The Molecular-Genetic Basis of Functional Hyperandrogenism and the Polycystic Ovary Syndrome
http://www.100md.com
内分泌进展 2005年第2期
Departments of Endocrinology (H.F.E.-M., M.L.-R.) and Molecular Genetics (J.L.S.M.), Hospital Ramón y Cajal, Madrid, Spain E-28034
Abstract
The genetic mechanisms underlying functional hyperandrogenism and the polycystic ovary syndrome (PCOS) remain largely unknown. Given the large number of genetic variants found in association with these disorders, the emerging picture is that of a complex multigenic trait in which environmental influences play an important role in the expression of the hyperandrogenic phenotype.
Among others, genomic variants in genes related to the regulation of androgen biosynthesis and function, insulin resistance, and the metabolic syndrome, and proinflammatory genotypes may be involved in the genetic predisposition to functional hyperandrogenism and PCOS.
The elucidation of the molecular genetic basis of these disorders has been burdened by the heterogeneity in the diagnostic criteria used to define PCOS, the limited sample size of the studies conducted to date, and the lack of precision in the identification of ethnic and environmental factors that trigger the development of hyperandrogenic disorders. Progress in this area requires adequately sized multicenter collaborative studies after standardization of the diagnostic criteria used to classify hyperandrogenic patients, in whom modifying environmental factors such as ethnicity, diet, and lifestyle are identified with precision.
In addition to classic molecular genetic techniques such as linkage analysis in the form of a whole-genome scan and large case-control studies, promising genomic and proteomic approaches will be paramount to our understanding of the pathogenesis of functional hyperandrogenism and PCOS, allowing a more precise prevention, diagnosis, and treatment of these prevalent disorders.
I. Introduction
II. Evidence Suggesting a Genetic Origin for Functional Hyperandrogenism and PCOS
A. Familial aggregation
B. Male phenotype
C. Twin studies
D. Environmental and other confounding factors
III. Classic Techniques Used in Molecular Genetic Studies
A. Linkage analysis
B. Case-control studies
IV. Studies in Pediatric and Adolescent Hyperandrogenism
V. Studies in Hyperandrogenic Adults
A. Genes involved in androgen biosynthesis, transport, and action, and their regulation
B. Genes involved in insulin resistance and associated disorders
C. Proinflammatory genotypes
D. Other candidate genes
VI. Hyperandrogenism, PCOS, and Survival Advantage
VII. Explanations for the Lack of Reproducible Association of Hyperandrogenism and PCOS with Molecular Genetic Abnormalities and Genomic Variants
A. Ascertainment issues
B. Involvement of environmental factors
C. Possible polygenic etiology for functional hyperandrogenism and PCOS
D. Limitations of the genetic techniques used to date
VIII. Future Perspective: Functional Hyperandrogenism and PCOS in the Age of "Omics"
IX. Summary
I. Introduction
POLYCYSTIC OVARY SYNDROME (PCOS) is one of the most common endocrinopathies in women of childbearing age (1). PCOS is characterized by increased ovarian and adrenal androgen secretion (2); hyperandrogenic symptoms such as hirsutism, acne, and/or alopecia; menstrual irregularity; and, in a significant proportion of patients, insulin resistance (3).
The presence of male sexual secondary characteristics in women has been recognized from ancient times, but it was not until 1921 when Achard and Thyers (4) reported the association of hyperandrogenic symptoms with abnormalities in glucose metabolism, highlighting the presence of polycystic ovaries in some of their patients. However, only after the description of seven cases of amenorrhea and bilateral polycystic ovaries by Stein and Leventhal in 1935 (5) was PCOS considered a separate entity that interested clinicians and researchers worldwide.
Although for many years the interest in PCOS has been focused on the cutaneous and reproductive manifestations of this disorder, the recent evidence suggests that metabolic and cardiovascular risk factors cluster in these patients (6, 7, 8). This evidence has renewed research efforts on hyperandrogenism and PCOS, including those directed toward the identification of the genetic and environmental factors involved in the pathogenesis of these prevalent conditions.
At present, there is no consensus on the criteria for the diagnosis of PCOS (Table 1). Most clinicians and researchers from the United States and from southern Europe use the criteria derived from the conference held at the National Institute of Child Health and Human Development (NICHD) in 1990: clinical and/or biochemical hyperandrogenism, menstrual dysfunction, and exclusion of specific etiologies (9). According to these criteria, the presence of polycystic ovaries on ultrasound examination is not needed for the diagnosis. On the contrary, most specialists from the other European countries, Asia, and Oceania rely mostly on the presence of polycystic ovaries on ultrasound examination for this diagnosis, whereas menstrual dysfunction is not required (10). A recent consensus workshop held in The Netherlands in 2003 under the auspices of the European Society for Human Reproduction and Embryology and the American Society for Reproductive Medicine proposed a revision of the criteria for the diagnosis of PCOS, of which two of the following three would be needed: 1) oligo- and/or anovulation; 2) clinical and/or biochemical signs of hyperandrogenism; and 3) polycystic ovaries, together with the exclusion of other etiologies such as congenital adrenal hyperplasia, androgen-secreting tumors, or Cushing’s syndrome (11, 12).
However, the term PCOS as used in the literature is not specific and has been applied to a wide range of hyperandrogenic women, including women with or without menstrual dysfunction or polycystic ovaries. For that reason, in the present review, we will use the more general term functional hyperandrogenism to define these patients presenting with androgen excess, including those diagnosed with PCOS according to any of the current definitions of the syndrome, in whom a specific etiology such as congenital adrenal hyperplasia or androgen-secreting tumors cannot be identified, and, whenever possible, we will describe the particular characteristics of the patients included in the different studies. Of note, we will not include idiopathic hirsutism in this definition, because this infrequent disorder, in which hyperandrogenemia, menstrual dysfunction, and ultrasonographic polycystic ovaries are not present, appears to be a separate entity in which increased skin activity of 5-reductase (resulting in increased conversion of testosterone into the more potent androgen dihydrotestosterone), androgen receptor (AR) polymorphisms, and altered local androgen metabolism contribute to the development of hirsutism (13).
Functional hyperandrogenism and PCOS cluster in first-degree relatives of patients (14) and are inherited together with insulin resistance and metabolic disorders (15, 16). During the past decades, the inheritance of these disorders has been the subject of intense research (17, 18, 19), but many questions remain unanswered.
The pattern of inheritance is still unknown. Initially, an autosomal dominant model, including premature balding as the male phenotype, was proposed (20, 21), but later studies did not confirm this hypothesis. The heterogeneity of the populations, the large number of candidate genes studied to date (22), and the difficulty inherent to identifying the molecular genetic mechanism leading to a complex metabolic disorder such as PCOS, in which environmental factors play a major role (23, 24), provide an explanation for the fact that the molecular genetic basis of functional hyperandrogenism and PCOS remains largely unknown despite significant efforts.
The purpose of this review is to provide a systematic evaluation of the studies conducted to date in functional hyperandrogenism and PCOS and to suggest priorities and new strategies that may contribute to understanding the pathogenesis of these disorders.
II. Evidence Suggesting a Genetic Origin for Functional Hyperandrogenism and PCOS
A. Familial aggregation (Table 2)
The familial aggregation of PCOS, hyperandrogenemia, and associated metabolic abnormalities suggests a genetic origin for functional hyperandrogenism and PCOS. Back in 1968, Cooper et al. (25) studied the families of 18 Caucasian women with polycystic ovaries and clinical and biochemical traits associated with PCOS, and the families of 18 paired control women. The incidence of oligomenorrhea and polycystic ovaries was increased in first-degree relatives of PCOS patients compared with controls, and males in these families had increased hairiness according to a questionnaire, suggesting an autosomal dominant pattern of inheritance (25). Givens and colleagues (26, 27, 28) published a series of family-based studies in patients presenting with hirsutism, oligomenorrhea, and increased ovarian size, and they found familial aggregation of hyperandrogenic symptoms (hirsutism and oligomenorrhea) and of metabolic disorders (diabetes mellitus, dyslipidemia, arterial hypertension, and atherosclerosis). These authors suggested both a maternal and paternal pattern of inheritance, in which the latter showed higher penetrance and expression (26, 27, 28). Of note, in some of these male subjects, oligospermia and increased LH secretion were found pointing to an X-linked pattern of inheritance.
Ferriman and Purdie (29) studied first-degree relatives of hirsute women presenting with or without enlarged ovaries in gynecography. Compared with a control group, patients had an increased prevalence of oligomenorrhea and infertility, and the prevalence of hirsutism was increased in their female first-degree relatives. Hague et al. (30) studied the families of 61 patients with ultrasonographic polycystic ovaries and hyperandrogenic symptoms and found that 67% of the mothers and 87% of the sisters of probands were affected. Lunde et al. (31) studied the families of 132 Norwegian women with polycystic ovaries previously treated by ovarian wedge resection, who also had two or more of the following symptoms: menstrual irregularity, hirsutism, infertility, and/or obesity. A control group of 71 women and their families was used for comparison (31). Clinical manifestations of hyperandrogenism were found in 31.4% of the female relatives of the patients compared with only 3.2% of the female relatives of the controls (31). Among male relatives, premature balding and increased hairiness were found in 19.7% of the relatives of the patients, but only in 6.5% of the relatives of the controls (31). Azziz and colleagues (14, 32), using NICHD criteria for the diagnosis of PCOS, found that PCOS was present in 35% of the mothers and 40% of the sisters of PCOS patients.
In addition to hyperandrogenism, insulin resistance clusters in the families of hyperandrogenic women. Legro et al. (33), also using also NICHD criteria for the definition of PCOS, found that 22% of the sisters of patients actually had PCOS, whereas a further 24% of the sisters of these patients presented with hyperandrogenemia and regular menstrual cycles. This bimodal pattern suggested a monogenic defect for hyperandrogenism (33). Additional studies from Legro et al. (34) recently demonstrated that in these families, insulin resistance is associated with hyperandrogenemia rather than with menstrual dysfunction. This suggests that insulin resistance and hyperandrogenemia share the same pathogenic mechanisms, whereas the presence of menstrual dysfunction needed for the diagnosis of PCOS may be only a matter of degree. Therefore, of PCOS sisters, those with PCOS and those presenting with hyperandrogenemia and regular menstrual cycles may actually have functional hyperandrogenism.
The finding of an increased prevalence of insulin resistance in families of PCOS patients has been replicated in the Turkish population. Yildiz et al. (35) recently studied 102 relatives of 52 PCOS patients defined by NICHD criteria and found that, compared with different population-based control groups matched for sex, age, and pre- or postmenopausal status with the relatives of PCOS patients, insulin resistance and disorders of carbohydrate metabolism were more frequent in the mothers, sisters, and brothers of PCOS patients, and that the mothers and sisters of PCOS women had increased serum androgen levels compared with the controls.
B. Male phenotype
The lack of a clearly defined male phenotype in families of PCOS patients has burdened the progress in the search for the genetic origin of functional hyperandrogenism and PCOS. Although initial studies suggested that male pattern premature balding was the male equivalent for PCOS (20, 29, 31, 36, 37), these findings have not been universally confirmed (38). More recently, increased serum dehydroepiandrosterone sulfate concentrations in the brothers of PCOS patients described above (39) and insulin resistance in the fathers and brothers of PCOS women (35) have been proposed as the male phenotype in PCOS families. However, there was a considerable overlap between male PCOS relatives and controls in serum dehydroepiandrosterone sulfate levels and indexes of insulin resistance, and additional studies are needed to establish the actual usefulness of these abnormalities as markers of the syndrome in male relatives of PCOS patients.
C. Twin studies
There are very few data regarding PCOS in twins. Jahanfar et al. (40) studied 34 (19 monozygotic and 15 dizygotic) twin pairs, in which PCOS diagnosis was based on ultrasonographic and biochemical findings. Although 11 pairs were discordant for the presence of polycystic ovaries, model-fitting analysis suggested that fasting insulin level, serum androstanediol glucuronide, and body mass index were significantly influenced by genetic factors (40). A later report of this study suggested a genetic origin for an unfavorable lipid profile, especially for increased circulating concentrations of lipoprotein (a), but only in twin pairs concordant for polycystic ovaries (41). These data point to a genetic component in the metabolic abnormalities associated with PCOS but suggest a polygenic etiology in which environmental influences play a significant role.
D. Environmental and other confounding factors
Familial aggregation of hyperandrogenism and PCOS strongly suggest a genetic origin for these disorders, but another possibility is that clustering of PCOS within families results from nongenetic inheritance related to certain environmental factors that are present in the affected families, and not in the families of unaffected women.
As exemplified in Fig. 1, insults during pregnancy may induce intrauterine growth retardation, which has been proposed to induce a thrifty phenotype in small for gestational age babies. These babies are predisposed to suffer from insulin resistance, which may result in hypertension, glucose intolerance, adrenal axis hyperactivity with relative cortisol excess, functional hyperandrogenism, and PCOS (42) later in life, especially when these individuals are exposed to environmental factors such as a sedentary lifestyle and a diet rich in saturated fat. These environmental factors may cluster in certain families because exercising and dieting are heavily influenced by parental habits.
The metabolic abnormalities associated with the thrifty phenotype can induce additional insult to the pregnancies of small for gestational age women, and the defect might be transmitted to another generation without the participation of any genetic abnormality. Therefore, the presence of more than one small for gestational age baby in a family may result from exposure of the mothers and their fetuses to the same unfavorable environmental conditions, rather than being related to genetic factors.
Yet it is important to highlight, as shown in Fig. 1, that if small for gestational age babies have healthy habits, insulin resistance and its consequences might be ameliorated, and, at least in theory, their fetuses will not be exposed to an unfavorable metabolic milieu during pregnancy, preventing nongenetic inheritance of these conditions. Nevertheless, intrauterine growth retardation may also be influenced by genetic variants, and the most probable scenario is that of an interaction between predisposing genetic abnormalities with unfavorable environmental conditions. Of note, even the influence of intrauterine growth restriction on the development of PCOS is debatable (43, 44, 45), considering that the presence of polycystic ovaries also has been related to above-average birth weight in babies born from obese, androgenized mothers (43, 44, 45).
There are very few data on the influence of environmental factors on the development of hyperandrogenism and PCOS, other than the triggering role for obesity on the development of these disorders (46). Although the prevalence of PCOS is similar in different countries, ethnic factors influence the clinical manifestations of the syndrome. In Caucasian premenopausal women, the reported prevalences of PCOS defined by NICHD criteria ranged from 4.7% in Alabama (47) to 6.5% in Spain (48) and 6.8% in the Greek island of Lesbos (49). Moreover, the prevalence in African-American women from Alabama (3.4%) was comparable with that in Caucasian women (47).
However, ethnicity influences the manifestations of PCOS. Carmina et al. (50) compared the clinical histories, physical examinations, ovarian morphology by ultrasound, insulin sensitivity, serum gonadotropin, and steroid profiles in 25 Japanese, 25 Italian, and 25 Hispanic-American women with PCOS. The three groups were homogeneous in terms of serum testosterone and adrenal androgen concentrations, insulin resistance, and polycystic ovaries, but Japanese women were less obese and did not present with hirsutism (50).
Dunaif et al. (51) found that insulin resistance is higher in Hispanic-Caribbean women with PCOS defined by NICHD criteria compared with Caucasian patients and with control women paired for age, weight, body composition, and ethnicity. Williamson et al. (52) recently reported a higher incidence of obesity, dyslipidemia, and infertility, as well as a higher degree of insulin resistance in Maori and Pacific Islander PCOS patients, defined by ultrasonography and clinical symptoms, compared with European patients. In the United Kingdom, the prevalence of polycystic ovaries and type 2 diabetes is increased in Indian subcontinent Asian women (53). Compared with Caucasian women, PCOS patients of southeastern Asian origin presented with oligomenorrhea and were diagnosed at younger ages; hirsutism, acne, acanthosis nigricans, and subfertility were more prevalent, and patients were more insulin resistant (54).
