Polycystic ovarian syndrome (PCOS) is a common endocrine disorder that is found in ≈4% of women of reproductive age1
and results in reduced fertility and a sevenfold increased risk for type 2 diabetes mellitus.2
The syndrome is characterized by hyperandrogenism and chronic anovulation. It is also associated with polycystic ovaries, hirsutism, obesity, and insulin resistance. The observation of familial aggregation of PCOS3
showed that it is consistent with a genetic basis for this disorder. The mode of inheritance is uncertain at this point, and the role of shared environmental factors such as diet and lifestyle in the presentation of the disease is unknown. Loci proposed and investigated as possible PCOS genes include CYP11A
, the insulin gene, and a region near the insulin receptor gene.6
The wide range of PCOS symptoms likely plays a significant role in the inability thus far to identify a specific gene mutation.
Single-nucleotide polymorphisms (SNPs) are DNA sequence variations that occur when a single nucleotide (A, T, C, or G) in the genome sequence is changed. Most SNPs, actually about two of every three SNPs, involve the replacement of cytosine (C) with thymine (T). SNPs occur every 100–300 bases along the human genome. SNPs are stable from an evolutionarily standpoint not changing much from generation to generation.
SNPs, which make up about 90% of all human genetic variation, occur every 100–300 bases along the 3-billion-base human genome. SNPs can occur in both coding (gene) and noncoding regions of the genome. Many SNPs have no effect on cell function, but scientists believe others could predispose people to disease or influence their response to a drug. SNP maps will help them to identify the multiple genes associated with such complex diseases as cancer, diabetes, vascular disease, and some forms of mental illness.
Several pathways have been implicated in the etiology of PCOS. These include the metabolic or regulatory pathways of steroid hormone synthesis,7
regulatory pathways of gonadotropin action,9
the insulin-signaling pathway,10
and pathways regulating body weight.13
Several genes from these pathways have been tested as candidate genes for PCOS.7
Although mutation analysis, linkage studies, and case-control association studies have been carried out with these candidate genes, evidence that any of them play a role in PCOS has not been replicated widely and is still inconclusive. These uncertainties are common in ‘complex’ genetic diseases, where identifying the contributing genes is made difficult by likely genetic heterogeneity, environmental contributions, and multiple etiologies. However, the mode of inheritance of PCOS has not been firmly established. Although some studies support a single dominant gene with high penetrance,20
others do not.23
A study was conducted by Urbanek et al24
on a carefully chosen collection of 37 candidate genes for linkage and association with PCOS or hyperandrogenemia in data from 150 families. The authors found that strongest evidence for linkage was with the follistatin gene.
Strong evidence for a link between the follistatin gene and PCOS has been recently found in a well-designed large-scale study. The pathway in which follistatin gene is involved is shown in as in KEGG database: Follistatin binds to activin and affects its functions, for example, stimulation of follicle-stimulating hormone (FSH) synthesis and secretion. Thus, it may play a role in the functional impairment of the FSH–granulosa cell axis in PCOS. Several genes involved in the biosynthesis of androgen, and action of insulin and gonadotrophin have been examined as candidate genes for PCOS. These genes include those for the cholesterol side-chain cleavage enzyme (CYP11A
), 17-hydroxylase/17,20-lyase (CYP17
), insulin, insulin receptor, and LH
. Two of them, CYP11A
and the insulin gene, and variable number of tandem repeats, have been proposed as predisposing genetic factors contributing to PCOS. However, neither of them have been widely accepted as a major cause for this syndrome. Recently, in a well-designed large-scale study, Urbanek et al24
tested for linkage and association between 37 candidate genes including those previously studied and PCOS. These genes were carefully selected from those involved in the action of androgen, gonadotrophin, and insulin, and also the regulation of obesity and energy. They found evidence for linkage only with the CYP11A
and follistatin genes. However, only the linkage with follistatin gene remained significant after correction for multiple testing. Nevertheless, this study apparently confirmed previous findings which demonstrated the linkage of CYP11A
gene with PCOS.
The pathway in which follistatin gene is involved is shown below as in KEG database.
Follistatin is a single-chain glycosylated polypeptide that can bind to activin with high affinity and neutralize its biological action of stimulating the secretion of FSH and increasing FSHß mRNA levels and may therefore, arrest folliculogenesis. Indeed, overexpression of follistatin in the transgenic mice resulted in the suppression of both serum concentrations of FSH and ovarian folliculogenesis, similar to the clinical features commonly found in PCOS patients. Follistatin is expressed in numerous tissues including the ovary, pituitary, adrenal cortex, and pancreas. The human cDNAs encoding follistatin have been cloned. There is a single follistatin gene that can generate two mature mRNA transcripts by alternative splicing, thus encoding proteins of 315 (FS-315) and 288 (FS-288) amino acid residues, respectively. The FS-288, a carboxy-truncated variant with increased biological potency, was found to bind strongly to heparin sulphate proteoglycans of the cell membrane, whereas FS-315 had little or no such binding affinity. Furthermore, in the anterior pituitary cells, FS-288 was more potent in suppressing FSH release. This cell-associated protein was also found to accelerate the uptake of activin into pituitary cells, leading to an increase in its degradation by lysosomal enzymes and thus playing a role in the activin clearance system.
In view of this, it was thought that it would be worthwhile to explore the role of follistatin gene and its linkage with PCOS. The objectives of the work are retrieval of follistatin gene and protein, analysis of follistatin gene and protein, and SNP studies on follistatin to study SNP association with PCOS. Hence, in this work, SNPs in the FST gene were studied to determine if genetic variation is associated with susceptibility to PCOS or key phenotypic features of PCOS patients.