et al. (2000), defined 16 variants in the FST gene of potential relevance to PCOS, but considered these too rare to make a major contribution to susceptibility. The only common variant they studied was a single base pair change in the 3' untranslated region of the gene. Many SNPs have been discovered in the genome since the publication of these previous reports, including the region of the FST gene, and our data suggest that further study is warranted. Ours is the first study to provide a more extensive analysis of polymorphism in both coding and non-coding regions of the FST gene. The potential role for polymorphism from non-coding and regulatory regions of the follistatin gene region was acknowledged by Urbanek et al. (2000). The original observation of linkage in the FST region in affected sib pairs suggested that non-coding polymorphisms from FST and SNPs in sequence surrounding the gene may have a role in susceptibility to PCOS or the phenotypic markers associated with the disorder (Urbanek et al., 2000), possibly by altering transcription factor or micro-RNA (miRNA) binding sites. We could not find any evidence in the literature for a specific role of rs3797297 in regulating transcription or altering mRNA stability, but the field of miRNA is relatively new and further studies will be needed to determine whether this SNP or a polymorphism in LD with it is responsible for the observed association.
In this study, we found an association between two key PCOS phenotypes and polymorphism in the FST gene, but did not find a difference in allele frequencies between cases and controls. This is in agreement with previous reports that FST is not the main susceptibility locus in the majority of patients. However, our data show that polymorphism in the FST gene may yet play a role in the disease, possibly through gene x gene interactions. The significant associations identified between rs3797297 and phenotypic markers of PCOS may provide a clue to a possible role of this gene in the aetiology of the disease in a subset of PCOS subjects or alternatively may be representing linkage with another polymorphic locus in the chromosomal region of the FST gene. These are preliminary data and these associations need to be further defined by investigation of other populations of similar and different ethnicity before further studies can be justified to provide a deeper insight into the interaction between this gene region and PCOS.
In conclusion, polymorphisms in the FST gene are not associated with susceptibility to PCOS, but the SNP rs3797297 was associated with key androgenic phenotypes of PCOS in our Western Australian cohort and may be of importance in the pathology of the disease.
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Acknowledgements |
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The study was supported by a research grant from the Ada Bartholemew Medical Research Trust of the University of Western Australia, Western Australia. We thank Lisa Italiano, Helena Ching, Fiona Robinson, Joanna Wagner and Dr Andrea Cussons for assistance with patient recruitment and phenotype data collection.
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References |
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Submitted on November 28, 2006; accepted on January 5, 2007.
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