These ethnic differences in the clinical presentation of PCOS may be related to environmental factors such as diet, exercise, and lifestyle. Of note, even the content of polyunsaturated or monounsaturated fatty acids in the diet may influence the metabolic manifestations of the syndrome (55). Thus, because of differences in the environmental factors triggering the development of PCOS, the genes contributing to PCOS patients may also be different, depending on the population studied. These considerations may also apply to the discrepant results of previous studies regarding the association of genomic variants with PCOS.
III. Classic Techniques Used in Molecular Genetic Studies
Before reviewing the molecular genetic studies conducted to date in functional hyperandrogenism and PCOS, we will provide a brief description of the techniques usually used for molecular genetic studies, focusing on the particular advantages and disadvantages of each technique.
A. Linkage analysis
Linkage analysis is a family-based molecular genetic technique that has permitted the identification of the genetic abnormalities leading to many monogenic Mendelian disorders (56). However, most complex metabolic disorders result from the interaction between multiple genes and environmental factors, making their identification much more difficult than in disorders with a Mendelian pattern of inheritance (57, 58, 59, 60, 61, 62, 63).
In linkage analysis, markers frequently located at intergenic regions of DNA are studied in multiple members of affected families. The cosegregation of alleles with a disease phenotype is studied, and the linkage of the disease with any marker focuses the search for genomic abnormalities to the DNA regions close to the polymorphic marker segregating with the disorder.
Linkage analysis is based on the meiotic recombination that occurs during ovogenesis and spermatogenesis. The closer two genomic markers are, the more seldom these markers are separated during meiotic recombination. The probability of recombination between two markers is an estimation of the distance between them, and is expressed in centimorgans (cM). Two markers are defined to be 1 cM apart if they become separated by recombination in 1% of meioses. One centimorgan is equivalent on average to 106 bp of physical distance. The markers usually studied in linkage studies include microsatellites and single nucleotide polymorphisms (SNPs).
Classical or parametric linkage analysis requires the specification of a model for the disease or trait a priori, in terms of allelic frequencies, penetrance, and pattern of inheritance, and the result is expressed by the LOD score (64). The LOD score is the base 10 logarithm of the odds that two markers (one being the disease-causing or disease-associated genomic variant) are truly linked, divided by the odds that the observed set of data may result from chance if the markers are unlinked. Usually, significant positive proof of genetic linkage requires a LOD score of 3.0 or greater, corresponding to odds favoring linkage of at least 1000:1 at a given specified recombination frequency. Because the pattern of inheritance of functional hyperandrogenism and PCOS is complex and uncertain, nonparametric or model-free linkage analysis is required to ensure the power to detect linkage (22). In nonparametric linkage analysis, multiple DNA markers are obtained from siblings and, if possible, from their parents, and allele sharing between relatives is investigated (64). Allele sharing is defined as identity-by-state (having the same DNA sequence) or as identity-by-descent (when two alleles come from the same ancestral allele) (64). Nonparametric linkage scores use a similarity statistic defined by the average of the possibilities that relatives are identity-by-state (64). However, the number of meioses assessed, disease penetrance, knowledge of the marker coupling phase, and other relevant variables must be considered before reaching a clinically significant conclusion (65).
There are several factors that may limit the usefulness of linkage studies including, among others, the uncertainty about the pattern of inheritance, a variable penetrance of the genetic defect, the delay in the debut of the disease, possible errors in clinical diagnosis, gene to gene interactions, interaction with environmental factors, and the limited resolution of these techniques. The sample size needed to detect a particular effect varies inversely with the square of the effect (62), and therefore genes that contribute modestly to the phenotype may be missed because of the extremely large sample size needed to demonstrate the association. This is especially important given that PCOS affects women in reproductive age and is associated with reduced fertility, limiting the pedigrees and the generations studied to two (mothers and daughters) in most of the linkage analyses performed to date, resulting in small sample sizes within families (66, 67).
The resolution of linkage analysis may be improved using tests such as affected sibling-pair identity by descent analysis or the transmission disequilibrium test (68), allowing the detection of genes of modest effects that might be otherwise missed by linkage analysis. The transmission disequilibrium test involves genotyping the parents of the proband and the proband, and it determines whether or not the parent heterozygous for the putative allele transmits it more often to their affected children than other allele(s). These tests are frequently used to confirm the linkage already established but may also be useful when used without previous evidence for linkage (69).
Linkage with a polymorphic marker only serves to focus the search of the genomic abnormality associated with the disorder to an area of approximately 1–2 cM, and locating the precise genetic defect may involve studying as many as 30–100 genes in the linked area. After obtaining linkage with a genomic region, the search of the particular genomic abnormality involved can be accomplished by fine-mapping of that region using additional markers, and by a candidate gene approach searching for variants in biologically appealing genes located there. This approach has been facilitated by the growing number of genes identified all across the genome, but frequently the demonstration that a gene has a causative role in a disease requires direct sequencing of the whole gene in affected and nonaffected individuals (70).
B. Case-control studies
These studies look for specific genetic markers or alleles that are more frequent in affected individuals (cases) compared with unaffected subjects from the same population (controls). These studies are focused on association of a genetic variant with a disease in a population and not on the mode of inheritance of a trait (71, 72). Association studies are preferable for finding "susceptibility" loci, low-risk alleles that are often found in relatively high frequencies in the general population, and do not result in robust signals in family-based studies (73). A positive association indicates an increased risk for the disease in subjects carrying the at-risk allele, which may have a causative role in the disorder or be in linkage disequilibrium with the gene actually causing the disease. Population stratification is a major problem in case-control studies, because the higher prevalence of particular alleles in different ethnic groups might bias the results and lead to false-positive associations. Moreover, case-control studies are especially prone to type II errors, that is, ruling out an association that is actually present in the population because of the small sample sizes often used in these studies.
In summary, linkage studies and case-control studies are both valid approaches for the study of the genetics of functional hyperandrogenism and PCOS, provided that the sample sizes of these studies are sufficiently large, the diagnostic criteria used to define affected and nonaffected subjects are clearly defined, and both ethnicity and differences in environmental factors are carefully considered.
IV. Studies in Pediatric and Adolescent Hyperandrogenism
Functional hyperandrogenism and hyperinsulinism may be detected early in life in affected women, even before pubertal development. As stated above, the presence of low birth weight in small for gestational age newborns, resulting from an unfavorable environment during intrauterine life and/or to defective maternal nutrition, has been related to the development of insulin resistance (74), disorders of glucose tolerance (75), hypertension, and cardiovascular disease (76, 77, 78, 79) later in life.
The association of low birth weight with the development of hyperinsulinism and type 2 diabetes mellitus has been confirmed in different populations (80, 81), and these children present with an accelerated growth and increased body mass index during adolescence.
Although environmental factors may be an important contributor to the restriction of fetal growth in small for gestational age newborns, genetic factors may also be involved in this association (82). According to this hypothesis, affected fetuses might suffer insulin resistance because of the influence of several unknown genes or genomic variants. Insulin resistance would lead to restriction of intrauterine growth and abnormal fetal vascular development, ultimately leading during adult life to increased vascular resistance and endothelial dysfunction. The compensatory increase in circulating insulin concentrations would contribute to the abdominal deposition of fat, and inflammatory cytokines and other mediators secreted by abdominal adipocytes would perpetuate insulin resistance and endothelial dysfunction, leading to abnormal glucose tolerance, atherosclerosis, and cardiovascular disease (82).
However, the analysis of the influence of genomic variants on the association of birth weight and insulin resistance has yielded conflicting results. Dunger et al. (83) analyzed a variable number of tandem repeats (VNTR) polymorphism in the insulin gene (INS) at 11p15.5. This VNTR polymorphism consists of a repeated sequence of 14 to 15 bp (ACAGGGGTGTGGGG), and is located at –596 bp of the start of transcription site (84). Among Caucasians, these alleles have been typed as class I (small, with 28–44 repeats, frequency approximately 70%), class II (intermediate, rare), and class III (large, with 138–159 repeats, frequency approximately 30%). The VNTR polymorphism in the INS promoter regulates the transcriptional rate of the gene (85) and probably that of the gene encoding IGF-II (84, 85, 86, 87, 88, 89). Newborns homozygous for class III alleles, which are those associated with increased transcription of the INS, presented with increased head circumference, length, and weight at birth as compared with babies homozygous for class I alleles, possibly favoring newborn survival. On the contrary, data from the same researchers suggested that the maternally inherited 16,189 mitochondrial DNA variant, which increases the risk for type 2 diabetes mellitus, was associated with restrained fetal growth, thereby favoring maternal survival by decreasing birth-related morbidity and mortality (90). Therefore, other genetic and environmental factors are possibly involved in the relationship between birth weight and the development of insulin resistance and type 2 diabetes mellitus in adulthood.
Given the association of insulin resistance with functional hyperandrogenism, Ibá?ez et al. (91) have proposed that low birth weight is related to the development of premature pubarche, and of functional hyperandrogenism during adolescence, insulin resistance being the underlying disorder common to both conditions. Retrospective analysis of a cohort of patients presenting with premature pubarche showed, especially in those girls who developed hyperinsulinemia and functional ovarian hyperandrogenism at adolescence, a lower birth weight compared with a control group, suggesting that low birth weight, premature pubarche, and functional hyperandrogenism are different manifestations of a unique disorder originating during prenatal life (92, 93, 94, 95), but becoming clinically apparent peripubertally (42).
Additional analysis of this cohort showed that adolescent girls who had anovulation and higher androgen levels were those with lower birth weight (96) and also confirmed the association of adrenal androgen excess, low birth weight, premature pubarche, and hyperinsulinemia (97).
In conceptual agreement, an exaggerated adrenal response to adrenocorticotropin stimulation, similar to that described in women with functional hyperandrogenism and PCOS, has been found in girls with premature pubarche, suggesting that this disorder can be considered an early manifestation of the syndrome (98). However, the relationship of low birth weight with hyperandrogenism and PCOS has not been universally confirmed. Jaquet et al. (99) compared a large population of young women born with intrauterine growth retardation with age-matched healthy controls, and although low birth weight was associated with insulin resistance, androgen levels were similar in both groups of women. Moreover, birth weight had no influence on the presence of hyperandrogenic symptoms suggestive of PCOS in a large cohort of Finnish women recently reported (100).
Given the association of functional hyperandrogenism and insulin resistance in children and adolescents, several genomic variants related to insulin resistance and the metabolic syndrome have been studied in these patients, mostly by the group of Ibánez in Spain and the group of Witchel in Pittsburgh.
The VNTR locus at the INS has been studied in girls with a history of premature pubarche (101). Although class I and class III alleles were equally distributed in patients and controls, patients carrying class I alleles presented with lower birth weight and lower insulin sensitivity compared with patients homozygous for class III alleles (101).
The common Gly972Arg variant in the gene encoding the insulin receptor substrate 1 (IRS-1), which has been shown to influence insulin resistance and glucose tolerance in adult PCOS patients (102, 103), has also been studied in adolescents (104). The frequencies of heterozygosity for the Gly972 allele were 31% among girls with a history of premature pubarche, 40% among girls with hyperinsulinemic ovarian hyperandrogenism, and only 19% among healthy control subjects. Carriers of Gly972 alleles presented with decreased sex hormone-binding levels (104).
The human homolog for the sorbin and SH3-domain-containing-1 gene (SORBS1), which encodes for an important signaling molecule in insulin-stimulated glucose uptake in the mouse, might play a role in human disorders with insulin resistance. The Ala228 allele of the Thr228Ala polymorphism of SORBS1 is a protective factor for both obesity and diabetes (105). However, alleles of the Thr228Ala polymorphism of SORBS1 were equally distributed in a multiethnic group of healthy adolescents compared with those presenting with premature pubarche and/or functional hyperandrogenism (106).
The Arg64 allele of the Trp64Arg polymorphism in the ?3-adrenergic receptor gene is associated with abdominal obesity and resistance to insulin and may contribute to the early onset of type 2 diabetes mellitus (107). However, Witchel et al. (108) found no differences in the distribution of Trp64Arg alleles in the girls presenting with premature pubarche and/or functional hyperandrogenism described above compared with healthy adults, and neither was an influence of this polymorphism on the body mass index of these girls. Similarly, the study of the common Pro12Ala polymorphism in the gene encoding the peroxisome proliferator-activated receptor-2 (PPAR-2), which influences insulin sensitivity in Caucasians (109), has been studied in children with premature pubarche and in adolescent hyperandrogenic girls, but no association was found with any of the PPAR-2 alleles and hyperandrogenism (110).
In addition to genes related to insulin resistance, those encoding for the steroidogenic enzymes involved in androgen biosynthesis and the genes encoding several molecules involved in androgen metabolism and action have been considered candidate genes to explain premature pubarche and adolescent hyperandrogenism.
Witchel and colleagues from Pittsburgh conducted a series of studies focused on the steroidogenic enzymes involved in androgen synthesis, which are shared by the adrenal and the ovaries. These authors have found that children with premature pubarche and adolescent girls with hyperandrogenism were heterozygous for mutations in CYP21, the gene encoding for 21-hydroxylase, and in the gene encoding 3?-hydroxysteroid-dehydrogenase (HSD3B2), more frequently than nonhyperandrogenic controls (111, 112, 113). In these patients, CYP17 mutations were ruled out as the cause of androgen excess (114). However, because congenital adrenal hyperplasia is an autosomal recessive disease requiring mutations in both alleles–in homozygosity or double heterozygosity–of CYP21 or of HSD3B2, it is unclear how carrying only one defective allele of these genes in these clinically symptomatic heterozygotes contributes to the hyperandrogenic phenotype (113). Moreover, defective CYP21 and HSD3B2 alleles were found only in a minority of these patients, and the authors concluded that other genomic variants were also involved in the pathogenesis of these disorders (111, 112, 113).
To further explore this possibility, Witchel et al. (115) evaluated the prevalence of the common variants and mutations in CYP21, HSD3B2, IRS-1, ?3-adrenergic receptor gene described above, and the glucocorticoid receptor gene within their series of children with premature pubarche and adolescent hyperandrogenism. They found that the prevalence of these variants and mutations was clearly increased when compared with 15 healthy controls, suggesting that the occurrence of multiple sequence variants in these genes might contribute to the development of hyperandrogenism.
Finally, the AR and uridine diphosphate-glucuronyltransferase 2B15, an enzyme involved in androgen inactivation, have been considered candidate genes for premature pubarche and adolescent hyperandrogenism. The study of the AR gene CAG repeat polymorphism suggested that shorter AR gene CAG numbers, indicative of increased androgen sensitivity, were associated with premature pubarche (116), whereas the allele frequencies of the D85Y polymorphism in uridine diphosphate-glucuronyltransferase 2B15 gene were similar in patients and in controls (117).
In summary, studies conducted to date in girls with premature pubarche and adolescent hyperandrogenism suggest a polygenic etiology for these disorders, which can be considered predictors of functional hyperandrogenism and PCOS later in life.
V. Studies in Hyperandrogenic Adults
Most if not all the studies conducted to date to elucidate the genetics of functional hyperandrogenism and PCOS have used a candidate gene approach, because no genome-wide scan has been conducted comparable with those done for other complex metabolic disorders, such as type 2 diabetes mellitus. Although many genes have been considered candidates to explain PCOS inheritance, most studies have included genes related to androgen biosynthesis and action and their regulation, genes involved in insulin resistance and associated disorders, and lately genes involved in chronic inflammation and atherosclerosis.
A. Genes involved in androgen biosynthesis, transport, and action, and their regulation (Table 3)
1. CYP17.
The limiting step in androgen biosynthesis in the ovary and adrenal gland is an enzyme termed P450c17, which possesses both 17-hydroxylase and 17,20-lyase activities. Therefore, P450c17 may catalyze the conversion of pregnenolone and progesterone into 17-hydroxypregnenolone and 17-hydroxyprogesterone, respectively, and of these steroids into dehydroepiandrosterone and androstenedione, although in humans only the conversion of 5 steroids has been demonstrated (118).
Rosenfield et al. (2, 119, 120) proposed several years ago that women with functional hyperandrogenism and PCOS had an exaggerated adrenal and ovarian responsiveness, and that increased activity of P450c17 was responsible for the enhanced androgen synthesis and secretion. They proposed the increase in serum 17-hydroxyprogesterone in response to the GnRH analog nafarelin as the marker of functional ovarian hyperandrogenism (119). Studies using adrenal stimulation with ACTH and ovarian stimulation and suppression with the long-acting GnRH triptorelin also suggested that most hyperandrogenic women have increased P450c17 activity in the adrenal and ovary (121, 122, 123), and that the increased adrenal P450c17 activity was not influenced by ovarian function (122). Moreover, P450c17 expression and activity are increased in ovarian theca cells from PCOS women, defined by NICHD criteria, compared with those from nonhyperandrogenic controls (124).
Given these findings, the gene encoding the P450c17 enzyme, CYP17, was considered a candidate gene in early studies. CYP17 is located in chromosome 10q24.3 (125, 126), and its promoter contains a T/C SNP at –34 bp from the start of transcription site that might modulate enzyme activity. Some studies suggested that this polymorphism was associated with the presence of polycystic ovaries on ultrasound (125), and PCOS patients homozygous for C alleles of this polymorphism presented with increased serum testosterone levels (127, 128). Other studies failed to confirm these observations, suggesting that this base change is a polymorphism without functional consequences for the development of polycystic ovaries and hyperandrogenism (129, 130, 131). Also, no evidence for linkage or association was found between PCOS, defined by NICHD criteria, and the CYP17 locus in a family-based study that included mostly families of European ancestry from the United States (132). Moreover, no abnormalities were found after single-strand conformational polymorphism analysis of the entire coding region of CYP17 in a small sample of PCOS patients with or without exaggerated 17-hydroxyprogesterone response to a GnRH analog (133), ruling out CYP17 as a major candidate gene for the pathogenesis of PCOS and functional hyperandrogenism. However, posttranscriptional hyperphosphorylation of the serine residues of P450c17 by a defective serine kinase might increase the 17,20-lyase activity of this enzyme, contributing to hyperandrogenism (118, 134). Confirmation of this hypothesis is still pending (135).
2. CYP11A.
Ovarian theca cells from women with PCOS defined by NICHD criteria overexpress all the steroidogenic enzymes involved in androgen biosynthesis (136, 137), and these cells secrete increased amounts of progesterone, 17-hydroxyprogesterone, testosterone, and androstenedione compared with theca cells from nonhyperandrogenic women (136, 138).
The initial step in adrenal and ovarian steroidogenesis is the conversion of cholesterol into progesterone, which is catalyzed by the cholesterol side chain cleavage enzyme. The CYP11A gene, located at 15q24, encodes the cholesterol side chain cleavage enzyme and has therefore been considered a candidate gene for functional hyperandrogenism and PCOS (139). It has been proposed that a VNTR polymorphism, consisting in repeats of a (tttta)n pentanucleotide at –528 bp from the ATG start of translation site in the CYP11A promoter, plays a role in the pathogenesis of PCOS (140). Evidence for linkage with the CYP11A locus was found in 20 pedigrees presenting with PCOS, based mostly on the presence of polycystic ovaries or male pattern premature balding, and the absence of the more common four-repeats allele (this VNTR appears with four, six, eight, and nine repeats in Caucasians) was associated with hirsute PCOS patients and with higher serum testosterone levels (140). Diamanti-Kandarakis et al. (141), using NICHD criteria for the definition of PCOS, confirmed its association with absence of four-repeat alleles in Greek patients. In women from the United States, nine-repeat alleles were more frequent in PCOS patients, defined by oligomenorrhea and polycystic ovaries, and four- and six-repeat alleles were more frequent in controls, but these allelic differences did not influence CYP11A expression in theca cells (142).
Other studies failed to demonstrate linkage with the CYP11A locus in PCOS patients defined by NICHD criteria (132) or association of CYP11A VNTR alleles with functional hyperandrogenism (143). Moreover, in a small sample of hirsute hyperandrogenic patients from Spain (144), no consistent genomic abnormalities have been found in the entire CYP11A coding region, nor in the genes encoding the steroidogenic acute regulatory protein, steroidogenic factor-1, and dosage-sensitive sex reversal-adrenal hypoplasia gene on the X chromosome gene-1 (DAX-1), which are also involved in the first step of steroidogenesis and its regulation. The family-based study cited above failed also to demonstrate linkage or association with the steroidogenic acute regulatory protein (132). Finally, Gaasenbeek et al. (145) recently failed to confirm any influence of CYP11A VNTR alleles on polycystic ovaries and on serum testosterone levels in a series of experiments involving a large number of subjects, concluding that the existence of associations between CYP11A promoter variation and androgen-related phenotypes had been substantially overestimated in previous studies, including their own preliminary report cited above (140).
3. CYP21.
An exaggerated serum 17-hydroxyprogesterone response to ACTH stimulation is a common finding in women with PCOS or functional hyperandrogenism (121, 146). This finding prompted several groups to study CYP21, which encodes the 21-hydroxylase enzyme catalyzing the conversion of 17-hydroxyprogesterone into 11-deoxycortisol; increased serum 17-hydroxyprogesterone levels are the biochemical marker for 21-hydroxylase deficiency. This type of congenital adrenal hyperplasia is an autosomal recessive disease resulting from homozygosity or double heterozygosity for missense or nonsense mutations in CYP21 and is characterized by hyperandrogenism with or without mineralocorticoid deficiency.
However, whether or not heterozygous CYP21 mutations influence functional hyperandrogenism and PCOS is still matter of debate. As stated above, girls presenting with premature adrenarche and adolescent hyperandrogenism carry CYP21 mutations more frequently than nonhyperandrogenic controls (112). Clinically symptomatic heterozygotes for CYP21 mutations present with a phenotype that resembles that of PCOS (113), but it is unclear whether all carriers of CYP21 mutations have an increased risk of having PCOS. Moreover, in hyperandrogenic women carrying CYP21 mutations there is no clear concordance between the CYP21 genotype and the functional origin of androgen excess (147).
Finally, the presence of polycystic ovaries in ultrasound scans has been associated with increased frequency of DRW6 and decreased DR7 human leukocyte antigen haplotypes, whereas 21-hydroxylase deficiency is associated with the Bw47, B14, or DR1 haplotypes (148). Moreover, in families of patients with 21-hydroxylase deficiency, polycystic ovaries segregate independently from adrenal dysfunction (149).
4. HSD3B2.
This enzyme catalyzes the conversion of 5 steroids into 4 steroids in the adrenal and in the ovary. Mutations in HSD3B2 result in a rare form of classic congenital adrenal hyperplasia, causing various degrees of salt wasting in both sexes and incomplete masculinization of the external genitalia in genetic males because both adrenal and ovarian steroidogenesis are severely impaired (150). Biochemically, this disorder is characterized by a marked increase in serum 17-hydroxypregnenolone and dehydroepiandrosterone concentrations (150).
Mild increases in these steroid precursors, and in the ratio of 5 to 4 steroids, are not infrequent in hyperandrogenic patients, and hyperandrogenic patients presenting with increased 17-hydroxypregnenolone concentrations and/or increased 5 to 4 ratios after adrenal stimulation with cosyntropin were initially considered to have a nonclassic form of HSD3B2 deficiency (151, 152, 153). However, these mild increases in serum 17-hydroxypregnenolone concentrations and in the 5 to 4 ratios were interpreted by others as an exaggerated adrenal response to cosyntropin stimulation without a genetic origin (154). Molecular analysis of HSD3B2 of hyperandrogenic patients presenting with increased 5 steroids and increased 5 to 4 ratios revealed no abnormalities (155, 156), ruling out the existence of a nonclassic form of HSD3B2 deficiency in these women. In conceptual agreement, a marker close to the HSD3B2 locus was not in linkage or association with PCOS, defined by NICHD criteria, in the American family-based study cited above (132).
5. 17?-Hydroxysteroid dehydrogenases.
17?-Hydroxysteroid dehydrogenase type III is also known as 17-ketosteroid reductase. This enzyme catalyzes the conversion of androstenedione into testosterone in the testis. Up to 20 mutations in HSD17B3 have been identified as the cause of male pseudohermaphroditism because of testosterone deficiency (157). Pang et al. (158) hypothesized that genetic females with ovarian 17-ketosteroid reductase deficiency would probably have a normal female phenotype at birth and normal pubertal breast development but would present at puberty with virilization and menstrual disorders because of the sudden increase of the ovarian secretion of androstenedione. However, no mutations have been identified in HSD17B3 in Caucasian and African-American women presenting with PCOS defined by NICHD criteria (159). Also, no evidence for linkage or association with the type 1, type 2, and type 3 17?-hydroxysteroid dehydrogenase loci was found in the American family-based study cited previously (132).
6. Aromatase.
This enzyme, encoded by CYP19, is responsible for the conversion of C19 steroids (androgens) into C18 steroids (estrogens). Aromatase activity may be decreased in granulosa cells and follicles from women with PCOS, and the possible androgen excess resulting from this decreased activity might contribute to abnormal follicle development (160, 161). Although mutations in CYP19 have been associated with multicystic ovaries in a case report, the clinical picture was that of hypergonadotropic hypogonadism (162). Moreover, no evidence for linkage of CYP19 with PCOS was found in studies conducted in the United Kingdom (140) and in the United States (132).
7. 11?-Hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase.
These enzymes regulate the inactivation of cortisol into cortisone at the tissue level. 11?-Hydroxysteroid dehydrogenase type 1 converts cortisol into cortisone directly, and endoluminal hexose-6-phosphate dehydrogenase regenerates reduced nicotinamide adenine dinucleotide phosphate in the endoplasmic reticulum, thereby influencing the directionality of 11?-hydroxysteroid dehydrogenase type 1 activity. Mutations in the genes encoding 11?-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase have been recently shown to cause cortisone reductase deficiency (163), and because the phenotypic features of affected women resemble those of PCOS, these genes should be considered candidates to explain the pathogenesis of PCOS. However, this possibility has not been explored in any series of patients with functional hyperandrogenism and PCOS to date.
8. Gonadotropins.
In as many as 40% of PCOS patients, LH hypersecretion is present (164). The gene encoding the ?-subunit of LH (LH?), which is responsible for LH specificity, has been explored in PCOS and hyperandrogenic women. Initially, two point mutations, Trp8Arg and Ile15Thr, were identified as the cause of an immunologically abnormal LH? molecule (165). These mutations induce structural changes in the mutant LH? molecule (166) and have a 15% prevalence worldwide, although with significant differences depending on the population studied (167). The resulting LH? molecule has an increased in vitro activity and a decreased in vivo half-life compared with the wild-type molecule (168).
This LH? variant has been associated with increased serum testosterone, estrogen, and SHBG levels in healthy women, but not in PCOS patients (169). Furthermore, the prevalence of this LH? variant is reduced in obese women with PCOS, suggesting a protective role against the development of hyperandrogenic symptoms in these women and a possible usefulness in determining the risk for PCOS in obese women (170, 171). However, no association with PCOS, hyperandrogenism, or serum androgen or estrogen concentrations has been found in other studies (172, 173).
Other variants in the LH? gene might influence hyperandrogenism and PCOS. A Gly102Ser mutation in exon 3 has been related to menstrual disorders in Chinese women (172, 174). Finally, in Japanese patients with ovulatory disorders, several SNPs in the promoter of the LH? gene (–894C/T, –1018G/C, –1036C/A, –1098C/T, and –1423C/T) are more frequent than in normal ovulatory women (175).
Polymorphisms in the FSH ?-subunit gene have also been reported to influence PCOS. In Chinese women, homozygosity for a thymine-cytosine substitution in exon 3 (codon 76, TAT to TAC) has been found more frequently in PCOS patients, as defined by oligomenorrhea and polycystic ovaries, compared with nonhyperandrogenic women, especially in obese patients, and correlating with higher serum androgen concentrations (176). In these women, two SNPs in the gene encoding the FSH receptor, Thr307Ala and Ser680Asn, showed similar allele distribution among PCOS patients and controls (177). Moreover, the search for mutations in exons 6, 7, 9, and 10 of the FSH receptor gene, which have been shown previously to inactivate the receptor, yielded negative results in Japanese patients with oligomenorrhea and polycystic ovaries (178). This locus was not in linkage or association with PCOS in a family-based study conducted in the United States (132). Finally, mutations in the gene encoding the GnRH receptor have not been found in PCOS patients, defined by NICHD criteria (179).
9. Dopamine receptor.
Given that dopamine is involved in the hypothalamic control of gonadotropin secretion, the association between polymorphisms in the dopamine receptor and disorders of ovulation and fertility has been studied. Legro et al. (180) reported that SNPs in exons 5 and 6 of the gene encoding the type 2 dopamine receptor influenced serum gonadotropin and prolactin levels, as well as parity and prevalence of miscarriage in women of Hispanic ancestry. In these women, a genomic variant in the type 3 dopamine receptor was associated with hyperandrogenic chronic anovulation and resistance to clomiphene citrate (181), but this association was not confirmed in a large series of non-Hispanic women (182).
10. SHBG.
SHBG regulates the access of testosterone and estradiol to target tissues. Decreased SHBG concentrations are characteristic of hyperandrogenic women, contributing to increased tissue androgen availability (183). Despite the fact that evidence for linkage or association was not found between a marker close to the SHBG locus and PCOS in a family-based study conducted in the United States (132), a missense Pro156Leu mutation in the SHBG gene, resulting in abnormal glycosylation and secretion of a SHGB mutant that retains binding capacity (184), has been detected in a women presenting with severe hyperandrogenism during pregnancy. However, it is seldom found in unselected hyperandrogenic women (184).
Recently, an association between a (TAAAA)n polymorphism in the promoter of the SHBG gene and PCOS has been reported (185). Longer alleles (more than eight repeats) were frequent in Greek PCOS patients, defined by NICHD criteria, whereas nonhyperandrogenic women presented with a higher frequency of shorter alleles (185). Furthermore, in the PCOS group, carriers of the longer allele genotypes had lower SHBG levels than in those with shorter alleles (185). Therefore, this polymorphism might be related to the low SHBG levels characteristic of PCOS, facilitating androgen availability to target tissues. Cousin et al. (186) studied the (TAAAA)n polymorphism in hirsute women and found strong disequilibrium linkage with an Asp327Asn SNP in exon 8 of SHBG, 327Asn alleles being associated with eight-repeat (TAAAA)n alleles, and resulting in increased serum SHBG levels when compared with subjects homozygous for 327Asp alleles. Moreover, longer (TAAAA)n alleles resulted in decreased serum SHBG levels when compared with six-repeat alleles (186), in conceptual agreement with the results in PCOS women described above (185).
11. Glucocorticoid receptor.
Mutations in the glucocorticoid receptor gene result in a compensatory increase in circulating ACTH, resulting in excess secretion of adrenal androgens. Therefore, the glucocorticoid receptor has been studied as a candidate gene for functional hyperandrogenism and PCOS. However, genomic abnormalities and polymorphisms in the glucocorticoid receptor gene do not appear to influence the pathogenesis of functional hyperandrogenism (187, 188) and PCOS (189).
12. Steroid 5-reductase (SRD5A).
This enzyme catalyzes the conversion of testosterone into the more potent androgen dihydrotestosterone. SRD5A1 and SRD5A2 are the two isoforms of the enzyme. Mutations in SRD5A2 result in male pseudohermaphroditism (190, 191). Because total SRD5A activity is increased in polycystic ovaries (192), the genes encoding the SRD5A isoforms can be considered candidate genes for functional hyperandrogenism and PCOS. To date, no data have been published regarding the possible involvement of molecular genetic variants in these genes in hyperandrogenic disorders.
13. AR.
The AR is a member of the superfamily of ligand-activated transcription factors that regulate many biological processes and is encoded by a gene located at Xq11–12 (193, 194). The AR has three functional domains responsible for transactivation, for ligand binding, and for binding to DNA. Exon 1 contains a VNTR polymorphism consisting of (CAG)n repeats, which encodes for a polyglutamine tract in the N-terminal transactivation domain of the AR protein (195).
The number of CAG repeats in the normal population varies between 11 and 31, with 20 repeats being the most frequent (196). The number of CAG repeats is inversely related to the transactivation of the AR and its activity (197). Thus, the decreased number of CAG repeats has been proposed to increase androgen activity at target tissues, favoring hirsutism (198), premature pubarche, and ovarian hyperandrogenism (116) in women, infertility in men (199), as well as androgen-dependent skin disorders in both men and women (200). A decreased number of AR gene CAG repeats has been suggested to explain the normal serum androgen levels found in some women with polycystic ovaries, infertility, and oligomenorrhea, in whom the hyperandrogenic symptoms would result from the intrinsic increase in the AR activity (201).
One of the two X chromosomes in every cell of a woman undergoes inactivation, a process that involves methylation of DNA and occurs in a random fashion. Skewed inactivation of X chromosome with the larger CAG repeat, favoring expression of the shorter allele, has been proposed to play a role for idiopathic hirsutism and PCOS (202, 203). However, we have shown that both the number of CAG repeats and the prevalence of skewed X chromosome inactivation were equally distributed in hirsute patients with or without hyperandrogenemia and in healthy women (204). Moreover, no evidence for linkage or association with PCOS was found with the AR locus in the family-based study conducted in the United States cited above (132).
14. Aldosterone synthetase.
The renin-angiotensin system may be hyperactive in women with PCOS (205, 206). Recently, a –344T/C SNP in the promoter of the gene encoding aldosterone synthetase, CYP11B2, has been proposed to influence the pathogenesis of PCOS, because C alleles are more frequent in these patients than in healthy controls (207). Interestingly, women homozygous for C alleles presented with increased plasma renin activity and increased serum angiotensin II, aldosterone, and testosterone levels compared with women homozygous for T alleles, suggesting that this polymorphism influences the activity of aldosterone synthetase and also contributes to androgen excess (207).
B. Genes involved in insulin resistance and associated disorders (Table 4)
1. Insulin receptor gene (INSR).
The presence of insulin resistance is common in PCOS (3) and is frequent in other hyperandrogenic patients (208, 209, 210). Therefore, the genes encoding the INSR and those encoding several molecules involved in postreceptor signaling have been studied in hyperandrogenic patients.
The INSR is a heterotetrameric glycoprotein consisting of two - and two ?-subunits, and it is encoded by a gene located at chromosome 19. A marker relatively close (1 cM) to INSR, D19S884, has been reported in association with PCOS, defined by NICHD criteria, using the transmission disequilibrium test in a family-based study (132), and in a case-control study (211), both conducted in the United States. However, this association was not confirmed in PCOS patients from Spain and Italy using a case-control design (212). In conceptual agreement with the latter, the 22 exons of INSR have been sequenced in PCOS patients with negative results (213). INSR contains several polymorphisms, yet most of them are silent polymorphisms or are located in intronic regions and are present with similar frequencies in patients with polycystic ovaries and hyperandrogenism and in controls (214).
Recently, Siegel et al. (215) have observed a C/T SNP at the tyrosine kinase domain of INSR associated with PCOS defined by NICHD criteria. This SNP could be a susceptible variant for PCOS, or it could be in linkage disequilibrium with another INSR polymorphism; such an association must be confirmed in future studies.
Studying cultured skin fibroblasts and muscle samples from PCOS patients defined by NICHD criteria, Dunaif et al. (216) found increased INSR serine phosphorylation, which decreases its protein tyrosine kinase activity, in as many as 50% of the cases. Subsequent studies in these patients confirmed in vivo the defect in insulin signaling, using serial skeletal muscle biopsies obtained during euglycemic glucose clamp studies (217). However, the search for mutation in the tyrosine kinase domain of the INSR gene did not show abnormalities other than polymorphisms in exon 17, which were not associated with insulin resistance (218). Therefore, the still unidentified factor responsible for the increased phosphorylation of the serine residues of the INSR appears to be extrinsic to the receptor and might also contribute to the increased serine phosphorylation of P450c17 also found in some PCOS patients (118).
2. IRS-1 and IRS-2.
After insulin binding, autophosphorylation of tyrosine residues results in the activation of the INSR, and tyrosine kinase activity phosphorylates intracellular substrates such as IRS-1 and IRS-2 (3). PCOS women present a defect in insulin receptor signaling characterized by a decreased IRS-1-associated phosphatidylinositol 3-kinase activity (217). Two common SNPs in the genes encoding insulin-receptor substrates, Gly972Arg in IRS-1 and Gly1057Asp in IRS-2, are susceptibility genes for type 2 diabetes mellitus (219, 220); they have been studied in PCOS patients, despite the fact that evidence for linkage or association with PCOS was not found with IRS-1 in a family-based study conducted in the United States (132).
Carriers of Arg972 IRS-1 alleles presented with increased fasting insulin levels compared with women homozygous for Gly972 alleles, whereas carriers of Asp1057 IRS-2 alleles presented with increased glucose and insulin levels 2 h after an oral glucose load and had an increased prevalence of glucose intolerance compared with subjects homozygous for Gly1057 alleles (102). However, a subsequent study in a larger series of PCOS patients showed only the effect of the Gly1057Asp polymorphism in IRS-2 on glucose tolerance, and no effect of the Gly972Arg polymorphism in IRS-1 (103). Surprisingly, the effect was just the opposite found previously, because the 2-h glucose values were actually increased in subjects homozygous for Gly1057 alleles when compared with carriers of Asp1057 alleles (103). Additional studies are needed to confirm the influence of these polymorphisms on glucose tolerance and insulin resistance in PCOS.
3. INS.
The presence of pancreatic ?-cell dysfunction in women presenting with PCOS appears to have a genetic origin (221). Therefore, INS has been studied in women with PCOS and functional hyperandrogenism. Waterworth et al. (222) found that women with menstrual disturbances and/or hirsutism and polycystic ovaries, who were homozygous for class III alleles, were more frequently anovulatory and had increased body mass index and fasting insulin compared with women homozygous for class I alleles. Moreover, class III alleles were associated with symptomatic women (222, 223). Paternal transmission of class III alleles from heterozygous fathers to anovulatory PCOS patients is more frequent than maternal transmission of the allele (222, 223, 224), and class III alleles predisposed these patients to both PCOS and type 2 diabetes mellitus. However, later case-control studies in European Caucasian women, conducted outside the United Kingdom, have failed to reproduce these results (225, 226), and the INS locus was not associated with PCOS in a linkage study in American PCOS patients (132).
4. IGF system.
IGFs, their receptors, binding proteins, and proteases are important for the normal development of the ovary (227). IGFs stimulate ovarian cellular mitosis and steroidogenesis, inhibit apoptosis, and might be related to the development of functional hyperandrogenism and PCOS (228).
No evidence for linkage with PCOS was found for markers close to the genes encoding IGF-I and IGF-binding proteins 1 and 3 in a family study conducted in the United States (132). San Millán et al. (229) recently found an association of PCOS with homozygosity for G alleles of the ApaI polymorphism in IGF-II, but not with a dinucleotide polymorphism in IGF-I, a trinucleotide polymorphism in the IGF-I receptor, or with an ACAA-insertion/deletion polymorphism at the 3' nontranslated region of the IGF-II receptor, previously described (230, 231, 232). G alleles of the ApaI polymorphism in IGF-II have been attributed to increased IGF-II mRNA in leukocytes compared with A alleles (233), and possibly result in increased liver IGF-II expression and secretion (234). Given that IGF-II stimulates adrenal (235, 236) and ovarian (237) androgen secretion, the increased frequency of homozygosity for these alleles might contribute to hyperandrogenism in some PCOS patients, provided we assume that G alleles may increase IGF-II expression at the ovary, as reported for other tissues. In the same case-control study involving PCOS patients cited above (229), we found that subjects homozygous for 90-bp alleles of a trinucleotide repeat polymorphism in the gene encoding IGF-I receptor had increased fasting glucose levels and fasting insulin resistance index compared with subjects carrying 93-bp alleles, but no association of any genotype with PCOS.
5. PPAR-2.
Activation of PPAR- by using the insulin sensitizer drugs, thiazolidinediones, has been one of the most important advances for the treatment of type 2 diabetes mellitus in past years. As described above, insulin resistance is a common finding in hyperandrogenic patients, and thiazolidinediones improve insulin sensitivity, hyperandrogenism, and ovulation in women with PCOS (238, 239, 240, 241, 242, 243).
The Pro12Ala SNP in PPAR-2 has been studied in women with PCOS, despite the fact that evidence for linkage or association with PCOS was not found for a marker close to the PPAR-2 gene, in a family-based study conducted in the United States (132). Ala12 alleles of the PPAR-2 gene favor weight gain in obese adults (244) and in obese hyperandrogenic girls and adolescents (110). Also, Ala12 alleles preserve insulin sensitivity in Caucasian men (109) and in Caucasian women presenting with PCOS defined by NICHD criteria (245). Recently, a marginally significant decrease in the frequency of the Ala12 allele has been reported in women with polycystic ovaries from Finland (246), but this result has not been confirmed in a small case-control study of PCOS patients conducted in Spain (229) or in a recently published study in Italian PCOS patients defined by NICHD criteria and ultrasonography (247). In the latter, a silent C to T substitution at position 142 in exon 6 was differentially distributed in PCOS patients and controls, T alleles being more frequent in women with PCOS (247). This silent polymorphism was not in linkage disequilibrium with the Pro12Ala polymorphism, but the possibility of an association with other unknown genomic variant in the PPAR-2 gene was not explored (247). Nevertheless, both Ala12 and T142 alleles are relatively uncommon (less than 20%) in normal and hyperandrogenic populations (229, 245, 246, 247), and therefore their putative influences on insulin sensitivity and/or hyperandrogenism would be restricted to a small number of PCOS patients.
6. Paraoxonase (PON1).
We have recently explored the –108C/T, Leu55Met, and Gln192Arg polymorphisms in the gene encoding serum PON1 in PCOS patients defined by NICHD criteria. The PON1 gene is expressed mainly in the liver and encodes for serum PON1, which is an antioxidant high-density lipoprotein-associated enzyme. Liver PON1 mRNA expression is influenced by genetic and environmental factors, and both androgens and proinflammatory mediators decrease liver PON1 expression (248). Interestingly, both androgen excess and proinflammatory genotypes contribute to the pathogenesis of PCOS (249, 250, 251).
Homozygosity for T alleles of the –108C/T polymorphism in PON1 was more frequent in patients compared with nonhyperandrogenic women (229). As expected from the association with PCOS, subjects homozygous for –108T alleles of PON1 presented with increased hirsutism scores, total testosterone, and free testosterone and androstenedione concentrations compared with carriers of –108C alleles (229). Moreover, in a logistic regression model, homozygosity for –108T alleles of PON1 was associated with a 7.1 odds ratio (95% confidence interval, 2.1–23.8) of having PCOS (229).
The –108C/T polymorphism is responsible of approximately 23% of PON1-expression levels in some cell systems, in which –108TT constructs showed reduced PON1 expression compared with –108CC constructs (252). We thus speculated that homozygosity for –108T alleles, hyperandrogenism, and proinflammatory genotypes might contribute to reduced PON1 expression, resulting in a higher oxidative stress in these women. The latter has been found in PCOS patients (253).
Oxidative stress may impair insulin action (254). Therefore, reduced serum PON1 activity might contribute to the insulin resistance of PCOS patients. This hypothesis is supported by the finding of reduced serum PON1 activity in other insulin-resistant disorders such as type 2 diabetes mellitus (255, 256) and cardiovascular atherosclerotic disease (257, 258). If confirmed in future studies, the association of homozygosity for –108T alleles of PON1 with PCOS might contribute to explaining the insulin resistance and the increased risk for atherosclerosis associated with this syndrome (259).
In our study (229), subjects homozygous for Met55 alleles presented with increased body mass index and indexes of insulin resistance compared with carriers of Leu55 alleles, further suggesting the involvement of PON1 in PCOS, despite the fact that the Leu55Met and Gln192Arg polymorphisms in PON1 were not associated with PCOS.
7. Human homolog for the sorbin and SH3-domain-containing-1 gene (SORBS1).
In addition to the studies in adolescents cited above (106), we have recently studied the Thr228Ala polymorphism in adult PCOS patients. Allele frequencies were similar in PCOS patients and nonhyperandrogenic women, but carriers of Ala228 alleles of SORBS1 presented with increased body mass index compared with subjects homozygous for 228T alleles (229), in conceptual agreement with a large study conducted in Europe (260).
8. Calpain-10.
This enzyme is a cysteine protease that plays a role in insulin secretion and action (261). The 112/121-haplotype combination of University of Chicago single nucleotide polymorphism (UCSNP)-43, UCSNP-19, and UCSNP-63 polymorphisms in the gene encoding calpain-10, located at 2q37.3, has been reported to increase the risk for diabetes (262). Ehrmann et al. (263) found no association between this haplotype and any of the phenotypic features of PCOS in Caucasian nondiabetic PCOS patients, defined by NICHD criteria, whereas the 112/121-haplotype was significantly associated with higher insulin levels in response to an oral glucose tolerance test in African-American, nondiabetic PCOS women. Moreover, when considering Caucasian and African-American, nondiabetic PCOS patients as a whole, the 112/121 haplotype was associated with a 2-fold increase in susceptibility to PCOS.
However, the association of calpain-10 SNPs with PCOS, as defined by polycystic ovaries, hyperandrogenism, and/or anovulation, was not confirmed by Haddad et al. (264) in 330 PCOS patients from the United Kingdom. We have studied three common polymorphisms in the calpain-10 gene in 97 Spanish hyperandrogenic patients and 37 controls, including UCSNP-43 (265). C alleles at the UCSNP-45 locus were associated with idiopathic hirsutism, but neither the UCSNP-43 nor the UCSNP-44 was associated with hyperandrogenism or PCOS (265). However, in a different population from the south of Spain, González et al. (266, 267) recently reported an association between PCOS and USCNP-44. Additional studies are needed to clarify this issue, especially because the physiological roles of calpain-10 remain mostly unknown.
9. Genes encoding for other molecules related to insulin resistance and associated disorders.
Among other genes tested, no association has been reported in PCOS with genomic variants in the genes encoding glycogen synthetase (268), resistin (269), leptin and its receptor (270), apoprotein E (271), or with variants in the genes of plasma cell differentiation antigen glycoprotein, protein tyrosine phosphatase 1B, and adiponectin (229).
C. Proinflammatory genotypes (Table 5)
Chronic inflammation is involved in the development of metabolic syndrome and cardiovascular disease (272, 273), and serum inflammatory markers cluster in patients with cardiovascular disease, suggesting a role in the pathogenesis of atherosclerosis (272, 273).
Inverse correlations have been reported between indexes of insulin sensitivity and inflammatory markers such as circulating levels of TNF- (274), soluble type 2 TNF receptor (TNFR2) (275), IL-6 (276), C-reactive protein (CRP) (276), and soluble intercellular cell adhesion molecule-1 (276).
Adipose tissue plays a central role in the relationship between cytokines and insulin resistance. The expression of TNF- and TNFR2 in adipose tissue is increased in obesity (277, 278). TNF- expression correlates with indexes of insulin resistance and decreases with weight loss in parallel with the improvement in insulin sensitivity (277). Similar results have been reported for IL-6 (279). Moreover, inflammatory cytokines may induce insulin resistance by direct actions on insulin-signaling postreceptor molecules (280) or by inducing central obesity through activation of the hypothalamic-pituitary-adrenal axis (281).
Because obesity and insulin resistance are common findings in hyperandrogenic women (3), chronic inflammation might be involved in the pathogenesis of functional hyperandrogenism and PCOS. In animal models in which polycystic ovaries were induced by neonatal administration of estradiol, the production of TNF- and IL-6 was increased in ovaries and in peritoneal macrophages (282). The concentrations of IL-12 were decreased, and production of IL-13 and the number of activated lymphocytes were increased in follicular fluid of women with PCOS (283).
The study of serum inflammatory markers in PCOS has resulted in conflicting reports. Increased CRP levels have been reported in PCOS patients defined by NICHD criteria (284, 285). Similarly, increased serum IL-6 (286) and TNF- (287, 288, 289) concentrations have been reported in women with PCOS or functional hyperandrogenism.
However, we have recently reported that obesity, and not PCOS, appears to be the major determinant of the increase in serum CRP and IL-6 in premenopausal women, and this and other inflammatory markers such as serum TNF-, soluble TNFR2, or soluble intercellular cell adhesion molecule-1 are not increased by PCOS when controlling for confounding factors such as smoking and obesity (290). Similar findings have been published recently by Mohlig et al. (291). These results cast doubt upon the usefulness of these serum inflammatory molecules as markers of the inflammatory process associated with hyperandrogenism. On the contrary, another novel inflammatory marker of cardiovascular risk, IL-18, is increased in serum both by obesity and by PCOS, suggesting that this molecule may be a useful marker of inflammation in PCOS patients (292).
Given that proinflammatory genotypes influence obesity, type 2 diabetes mellitus, and insulin resistance-related disorders (273), over the past years our group has studied genomic variants in the genes encoding several inflammatory mediators and their receptors. Some of these variants are associated with functional hyperandrogenism and PCOS.
1. TNF-.
TNF- induces reproductive changes that closely resemble those found in patients with PCOS and functional hyperandrogenism. TNF- facilitates the effects of insulin and IGF-I on the ovary in a dose-dependent and additive fashion (293), stimulating proliferation and steroidogenesis in rat theca cells in vitro (293, 294). Moreover, TNF- may be involved in apoptosis and anovulation in the rat ovary (295).
We have recently studied serum TNF- levels and nine common polymorphisms (–1196C/T, –1125G/C, –1031T/C, –863C/A, –857C/T, –316G/A, –308G/A, –238G/A, and –163G/A) in the TNF- gene in 60 hyperandrogenic women and 27 healthy controls matched for body mass index (288). As a group, hyperandrogenic patients presented with increased serum TNF- levels, but this increase was only present in lean patients when compared with lean controls, and not in obese patients (288).
No differences between patients and controls were found in the allele frequencies of any of the polymorphisms studied (288). In conceptual agreement, –308G/A alleles were equally distributed between patients with polycystic ovaries and hyperandrogenic symptoms and controls in other studies (296, 297), and similar results were reported for the –805C/T polymorphism in the TNF- gene (298). However, when considering patients and controls as a whole in our series, carriers of –308A alleles presented with increased serum androgen and 17-hydroxyprogesterone levels before and after stimulation with the GnRH analog leuprolide (288). Therefore, polymorphisms in the TNF- gene do not appear to play a major role in the pathogenesis of functional hyperandrogenism and PCOS but might be a modifying factor for phenotypic traits associated with these disorders.
2. TNFR2 gene (TNFRSF1B).
TNFR2 mediates most of the metabolic effects of TNF- (299). We have recently studied serum soluble TNFR2 levels and several polymorphisms in the TNFRSF1B in women with functional hyperandrogenism, including PCOS defined by NICHD criteria (250).
TNFRSF1B has been studied in several metabolic disorders. The 1690T/C variant in exon 10 has been described to influence body mass index and insulin resistance (300). The CA-repeat polymorphism in intron 4 and the Met196Arg polymorphism in exon 6, which are in strong linkage disequilibrium, influence serum lipid levels (301, 302, 303) and diastolic blood pressure (302); linkage studies suggest that the TNFRSF1B locus is associated with hypertension (302) and familial combined hyperlipidemia (301). Moreover, the CA-repeat polymorphism has been recently proposed as a contributing factor to coronary artery disease (304).
In our series, the uncommon 196Arg allele of the Met196Arg (676T/G) polymorphism in exon 6 of TNFRSF1B was more frequent in patients with PCOS compared with healthy controls (250). When the study was extended to include Italian subjects, this variant was more frequent not only in PCOS patients but also in women with hyperandrogenic hirsutism and regular menstrual cycles (250). However, the Met196Arg polymorphisms did not influence any phenotypic trait associated with hyperandrogenism, insulin resistance, or obesity when studying patients and controls separately (250).
We also studied three SNPs in the 3'-untranslated region of TNFRSF1B in exon 10, 1663G/A, 1668T/G, and 1690T/C, which were not associated with hyperandrogenism (250). Serum soluble TNFR2 levels were not increased in hyperandrogenic women compared with controls, but they were influenced by the interaction between the 1663G/A and 1668T/G variants. We hypothesized that the Met196Arg variant in TNFRSF1B might contribute to PCOS by modulating TNF- actions at target tissues.
3. IL-6.
Among cytokines, IL-6 circulates in plasma and acts in distant tissues (305). TNF- stimulates IL-6 secretion by adipocytes, and mounting evidence suggests that IL-6 is also implicated in insulin resistance and associated syndromes (273, 306, 307, 308). Although serum IL-6 levels are not increased in women presenting with functional hyperandrogenism and PCOS (249, 290), IL-6 concentrations are increased in peritoneal fluid in clomiphene-resistant, anovulatory PCOS patients, suggesting a role in the pathogenesis of hyperandrogenic disorders (309).
We recently studied four common polymorphisms in the promoter of the IL-6 gene (–597G/A, –572G/C, –373A(n)T(n), and –174G/C) in 85 hyperandrogenic patients and 25 healthy women (249). The –597G/A and –174G/C variants were in strong disequilibrium linkage. When considering the three biallelic SNPs, five haplotypes were found (relative frequencies in parentheses): GGG (0.505), AGC (0.377), GGC (0.059), GCG (0.055), and GCC (0.005). The frequency of the GGG haplotype was increased in patients (0.559) compared with controls (0.320), and conversely, the frequency of the AGC haplotype was reduced in patients (0.318) compared with controls (0.580) (P < 0.02). Homozygosity and heterozygosity for –597G and –174G alleles were more frequent in controls, and controls carrying these alleles presented with increased serum IL-6, cortisol, 11-deoxycortisol, and 17-hydroxyprogesterone levels, and a tendency toward increased serum total testosterone levels compared with controls homozygous for –597A and –174C alleles. These findings suggest a protective role for the latter against IL-6 excess, adrenal hyperactivity, and hyperandrogenism. The –572G/C and –373A(n)T(n) were not associated with hyperandrogenism or with any androgen-related phenotypic trait (249). In conceptual agreement, Mohlig et al. (291) recently reported that the heterozygous –174G/C genotype in PCOS patients was associated with lower serum androstenedione levels.
4. IL-6 receptor.
This is a heterodimeric receptor consisting of two membrane-bound glycoproteins: an 80-kDa IL-6 binding unit and a 130-kDa IL-6 signal transducer (IL-6 receptor ? or gp130). gp130 is a transducer chain shared by other cytokines and is responsible for signal transduction of the chain-ligand complex through the Janus kinase/signal transducer and activator of the transcription pathway (310).
We have recently studied common polymorphisms in both subunits of the IL-6 receptor in a series of 145 hyperandrogenic women and 45 controls from Spain (251). The uncommon Arg148 allele of the Gly148Arg polymorphism in the gp130 gene was more frequent in controls compared with hyperandrogenic patients, and controls carrying Arg148 alleles had lower 11-deoxycortisol and 17-hydroxyprogesterone concentrations, a lower response of androstenedione to 1–24 adrenocorticotropin, and an almost statistically significant decrease in free testosterone levels, suggesting that, as occurred for the IL-6 polymorphisms described above, Arg148 alleles in the gp130 gene have a protective effect against androgen excess and adrenal hyperactivity (251). When considering patients and controls as a whole, a microsatellite CA-repeat polymorphism in the 80-kDa IL-6 binding unit locus was associated with obesity. The frequency of the common 149-bp allele was markedly increased in obese women compared with controls, further supporting the involvement of inflammatory genotypes in obesity and related syndromes (251).
Overall, our studies over the past years suggest that chronic inflammation underlies the pathogenesis of functional hyperandrogenism and PCOS, as has been proposed for other disorders associated with insulin resistance, and that proinflammatory genotypes may be involved.
D. Other candidate genes
1. Follistatin.
This protein binds to activin, inhibiting its action both in vivo and in vitro (311). Activin and follistatin are expressed in multiple tissues, including pituitary, ovary, adrenal, and pancreas (312, 313). In the ovary, activin promotes follicular development and inhibits androgen secretion by theca cells (314). Conversely, transgenic mice overexpressing follistatin present with follicular arrest and infertility (315), characteristics frequently found in hyperandrogenic women.
However, the expression of human inhibin/activin subunit, follistatin, type 2 activin receptor mRNAs, and their encoded proteins in ovarian follicles from PCOS patients suggested that an increase in the availability of activin, relative to inhibin, was actually present in the arrested follicles in PCOS patients (316). Moreover, the location of the mRNAs of the follistatin subunits is not altered in PCOS (317).
Initial molecular genetic studies in humans by Urbanek et al. (132) suggested evidence for linkage between the follistatin locus and PCOS, as cited above. Subsequent studies by these authors (318) have failed to confirm the involvement of the follistatin gene with PCOS in a large multiethnic study. The coding regions and some introns of the follistatin gene were sequenced, disclosing at least 17 polymorphisms; however, 16 of them were rare, making a significant contribution of these variants to the pathogenesis of hyperandrogenism unlikely (318). Moreover, the only common polymorphism found, located in exon 6 but not translated, was not associated with PCOS when correcting for multiple testing, and the authors concluded that contributions to the etiology of PCOS from the follistatin gene, if any, are probably small (318).
In conceptual agreement, no mutations in the follistatin gene have been found in Chinese PCOS patients defined by menstrual dysfunction, hyperandrogenism, and polycystic ovaries (319), and the only mutation found in our series of patients from Spain, a silent G951A variant, was equally distributed in PCOS patients and in healthy women (320).
2. Thrombophilic factors.
Women with PCOS have increased miscarriage rates, as happens in women with inherited thrombophilic conditions. The secretion of plasminogen activator inhibitor-1 (PAI-1) in adipose tissue is enhanced by inflammatory cytokines and by insulin (321, 322, 323). In agreement, the increased circulating PAI-1 levels found in PCOS patients decrease after treatment with insulin sensitizers (324). Homozygosity for 4G alleles of the –675 4G/5G polymorphism in the gene encoding PAI-1, which modulates PAI-1 activity, have been reported in association with obesity (325) and PCOS (326). We have found a similar trend in our series on Spanish women, although the difference in allele frequencies did not reach statistical significance, possibly because of small sample size (229).
Other genetic abnormalities associated with thrombophilia have been studied in PCOS. Tsanadis et al. (327) recently reported that the prevalence of antithrombin III, protein S and protein C deficiencies, factor V Leiden, prothrombin G20210A factor, and methylene tetrahydrofolate reductase 677C/T mutations is not increased in Greek patients with polycystic ovaries, menstrual dysfunction, and hyperandrogenism when compared with nonhyperandrogenic controls. Similar results have been found for the methylene tetrahydrofolate reductase variant in Italian women (328).
3. Microsomal epoxide hydrolase.
Two SNPs, Tyr113His and His139Arg, in the gene encoding the detoxifying enzyme microsomal epoxide hydrolase have been studied in women with PCOS, defined by the presence of polycystic ovaries and hyperandrogenic symptoms (329). Although none of the polymorphisms was associated with PCOS, the presence of the His113-Arg139 haplotype was associated with an odds ratio for PCOS of 2.28 and 95% confidence interval of 1.1–4.8 (329). However, it is unclear how changes in the activity of this enzyme might relate to functional hyperandrogenism and PCOS, and therefore, this result should be considered with caution unless confirmed in future studies.
4. Bone morphogenetic proteins.
The intraovarian bone morphogenetic protein system is involved in the control of granulosa cell proliferation and cytodifferentiation and plays a role in oocyte development (330). The genes encoding the growth differentiation factor 9 and bone morphogenetic protein 15 have been studied in Japanese women with polycystic ovaries, but no missense mutations have been found in these women (331).
VI. Hyperandrogenism, PCOS, and Survival Advantage
The increasing prevalence of complex metabolic disorders in developed countries raised the possibility that, from an evolutionary perspective, the pathogenetic mechanisms underlying these disorders might have provided survival advantages (332). However, these previously beneficial mechanisms may lead to disease with prolonged life expectancy or when these subjects are exposed to the present lifestyle in Western countries.
As suggested by Witchel et al. (333) for congenital adrenal hyperplasia, which is one of the most common inherited disorders with carrier frequencies of approximately 10% in all world populations studied to date and a relatively common cause of hyperandrogenism in children and adults, the presence of hyperandrogenism in women might have provided survival advantage for these women and their children.
The rapid maturation of the reproductive axis found in these subjects, together with the increase in assertive behavior resulting from increased androgen secretion, might be advantageous during times of environmental stress (333, 334, 335). Moreover, the relative infertility of these women could increase the interval between pregnancies, decreasing the birth rate and favoring maternal and infant survival (333). In addition to the lower rate of pregnancies secondary to oligoovulation, pregnancies may also occur at an older age, favoring the survival of these women. In agreement, an early beginning of fecundity is associated with higher mortality rates in animal models (335).
Yet survival advantage may also contribute to explaining the association of hyperandrogenism with other disorders related to insulin resistance. Human metabolism may be genetically adapted to the dominant conditions that have predominated for ages: near-continuous physical activity, a diet rich in carbohydrates and proteins yet poor in fat, and long periods of famine or food shortage (336, 337, 338). Survival was therefore favored by a combination of thrifty genotypes and phenotypes, in which insulin resistance played a central role (Fig. 2).
Insulin resistance increases glucose availability for brain metabolism. It also increases salt and water retention and sympathic tone and induces endothelial dysfunction, favoring an increase in blood pressure, obviously beneficial when trauma occurs. Similarly, the increased coagulability and decreased fibrinolysis associated with insulin resistance are defensive mechanisms against bleeding. But more important is that insulin resistance favors obesity, protecting against starvation, and obesity contributes to a proinflammatory state through the secretion of several cytokines, contributing to the defense against infection, and possibly to the development of functional hyperandrogenism and PCOS.
When the environmental conditions change and access to food is not restricted, significant trauma and epidemics seldom occur, and life expectancy increases markedly, these defensive mechanisms are no longer beneficial, and the price to pay is atherosclerosis and cardiovascular disease (Fig. 2).
For the reasons outlined above, it is not surprising that the genomic variants associated with obesity, type 2 diabetes mellitus, and other disorders in which insulin resistance plays a major role are frequently associated with functional hyperandrogenism and PCOS. However, genomic variants-associated insulin resistance may contribute to hyperandrogenism only indirectly, by inducing insulin resistance and hyperinsulinemia and/or by direct actions at the adrenal or the ovary. For example, TNF- induces insulin resistance by interfering with IRS-1-mediated insulin signaling (280) and also has reproductive actions that closely resemble those found in patients with PCOS and functional hyperandrogenism, as summarized above (293, 294).
Therefore, the precise elucidation of the mechanisms underlying these associations requires studies of the functional consequences of the genomic variants associated with hyperandrogenism in different target tissues, especially when some of the genomic variants appear to facilitate insulin resistance and hyperandrogenism, whereas others may protect against these disorders.
At present, the emerging picture for the molecular genetic mechanisms leading to functional hyperandrogenism and PCOS is that of a complex interaction between predisposing and protective genomic variants, and the strong impact of modifying environmental factors including diet, exercise, and lifestyle (Fig. 3).
VII. Explanations for the Lack of Reproducible Association of Hyperandrogenism and PCOS with Molecular Genetic Abnormalities and Genomic Variants
To date, most of the associations between genomic variants and functional hyperandrogenism or PCOS failed to replicate when studied in different populations. An immediate consideration is that most studies conducted to date have been modest with regard to the number of subjects included. A small sample size, leading to a lack of statistical power to detect the modest effects that genomic variants possibly play in the pathogenesis of a complex disorder such as functional hyperandrogenism, might explain the negative results found in many cases when efforts have been made to confirm previous studies. Although at present there are no doubts about the need of studies with large sample sizes, lack of consistency of the reported findings is not limited to hyperandrogenism and is a recurrent problem in the study of many complex metabolic disorders (339). Therefore, other factors might also contribute to the discrepancies observed when studying candidate genes for hyperandrogenism in different populations, summarized below.
A. Ascertainment issues
One of the essential requirements for the effectiveness of molecular genetic studies is a clear definition of the phenotype under study. This has not been the case for functional hyperandrogenism and PCOS. As an example, the linkage studies cited above (140, 222) of CYP11A and INS in women from the United Kingdom relied mostly on the use of ultrasonography for the diagnosis of PCOS, and this series included "ovulatory" PCOS patients. On the contrary, a linkage study conducted in the United States relied on the NICHD criteria (132), in which chronic anovulation is strictly required for the diagnosis of PCOS (9). Given the considerable differences in the criteria used to define the phenotype, it is unlikely that the genes associated with the disease would be the same in both studies.
Moreover, the spectrum of disorders covered by the criteria derived from the Rotterdam Consensus Workshop (11, 12) is even broader than those included with the NICHD definition: PCOS would be diagnosed not only in women presenting with hirsutism, hyperandrogenemia, and anovulation, but also in patients presenting with anovulation and polycystic ovaries but no clinical and/or biochemical evidence of hyperandrogenism. Therefore, these revised criteria are unlikely to be of significant advantage over previous ones with respect to molecular genetic studies. And to further complicate the search for PCOS-related genomic abnormalities in family-based studies, the male PCOS phenotype is still uncertain.
Because of the heterogeneous and syndromic nature of PCOS, it would be more helpful for molecular genetic studies to use criteria oriented toward the identification of specific clinical, reproductive, and metabolic traits associated with hyperandrogenism. This would require an important effort to identify and especially to standardize the methods used to identify these traits in large populations such as those needed for family-based or case-control designs.
Instead of trying to reach a consensus on how to diagnose precisely a disorder that is heterogeneous by definition, the practical issue would be to standardize the definitions and methods used to identify specific traits such as insulin resistance, overweight, visceral adiposity, dyslipidemia, hirsutism, acne, oligoovulation, and many others that characterize or are frequently found in hyperandrogenic women.
Given the apparently multigenic etiology for functional hyperandrogenism and PCOS, this approach would permit a more precise definition of the kind of hyperandrogenic patients included in the different molecular genetic studies. It would facilitate well-sized multicollaborative studies and thereby increase the probability of success in identifying genomic variants and abnormalities related to these particular traits that might be used as diagnostic and/or therapeutic targets in the future. It is hoped that scientific societies that address the study of hyperandrogenism, such as the recently created Androgen Excess Society (http://www.androgenexcesssociety.org), will serve as an adequate forum where these efforts in standardization could be made.
In addition to the problems of defining the PCOS phenotype, sampling issues also should be considered. For family-based studies, a common sampling procedure is to collect from an affected proband with at least one or two affected relatives, instead of the ideal, but rarely performed, procedure of selecting families from the population at random (340). Nonrandom ascertainment makes it more likely that families with multiple PCOS members enter the study than families with no affected relative, resulting in biased heritability estimates (340, 341). Another common source of error is that the affected status in relatives of the proband is typically determined on the basis of his or her report because of constraints in time and resources (340). Moreover, affected subjects may be more likely to be aware of the diagnosis in their relatives than nonaffected subjects (340), or more likely to misinterpret the symptoms of their relatives (340), such as considering the presence or absence of hirsutism as the equivalent of having or not having PCOS.
B. Involvement of environmental factors
The interactions between genetic and environmental factors are essential for the comprehension of the pathogenesis of common complex disorders (342). The precise knowledge of these interactions requires long-term studies analyzing the impact of different environmental factors in specific subgroups of patients, because controlling all the confounding environmental variables is extremely difficult (342).
Environmental factors such as weight gain may trigger the development of PCOS in predisposed women, as occurs in other complex metabolic disorders in which insulin resistance plays a major role. The identification and precise delimitation of the contribution of the environmental influences triggering the development of functional hyperandrogenism and PCOS may direct the search for the specific proteins and/or genomic variants involved to the metabolic pathways influenced by these environmental factors.
However, the environmental factors contributing to complex metabolic disorders may change depending on the population studied, because diet, exercise, and lifestyle have wide ethnic variations. Therefore, it should not be surprising that the genomic abnormalities contributing to these disorders may also change depending on the environmental conditions (i.e., the genes contributing to PCOS in obese sedentary women from a Western country are probably different from those involved in the PCOS phenotype of lean women from the Mediterranean area or from Asia). Finally, to further complicate the study of complex metabolic disorders, the phenotype changes during the life of the affected subjects as age advances. Therefore, different phenotypic traits may not be present when these women were phenotyped, but became apparent later in life, constituting one more confounding factor that is especially difficult to control in molecular genetic studies.
C. Possible polygenic etiology for functional hyperandrogenism and PCOS
It has been suggested that the phenotypic heterogeneity observed in PCOS patients, even within the same family, could be attributed to the interaction of a small number of genes with one another and with environmental factors (18, 22, 33). Furthermore, given the large number of genomic variants found associated with functional hyperandrogenism and PCOS to date, the emerging picture may be that of a complex metabolic disorder resulting from small predisposing or protecting effects arising from the interaction of multiple genomic variants and several environmental factors.
Even if a more precise definition for hyperandrogenic phenotypes was used, and that has not been the case, the classic requisite of replication of linkage or association of a genomic abnormality with monogenic diseases may not be applicable for complex disorders because the functional consequences of the genomic variant may be only apparent in certain populations or when a particular environmental factor is present (339). However, it is important that internal replication of findings is provided in larger series, because this might reveal false-positive associations reported in preliminary studies when limited sample sizes were studied.
Nowadays, it is technically feasible to genotype large series of individuals for multiple genomic variants, allowing whole-genome scans and high-throughput candidate gene analysis. These approaches will disclose reliable information of the relative contribution of these genomic variants to functional hyperandrogenism and PCOS and will facilitate the study of gene to gene and gene-environment interactions that probably contribute to the development of these prevalent disorders.
Finally, to avoid spurious associations from being considered causative of any disease-associated trait, every effort should be made to demonstrate in vivo or in vitro a functional consequence of the associated genomic variant that might reasonably account for the contribution of the variant to the disorder or its associated traits.
D. Limitations of the genetic techniques used to date
As discussed previously, the molecular genetic studies regarding PCOS and functional hyperandrogenism conducted to date had important limitations, especially because adequately sized whole-genome scans and large case-control association studies are still lacking, and therefore associations with genomic variants that have small effects on hyperandrogenism might have been missed by these studies.
Another source of confusion is the fact that quality control of genetic data has not been as strict as that applied to other methods used in clinical research or even in routine clinical practice. Genetic data are usually obtained from a single measure, and reliability and reproducibility of these analyses might be a problem, because genotyping errors may severely bias the estimates of genetic studies (339, 343). Although departure from the Hardy-Weinberg equilibrium may be useful for the detection of these genotyping errors (344), and although modern methods incorporate models of typing error (343, 345), efforts in standardization of these techniques should be made in the future (339).
VIII. Future Perspective: Functional Hyperandrogenism and PCOS in the Age of "Omics"
The suffix "omics" is being applied to recent technologies that are exponentially increasing our knowledge of human biology. Perhaps at present the most developed one is genomics.
Genomics aims to map, sequence, and analyze all the genes and their products in the genome, with the final intention of providing the complete and accurate DNA sequence of an organism (56, 346). The number of genes in the human genome is in the order of 35,000, and because of exon shuffling, alternative splicing, and posttranslational modifications, as many as 100,000 different proteins may be encoded in the human genome. Therefore, identification of all the genes and proteins of the human organism and their interactions requires an enormous effort.
Genomics is in constant evolution, and more specific subspecialties are being developed, such as functional genomics. Its aim is the identification of the biological functions of genes and their products, and how they interact with the environment in health and disease (347). To date, functional genomics has contributed to unravel the mechanisms of many diseases (348, 349, 350), and the genomic approach might be especially adequate for the study for complex polygenic disorders, given that traditional molecular genetic approaches have not been successful to date (351).
Genomic techniques, such as differential gene expression analyzed by DNA microarrays, allow the identification of genes that are differentially overexpressed or suppressed in patients compared with controls. This approach has the advantage over molecular genetic analyses in that the result integrates the presence of molecular genetic abnormalities with both gene-gene and gene-environment interactions. Therefore, the genes identified can be considered potential candidates to explain the disorder and also potential diagnostic and therapeutic targets. Recently, comparison of gene expression in cultured theca cells from PCOS patients and controls using DNA microarrays identified the genes encoding aldehyde dehydrogenase 6 and retinol dehydrogenase 2 as candidate genes for PCOS (352). These factors play a role in all-trans-retinoic acid biosynthesis and the transcription factor GATA6, which increase the expression of 17-hydroxylase (352), a characteristic of PCOS theca cells (137). In the near future, undergoing studies using DNA microarrays to compare the expression profiles of tissues such as adipose tissue and muscle, which are essential for the development of insulin resistance, will undoubtedly contribute to the identification of candidate genes in hyperandrogenic women.
But given that gene expression and protein concentration and activity are poorly correlated (353), modern techniques have been developed to study the "proteome" of a cell, tissue, or organism. Proteomics allows large-scale analysis of proteins, including their relative abundance, distribution, posttranslational modifications, functions, and interactions with other molecules (347). For example, it is possible to examine the expression of more than 1000 proteins by coupling mass spectrometry technology with several separation methods. Proteomic analysis of tissues involved in the pathogenesis of functional hyperandrogenism and PCOS, such as ovarian theca and granulosa cells, adrenal cortex, sc and omental adipose tissue, and muscle, are essential for our understanding of the complex interactions between the genome and the environment that underlie these disorders.
Ultimately, the identification of genes and proteins related to functional hyperandrogenism and PCOS, and their interactions with the environment, will be an essential step for the development of more precise diagnostic techniques, the identification of new therapeutic targets, and the identification of particular individuals that, because of their genetic background, may be predisposed to certain complications of the syndrome or respond differently to available treatments.
IX. Summary
Functional hyperandrogenism and PCOS appear to be complex multigenic disorders, arising from the interaction of predisposing and protective genetic variants that might have been selected during evolution because of a previous survival advantage, with environmental influences that play an important role in the expression of the hyperandrogenic phenotype.
Among others, genomic variants in genes pertaining to the regulation of androgen biosynthesis, insulin resistance, metabolic syndrome, and proinflammatory genotypes are involved in the genetic predisposition to functional hyperandrogenism and PCOS. Progress in this area requires adequately sized multicenter collaborative studies in which modifying environmental factors such as ethnicity, diet, and lifestyle are identified with precision.
In the future, classic molecular genetic techniques such as linkage analysis in the form of a whole-genome scan and large case-control studies, as well as modern genomic and proteomic approaches, will hopefully provide new insights into the pathogenesis of functional hyperandrogenism and PCOS, with the ultimate aim of improving the prevention, diagnosis, and treatment of these prevalent disorders.
Acknowledgments
This work was supported by Grants FIS 02/0741 and RGDM G03/212 from the Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, and by Grant CAM 08.6/0010/2001 from the Consejería de Educación, Comunidad de Madrid, Spain.
Footnotes
First Published Online November 23, 2004
Abbreviations: AR, Androgen receptor; CRP, C-reactive protein; CYP, cytochrome P450; gp130, gp130 subunit of IL-6 receptor; HSD, hydroxysteroid dehydrogenase; INS, insulin gene; INSR, insulin receptor gene; IRS, insulin receptor substrate; LH?, ?-subunit of LH; PAI-1, plasminogen activator inhibitor-1; PCOS, polycystic ovary syndrome; PON1, paraoxonase; PPAR-2, peroxisome proliferator-activated receptor-2; SORBS1, human homolog for the sorbin and SH3-domain-containing 1 gene; SNP, single nucleotide polymorphism; SRD5A, steroid 5-reductase; TNFR2, type 2 TNF receptor; VNTR, variable number of tandem repeats.
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Abstract
The genetic mechanisms underlying functional hyperandrogenism and the polycystic ovary syndrome (PCOS) remain largely unknown. Given the large number of genetic variants found in association with these disorders, the emerging picture is that of a complex multigenic trait in which environmental influences play an important role in the expression of the hyperandrogenic phenotype.
Among others, genomic variants in genes related to the regulation of androgen biosynthesis and function, insulin resistance, and the metabolic syndrome, and proinflammatory genotypes may be involved in the genetic predisposition to functional hyperandrogenism and PCOS.
The elucidation of the molecular genetic basis of these disorders has been burdened by the heterogeneity in the diagnostic criteria used to define PCOS, the limited sample size of the studies conducted to date, and the lack of precision in the identification of ethnic and environmental factors that trigger the development of hyperandrogenic disorders. Progress in this area requires adequately sized multicenter collaborative studies after standardization of the diagnostic criteria used to classify hyperandrogenic patients, in whom modifying environmental factors such as ethnicity, diet, and lifestyle are identified with precision.
In addition to classic molecular genetic techniques such as linkage analysis in the form of a whole-genome scan and large case-control studies, promising genomic and proteomic approaches will be paramount to our understanding of the pathogenesis of functional hyperandrogenism and PCOS, allowing a more precise prevention, diagnosis, and treatment of these prevalent disorders.
I. Introduction
II. Evidence Suggesting a Genetic Origin for Functional Hyperandrogenism and PCOS
A. Familial aggregation
B. Male phenotype
C. Twin studies
D. Environmental and other confounding factors
III. Classic Techniques Used in Molecular Genetic Studies
A. Linkage analysis
B. Case-control studies
IV. Studies in Pediatric and Adolescent Hyperandrogenism
V. Studies in Hyperandrogenic Adults
A. Genes involved in androgen biosynthesis, transport, and action, and their regulation
B. Genes involved in insulin resistance and associated disorders
C. Proinflammatory genotypes
D. Other candidate genes
VI. Hyperandrogenism, PCOS, and Survival Advantage
VII. Explanations for the Lack of Reproducible Association of Hyperandrogenism and PCOS with Molecular Genetic Abnormalities and Genomic Variants
A. Ascertainment issues
B. Involvement of environmental factors
C. Possible polygenic etiology for functional hyperandrogenism and PCOS
D. Limitations of the genetic techniques used to date
VIII. Future Perspective: Functional Hyperandrogenism and PCOS in the Age of "Omics"
IX. Summary
I. Introduction
POLYCYSTIC OVARY SYNDROME (PCOS) is one of the most common endocrinopathies in women of childbearing age (1). PCOS is characterized by increased ovarian and adrenal androgen secretion (2); hyperandrogenic symptoms such as hirsutism, acne, and/or alopecia; menstrual irregularity; and, in a significant proportion of patients, insulin resistance (3).
The presence of male sexual secondary characteristics in women has been recognized from ancient times, but it was not until 1921 when Achard and Thyers (4) reported the association of hyperandrogenic symptoms with abnormalities in glucose metabolism, highlighting the presence of polycystic ovaries in some of their patients. However, only after the description of seven cases of amenorrhea and bilateral polycystic ovaries by Stein and Leventhal in 1935 (5) was PCOS considered a separate entity that interested clinicians and researchers worldwide.
Although for many years the interest in PCOS has been focused on the cutaneous and reproductive manifestations of this disorder, the recent evidence suggests that metabolic and cardiovascular risk factors cluster in these patients (6, 7, 8). This evidence has renewed research efforts on hyperandrogenism and PCOS, including those directed toward the identification of the genetic and environmental factors involved in the pathogenesis of these prevalent conditions.
At present, there is no consensus on the criteria for the diagnosis of PCOS (Table 1). Most clinicians and researchers from the United States and from southern Europe use the criteria derived from the conference held at the National Institute of Child Health and Human Development (NICHD) in 1990: clinical and/or biochemical hyperandrogenism, menstrual dysfunction, and exclusion of specific etiologies (9). According to these criteria, the presence of polycystic ovaries on ultrasound examination is not needed for the diagnosis. On the contrary, most specialists from the other European countries, Asia, and Oceania rely mostly on the presence of polycystic ovaries on ultrasound examination for this diagnosis, whereas menstrual dysfunction is not required (10). A recent consensus workshop held in The Netherlands in 2003 under the auspices of the European Society for Human Reproduction and Embryology and the American Society for Reproductive Medicine proposed a revision of the criteria for the diagnosis of PCOS, of which two of the following three would be needed: 1) oligo- and/or anovulation; 2) clinical and/or biochemical signs of hyperandrogenism; and 3) polycystic ovaries, together with the exclusion of other etiologies such as congenital adrenal hyperplasia, androgen-secreting tumors, or Cushing’s syndrome (11, 12).
However, the term PCOS as used in the literature is not specific and has been applied to a wide range of hyperandrogenic women, including women with or without menstrual dysfunction or polycystic ovaries. For that reason, in the present review, we will use the more general term functional hyperandrogenism to define these patients presenting with androgen excess, including those diagnosed with PCOS according to any of the current definitions of the syndrome, in whom a specific etiology such as congenital adrenal hyperplasia or androgen-secreting tumors cannot be identified, and, whenever possible, we will describe the particular characteristics of the patients included in the different studies. Of note, we will not include idiopathic hirsutism in this definition, because this infrequent disorder, in which hyperandrogenemia, menstrual dysfunction, and ultrasonographic polycystic ovaries are not present, appears to be a separate entity in which increased skin activity of 5-reductase (resulting in increased conversion of testosterone into the more potent androgen dihydrotestosterone), androgen receptor (AR) polymorphisms, and altered local androgen metabolism contribute to the development of hirsutism (13).
Functional hyperandrogenism and PCOS cluster in first-degree relatives of patients (14) and are inherited together with insulin resistance and metabolic disorders (15, 16). During the past decades, the inheritance of these disorders has been the subject of intense research (17, 18, 19), but many questions remain unanswered.
The pattern of inheritance is still unknown. Initially, an autosomal dominant model, including premature balding as the male phenotype, was proposed (20, 21), but later studies did not confirm this hypothesis. The heterogeneity of the populations, the large number of candidate genes studied to date (22), and the difficulty inherent to identifying the molecular genetic mechanism leading to a complex metabolic disorder such as PCOS, in which environmental factors play a major role (23, 24), provide an explanation for the fact that the molecular genetic basis of functional hyperandrogenism and PCOS remains largely unknown despite significant efforts.
The purpose of this review is to provide a systematic evaluation of the studies conducted to date in functional hyperandrogenism and PCOS and to suggest priorities and new strategies that may contribute to understanding the pathogenesis of these disorders.
II. Evidence Suggesting a Genetic Origin for Functional Hyperandrogenism and PCOS
A. Familial aggregation (Table 2)
The familial aggregation of PCOS, hyperandrogenemia, and associated metabolic abnormalities suggests a genetic origin for functional hyperandrogenism and PCOS. Back in 1968, Cooper et al. (25) studied the families of 18 Caucasian women with polycystic ovaries and clinical and biochemical traits associated with PCOS, and the families of 18 paired control women. The incidence of oligomenorrhea and polycystic ovaries was increased in first-degree relatives of PCOS patients compared with controls, and males in these families had increased hairiness according to a questionnaire, suggesting an autosomal dominant pattern of inheritance (25). Givens and colleagues (26, 27, 28) published a series of family-based studies in patients presenting with hirsutism, oligomenorrhea, and increased ovarian size, and they found familial aggregation of hyperandrogenic symptoms (hirsutism and oligomenorrhea) and of metabolic disorders (diabetes mellitus, dyslipidemia, arterial hypertension, and atherosclerosis). These authors suggested both a maternal and paternal pattern of inheritance, in which the latter showed higher penetrance and expression (26, 27, 28). Of note, in some of these male subjects, oligospermia and increased LH secretion were found pointing to an X-linked pattern of inheritance.
Ferriman and Purdie (29) studied first-degree relatives of hirsute women presenting with or without enlarged ovaries in gynecography. Compared with a control group, patients had an increased prevalence of oligomenorrhea and infertility, and the prevalence of hirsutism was increased in their female first-degree relatives. Hague et al. (30) studied the families of 61 patients with ultrasonographic polycystic ovaries and hyperandrogenic symptoms and found that 67% of the mothers and 87% of the sisters of probands were affected. Lunde et al. (31) studied the families of 132 Norwegian women with polycystic ovaries previously treated by ovarian wedge resection, who also had two or more of the following symptoms: menstrual irregularity, hirsutism, infertility, and/or obesity. A control group of 71 women and their families was used for comparison (31). Clinical manifestations of hyperandrogenism were found in 31.4% of the female relatives of the patients compared with only 3.2% of the female relatives of the controls (31). Among male relatives, premature balding and increased hairiness were found in 19.7% of the relatives of the patients, but only in 6.5% of the relatives of the controls (31). Azziz and colleagues (14, 32), using NICHD criteria for the diagnosis of PCOS, found that PCOS was present in 35% of the mothers and 40% of the sisters of PCOS patients.
In addition to hyperandrogenism, insulin resistance clusters in the families of hyperandrogenic women. Legro et al. (33), also using also NICHD criteria for the definition of PCOS, found that 22% of the sisters of patients actually had PCOS, whereas a further 24% of the sisters of these patients presented with hyperandrogenemia and regular menstrual cycles. This bimodal pattern suggested a monogenic defect for hyperandrogenism (33). Additional studies from Legro et al. (34) recently demonstrated that in these families, insulin resistance is associated with hyperandrogenemia rather than with menstrual dysfunction. This suggests that insulin resistance and hyperandrogenemia share the same pathogenic mechanisms, whereas the presence of menstrual dysfunction needed for the diagnosis of PCOS may be only a matter of degree. Therefore, of PCOS sisters, those with PCOS and those presenting with hyperandrogenemia and regular menstrual cycles may actually have functional hyperandrogenism.
The finding of an increased prevalence of insulin resistance in families of PCOS patients has been replicated in the Turkish population. Yildiz et al. (35) recently studied 102 relatives of 52 PCOS patients defined by NICHD criteria and found that, compared with different population-based control groups matched for sex, age, and pre- or postmenopausal status with the relatives of PCOS patients, insulin resistance and disorders of carbohydrate metabolism were more frequent in the mothers, sisters, and brothers of PCOS patients, and that the mothers and sisters of PCOS women had increased serum androgen levels compared with the controls.
B. Male phenotype
The lack of a clearly defined male phenotype in families of PCOS patients has burdened the progress in the search for the genetic origin of functional hyperandrogenism and PCOS. Although initial studies suggested that male pattern premature balding was the male equivalent for PCOS (20, 29, 31, 36, 37), these findings have not been universally confirmed (38). More recently, increased serum dehydroepiandrosterone sulfate concentrations in the brothers of PCOS patients described above (39) and insulin resistance in the fathers and brothers of PCOS women (35) have been proposed as the male phenotype in PCOS families. However, there was a considerable overlap between male PCOS relatives and controls in serum dehydroepiandrosterone sulfate levels and indexes of insulin resistance, and additional studies are needed to establish the actual usefulness of these abnormalities as markers of the syndrome in male relatives of PCOS patients.
C. Twin studies
There are very few data regarding PCOS in twins. Jahanfar et al. (40) studied 34 (19 monozygotic and 15 dizygotic) twin pairs, in which PCOS diagnosis was based on ultrasonographic and biochemical findings. Although 11 pairs were discordant for the presence of polycystic ovaries, model-fitting analysis suggested that fasting insulin level, serum androstanediol glucuronide, and body mass index were significantly influenced by genetic factors (40). A later report of this study suggested a genetic origin for an unfavorable lipid profile, especially for increased circulating concentrations of lipoprotein (a), but only in twin pairs concordant for polycystic ovaries (41). These data point to a genetic component in the metabolic abnormalities associated with PCOS but suggest a polygenic etiology in which environmental influences play a significant role.
D. Environmental and other confounding factors
Familial aggregation of hyperandrogenism and PCOS strongly suggest a genetic origin for these disorders, but another possibility is that clustering of PCOS within families results from nongenetic inheritance related to certain environmental factors that are present in the affected families, and not in the families of unaffected women.
As exemplified in Fig. 1, insults during pregnancy may induce intrauterine growth retardation, which has been proposed to induce a thrifty phenotype in small for gestational age babies. These babies are predisposed to suffer from insulin resistance, which may result in hypertension, glucose intolerance, adrenal axis hyperactivity with relative cortisol excess, functional hyperandrogenism, and PCOS (42) later in life, especially when these individuals are exposed to environmental factors such as a sedentary lifestyle and a diet rich in saturated fat. These environmental factors may cluster in certain families because exercising and dieting are heavily influenced by parental habits.
The metabolic abnormalities associated with the thrifty phenotype can induce additional insult to the pregnancies of small for gestational age women, and the defect might be transmitted to another generation without the participation of any genetic abnormality. Therefore, the presence of more than one small for gestational age baby in a family may result from exposure of the mothers and their fetuses to the same unfavorable environmental conditions, rather than being related to genetic factors.
Yet it is important to highlight, as shown in Fig. 1, that if small for gestational age babies have healthy habits, insulin resistance and its consequences might be ameliorated, and, at least in theory, their fetuses will not be exposed to an unfavorable metabolic milieu during pregnancy, preventing nongenetic inheritance of these conditions. Nevertheless, intrauterine growth retardation may also be influenced by genetic variants, and the most probable scenario is that of an interaction between predisposing genetic abnormalities with unfavorable environmental conditions. Of note, even the influence of intrauterine growth restriction on the development of PCOS is debatable (43, 44, 45), considering that the presence of polycystic ovaries also has been related to above-average birth weight in babies born from obese, androgenized mothers (43, 44, 45).
There are very few data on the influence of environmental factors on the development of hyperandrogenism and PCOS, other than the triggering role for obesity on the development of these disorders (46). Although the prevalence of PCOS is similar in different countries, ethnic factors influence the clinical manifestations of the syndrome. In Caucasian premenopausal women, the reported prevalences of PCOS defined by NICHD criteria ranged from 4.7% in Alabama (47) to 6.5% in Spain (48) and 6.8% in the Greek island of Lesbos (49). Moreover, the prevalence in African-American women from Alabama (3.4%) was comparable with that in Caucasian women (47).
However, ethnicity influences the manifestations of PCOS. Carmina et al. (50) compared the clinical histories, physical examinations, ovarian morphology by ultrasound, insulin sensitivity, serum gonadotropin, and steroid profiles in 25 Japanese, 25 Italian, and 25 Hispanic-American women with PCOS. The three groups were homogeneous in terms of serum testosterone and adrenal androgen concentrations, insulin resistance, and polycystic ovaries, but Japanese women were less obese and did not present with hirsutism (50).
Dunaif et al. (51) found that insulin resistance is higher in Hispanic-Caribbean women with PCOS defined by NICHD criteria compared with Caucasian patients and with control women paired for age, weight, body composition, and ethnicity. Williamson et al. (52) recently reported a higher incidence of obesity, dyslipidemia, and infertility, as well as a higher degree of insulin resistance in Maori and Pacific Islander PCOS patients, defined by ultrasonography and clinical symptoms, compared with European patients. In the United Kingdom, the prevalence of polycystic ovaries and type 2 diabetes is increased in Indian subcontinent Asian women (53). Compared with Caucasian women, PCOS patients of southeastern Asian origin presented with oligomenorrhea and were diagnosed at younger ages; hirsutism, acne, acanthosis nigricans, and subfertility were more prevalent, and patients were more insulin resistant (54).
These ethnic differences in the clinical presentation of PCOS may be related to environmental factors such as diet, exercise, and lifestyle. Of note, even the content of polyunsaturated or monounsaturated fatty acids in the diet may influence the metabolic manifestations of the syndrome (55). Thus, because of differences in the environmental factors triggering the development of PCOS, the genes contributing to PCOS patients may also be different, depending on the population studied. These considerations may also apply to the discrepant results of previous studies regarding the association of genomic variants with PCOS.
III. Classic Techniques Used in Molecular Genetic Studies
Before reviewing the molecular genetic studies conducted to date in functional hyperandrogenism and PCOS, we will provide a brief description of the techniques usually used for molecular genetic studies, focusing on the particular advantages and disadvantages of each technique.
A. Linkage analysis
Linkage analysis is a family-based molecular genetic technique that has permitted the identification of the genetic abnormalities leading to many monogenic Mendelian disorders (56). However, most complex metabolic disorders result from the interaction between multiple genes and environmental factors, making their identification much more difficult than in disorders with a Mendelian pattern of inheritance (57, 58, 59, 60, 61, 62, 63).
In linkage analysis, markers frequently located at intergenic regions of DNA are studied in multiple members of affected families. The cosegregation of alleles with a disease phenotype is studied, and the linkage of the disease with any marker focuses the search for genomic abnormalities to the DNA regions close to the polymorphic marker segregating with the disorder.
Linkage analysis is based on the meiotic recombination that occurs during ovogenesis and spermatogenesis. The closer two genomic markers are, the more seldom these markers are separated during meiotic recombination. The probability of recombination between two markers is an estimation of the distance between them, and is expressed in centimorgans (cM). Two markers are defined to be 1 cM apart if they become separated by recombination in 1% of meioses. One centimorgan is equivalent on average to 106 bp of physical distance. The markers usually studied in linkage studies include microsatellites and single nucleotide polymorphisms (SNPs).
Classical or parametric linkage analysis requires the specification of a model for the disease or trait a priori, in terms of allelic frequencies, penetrance, and pattern of inheritance, and the result is expressed by the LOD score (64). The LOD score is the base 10 logarithm of the odds that two markers (one being the disease-causing or disease-associated genomic variant) are truly linked, divided by the odds that the observed set of data may result from chance if the markers are unlinked. Usually, significant positive proof of genetic linkage requires a LOD score of 3.0 or greater, corresponding to odds favoring linkage of at least 1000:1 at a given specified recombination frequency. Because the pattern of inheritance of functional hyperandrogenism and PCOS is complex and uncertain, nonparametric or model-free linkage analysis is required to ensure the power to detect linkage (22). In nonparametric linkage analysis, multiple DNA markers are obtained from siblings and, if possible, from their parents, and allele sharing between relatives is investigated (64). Allele sharing is defined as identity-by-state (having the same DNA sequence) or as identity-by-descent (when two alleles come from the same ancestral allele) (64). Nonparametric linkage scores use a similarity statistic defined by the average of the possibilities that relatives are identity-by-state (64). However, the number of meioses assessed, disease penetrance, knowledge of the marker coupling phase, and other relevant variables must be considered before reaching a clinically significant conclusion (65).
There are several factors that may limit the usefulness of linkage studies including, among others, the uncertainty about the pattern of inheritance, a variable penetrance of the genetic defect, the delay in the debut of the disease, possible errors in clinical diagnosis, gene to gene interactions, interaction with environmental factors, and the limited resolution of these techniques. The sample size needed to detect a particular effect varies inversely with the square of the effect (62), and therefore genes that contribute modestly to the phenotype may be missed because of the extremely large sample size needed to demonstrate the association. This is especially important given that PCOS affects women in reproductive age and is associated with reduced fertility, limiting the pedigrees and the generations studied to two (mothers and daughters) in most of the linkage analyses performed to date, resulting in small sample sizes within families (66, 67).
The resolution of linkage analysis may be improved using tests such as affected sibling-pair identity by descent analysis or the transmission disequilibrium test (68), allowing the detection of genes of modest effects that might be otherwise missed by linkage analysis. The transmission disequilibrium test involves genotyping the parents of the proband and the proband, and it determines whether or not the parent heterozygous for the putative allele transmits it more often to their affected children than other allele(s). These tests are frequently used to confirm the linkage already established but may also be useful when used without previous evidence for linkage (69).
Linkage with a polymorphic marker only serves to focus the search of the genomic abnormality associated with the disorder to an area of approximately 1–2 cM, and locating the precise genetic defect may involve studying as many as 30–100 genes in the linked area. After obtaining linkage with a genomic region, the search of the particular genomic abnormality involved can be accomplished by fine-mapping of that region using additional markers, and by a candidate gene approach searching for variants in biologically appealing genes located there. This approach has been facilitated by the growing number of genes identified all across the genome, but frequently the demonstration that a gene has a causative role in a disease requires direct sequencing of the whole gene in affected and nonaffected individuals (70).
B. Case-control studies
These studies look for specific genetic markers or alleles that are more frequent in affected individuals (cases) compared with unaffected subjects from the same population (controls). These studies are focused on association of a genetic variant with a disease in a population and not on the mode of inheritance of a trait (71, 72). Association studies are preferable for finding "susceptibility" loci, low-risk alleles that are often found in relatively high frequencies in the general population, and do not result in robust signals in family-based studies (73). A positive association indicates an increased risk for the disease in subjects carrying the at-risk allele, which may have a causative role in the disorder or be in linkage disequilibrium with the gene actually causing the disease. Population stratification is a major problem in case-control studies, because the higher prevalence of particular alleles in different ethnic groups might bias the results and lead to false-positive associations. Moreover, case-control studies are especially prone to type II errors, that is, ruling out an association that is actually present in the population because of the small sample sizes often used in these studies.
In summary, linkage studies and case-control studies are both valid approaches for the study of the genetics of functional hyperandrogenism and PCOS, provided that the sample sizes of these studies are sufficiently large, the diagnostic criteria used to define affected and nonaffected subjects are clearly defined, and both ethnicity and differences in environmental factors are carefully considered.
IV. Studies in Pediatric and Adolescent Hyperandrogenism
Functional hyperandrogenism and hyperinsulinism may be detected early in life in affected women, even before pubertal development. As stated above, the presence of low birth weight in small for gestational age newborns, resulting from an unfavorable environment during intrauterine life and/or to defective maternal nutrition, has been related to the development of insulin resistance (74), disorders of glucose tolerance (75), hypertension, and cardiovascular disease (76, 77, 78, 79) later in life.
The association of low birth weight with the development of hyperinsulinism and type 2 diabetes mellitus has been confirmed in different populations (80, 81), and these children present with an accelerated growth and increased body mass index during adolescence.
Although environmental factors may be an important contributor to the restriction of fetal growth in small for gestational age newborns, genetic factors may also be involved in this association (82). According to this hypothesis, affected fetuses might suffer insulin resistance because of the influence of several unknown genes or genomic variants. Insulin resistance would lead to restriction of intrauterine growth and abnormal fetal vascular development, ultimately leading during adult life to increased vascular resistance and endothelial dysfunction. The compensatory increase in circulating insulin concentrations would contribute to the abdominal deposition of fat, and inflammatory cytokines and other mediators secreted by abdominal adipocytes would perpetuate insulin resistance and endothelial dysfunction, leading to abnormal glucose tolerance, atherosclerosis, and cardiovascular disease (82).
However, the analysis of the influence of genomic variants on the association of birth weight and insulin resistance has yielded conflicting results. Dunger et al. (83) analyzed a variable number of tandem repeats (VNTR) polymorphism in the insulin gene (INS) at 11p15.5. This VNTR polymorphism consists of a repeated sequence of 14 to 15 bp (ACAGGGGTGTGGGG), and is located at –596 bp of the start of transcription site (84). Among Caucasians, these alleles have been typed as class I (small, with 28–44 repeats, frequency approximately 70%), class II (intermediate, rare), and class III (large, with 138–159 repeats, frequency approximately 30%). The VNTR polymorphism in the INS promoter regulates the transcriptional rate of the gene (85) and probably that of the gene encoding IGF-II (84, 85, 86, 87, 88, 89). Newborns homozygous for class III alleles, which are those associated with increased transcription of the INS, presented with increased head circumference, length, and weight at birth as compared with babies homozygous for class I alleles, possibly favoring newborn survival. On the contrary, data from the same researchers suggested that the maternally inherited 16,189 mitochondrial DNA variant, which increases the risk for type 2 diabetes mellitus, was associated with restrained fetal growth, thereby favoring maternal survival by decreasing birth-related morbidity and mortality (90). Therefore, other genetic and environmental factors are possibly involved in the relationship between birth weight and the development of insulin resistance and type 2 diabetes mellitus in adulthood.
Given the association of insulin resistance with functional hyperandrogenism, Ibá?ez et al. (91) have proposed that low birth weight is related to the development of premature pubarche, and of functional hyperandrogenism during adolescence, insulin resistance being the underlying disorder common to both conditions. Retrospective analysis of a cohort of patients presenting with premature pubarche showed, especially in those girls who developed hyperinsulinemia and functional ovarian hyperandrogenism at adolescence, a lower birth weight compared with a control group, suggesting that low birth weight, premature pubarche, and functional hyperandrogenism are different manifestations of a unique disorder originating during prenatal life (92, 93, 94, 95), but becoming clinically apparent peripubertally (42).
Additional analysis of this cohort showed that adolescent girls who had anovulation and higher androgen levels were those with lower birth weight (96) and also confirmed the association of adrenal androgen excess, low birth weight, premature pubarche, and hyperinsulinemia (97).
In conceptual agreement, an exaggerated adrenal response to adrenocorticotropin stimulation, similar to that described in women with functional hyperandrogenism and PCOS, has been found in girls with premature pubarche, suggesting that this disorder can be considered an early manifestation of the syndrome (98). However, the relationship of low birth weight with hyperandrogenism and PCOS has not been universally confirmed. Jaquet et al. (99) compared a large population of young women born with intrauterine growth retardation with age-matched healthy controls, and although low birth weight was associated with insulin resistance, androgen levels were similar in both groups of women. Moreover, birth weight had no influence on the presence of hyperandrogenic symptoms suggestive of PCOS in a large cohort of Finnish women recently reported (100).
Given the association of functional hyperandrogenism and insulin resistance in children and adolescents, several genomic variants related to insulin resistance and the metabolic syndrome have been studied in these patients, mostly by the group of Ibánez in Spain and the group of Witchel in Pittsburgh.
The VNTR locus at the INS has been studied in girls with a history of premature pubarche (101). Although class I and class III alleles were equally distributed in patients and controls, patients carrying class I alleles presented with lower birth weight and lower insulin sensitivity compared with patients homozygous for class III alleles (101).
The common Gly972Arg variant in the gene encoding the insulin receptor substrate 1 (IRS-1), which has been shown to influence insulin resistance and glucose tolerance in adult PCOS patients (102, 103), has also been studied in adolescents (104). The frequencies of heterozygosity for the Gly972 allele were 31% among girls with a history of premature pubarche, 40% among girls with hyperinsulinemic ovarian hyperandrogenism, and only 19% among healthy control subjects. Carriers of Gly972 alleles presented with decreased sex hormone-binding levels (104).
The human homolog for the sorbin and SH3-domain-containing-1 gene (SORBS1), which encodes for an important signaling molecule in insulin-stimulated glucose uptake in the mouse, might play a role in human disorders with insulin resistance. The Ala228 allele of the Thr228Ala polymorphism of SORBS1 is a protective factor for both obesity and diabetes (105). However, alleles of the Thr228Ala polymorphism of SORBS1 were equally distributed in a multiethnic group of healthy adolescents compared with those presenting with premature pubarche and/or functional hyperandrogenism (106).
The Arg64 allele of the Trp64Arg polymorphism in the ?3-adrenergic receptor gene is associated with abdominal obesity and resistance to insulin and may contribute to the early onset of type 2 diabetes mellitus (107). However, Witchel et al. (108) found no differences in the distribution of Trp64Arg alleles in the girls presenting with premature pubarche and/or functional hyperandrogenism described above compared with healthy adults, and neither was an influence of this polymorphism on the body mass index of these girls. Similarly, the study of the common Pro12Ala polymorphism in the gene encoding the peroxisome proliferator-activated receptor-2 (PPAR-2), which influences insulin sensitivity in Caucasians (109), has been studied in children with premature pubarche and in adolescent hyperandrogenic girls, but no association was found with any of the PPAR-2 alleles and hyperandrogenism (110).
In addition to genes related to insulin resistance, those encoding for the steroidogenic enzymes involved in androgen biosynthesis and the genes encoding several molecules involved in androgen metabolism and action have been considered candidate genes to explain premature pubarche and adolescent hyperandrogenism.
Witchel and colleagues from Pittsburgh conducted a series of studies focused on the steroidogenic enzymes involved in androgen synthesis, which are shared by the adrenal and the ovaries. These authors have found that children with premature pubarche and adolescent girls with hyperandrogenism were heterozygous for mutations in CYP21, the gene encoding for 21-hydroxylase, and in the gene encoding 3?-hydroxysteroid-dehydrogenase (HSD3B2), more frequently than nonhyperandrogenic controls (111, 112, 113). In these patients, CYP17 mutations were ruled out as the cause of androgen excess (114). However, because congenital adrenal hyperplasia is an autosomal recessive disease requiring mutations in both alleles–in homozygosity or double heterozygosity–of CYP21 or of HSD3B2, it is unclear how carrying only one defective allele of these genes in these clinically symptomatic heterozygotes contributes to the hyperandrogenic phenotype (113). Moreover, defective CYP21 and HSD3B2 alleles were found only in a minority of these patients, and the authors concluded that other genomic variants were also involved in the pathogenesis of these disorders (111, 112, 113).
To further explore this possibility, Witchel et al. (115) evaluated the prevalence of the common variants and mutations in CYP21, HSD3B2, IRS-1, ?3-adrenergic receptor gene described above, and the glucocorticoid receptor gene within their series of children with premature pubarche and adolescent hyperandrogenism. They found that the prevalence of these variants and mutations was clearly increased when compared with 15 healthy controls, suggesting that the occurrence of multiple sequence variants in these genes might contribute to the development of hyperandrogenism.
Finally, the AR and uridine diphosphate-glucuronyltransferase 2B15, an enzyme involved in androgen inactivation, have been considered candidate genes for premature pubarche and adolescent hyperandrogenism. The study of the AR gene CAG repeat polymorphism suggested that shorter AR gene CAG numbers, indicative of increased androgen sensitivity, were associated with premature pubarche (116), whereas the allele frequencies of the D85Y polymorphism in uridine diphosphate-glucuronyltransferase 2B15 gene were similar in patients and in controls (117).
In summary, studies conducted to date in girls with premature pubarche and adolescent hyperandrogenism suggest a polygenic etiology for these disorders, which can be considered predictors of functional hyperandrogenism and PCOS later in life.
V. Studies in Hyperandrogenic Adults
Most if not all the studies conducted to date to elucidate the genetics of functional hyperandrogenism and PCOS have used a candidate gene approach, because no genome-wide scan has been conducted comparable with those done for other complex metabolic disorders, such as type 2 diabetes mellitus. Although many genes have been considered candidates to explain PCOS inheritance, most studies have included genes related to androgen biosynthesis and action and their regulation, genes involved in insulin resistance and associated disorders, and lately genes involved in chronic inflammation and atherosclerosis.
A. Genes involved in androgen biosynthesis, transport, and action, and their regulation (Table 3)
1. CYP17.
The limiting step in androgen biosynthesis in the ovary and adrenal gland is an enzyme termed P450c17, which possesses both 17-hydroxylase and 17,20-lyase activities. Therefore, P450c17 may catalyze the conversion of pregnenolone and progesterone into 17-hydroxypregnenolone and 17-hydroxyprogesterone, respectively, and of these steroids into dehydroepiandrosterone and androstenedione, although in humans only the conversion of 5 steroids has been demonstrated (118).
Rosenfield et al. (2, 119, 120) proposed several years ago that women with functional hyperandrogenism and PCOS had an exaggerated adrenal and ovarian responsiveness, and that increased activity of P450c17 was responsible for the enhanced androgen synthesis and secretion. They proposed the increase in serum 17-hydroxyprogesterone in response to the GnRH analog nafarelin as the marker of functional ovarian hyperandrogenism (119). Studies using adrenal stimulation with ACTH and ovarian stimulation and suppression with the long-acting GnRH triptorelin also suggested that most hyperandrogenic women have increased P450c17 activity in the adrenal and ovary (121, 122, 123), and that the increased adrenal P450c17 activity was not influenced by ovarian function (122). Moreover, P450c17 expression and activity are increased in ovarian theca cells from PCOS women, defined by NICHD criteria, compared with those from nonhyperandrogenic controls (124).
Given these findings, the gene encoding the P450c17 enzyme, CYP17, was considered a candidate gene in early studies. CYP17 is located in chromosome 10q24.3 (125, 126), and its promoter contains a T/C SNP at –34 bp from the start of transcription site that might modulate enzyme activity. Some studies suggested that this polymorphism was associated with the presence of polycystic ovaries on ultrasound (125), and PCOS patients homozygous for C alleles of this polymorphism presented with increased serum testosterone levels (127, 128). Other studies failed to confirm these observations, suggesting that this base change is a polymorphism without functional consequences for the development of polycystic ovaries and hyperandrogenism (129, 130, 131). Also, no evidence for linkage or association was found between PCOS, defined by NICHD criteria, and the CYP17 locus in a family-based study that included mostly families of European ancestry from the United States (132). Moreover, no abnormalities were found after single-strand conformational polymorphism analysis of the entire coding region of CYP17 in a small sample of PCOS patients with or without exaggerated 17-hydroxyprogesterone response to a GnRH analog (133), ruling out CYP17 as a major candidate gene for the pathogenesis of PCOS and functional hyperandrogenism. However, posttranscriptional hyperphosphorylation of the serine residues of P450c17 by a defective serine kinase might increase the 17,20-lyase activity of this enzyme, contributing to hyperandrogenism (118, 134). Confirmation of this hypothesis is still pending (135).
2. CYP11A.
Ovarian theca cells from women with PCOS defined by NICHD criteria overexpress all the steroidogenic enzymes involved in androgen biosynthesis (136, 137), and these cells secrete increased amounts of progesterone, 17-hydroxyprogesterone, testosterone, and androstenedione compared with theca cells from nonhyperandrogenic women (136, 138).
The initial step in adrenal and ovarian steroidogenesis is the conversion of cholesterol into progesterone, which is catalyzed by the cholesterol side chain cleavage enzyme. The CYP11A gene, located at 15q24, encodes the cholesterol side chain cleavage enzyme and has therefore been considered a candidate gene for functional hyperandrogenism and PCOS (139). It has been proposed that a VNTR polymorphism, consisting in repeats of a (tttta)n pentanucleotide at –528 bp from the ATG start of translation site in the CYP11A promoter, plays a role in the pathogenesis of PCOS (140). Evidence for linkage with the CYP11A locus was found in 20 pedigrees presenting with PCOS, based mostly on the presence of polycystic ovaries or male pattern premature balding, and the absence of the more common four-repeats allele (this VNTR appears with four, six, eight, and nine repeats in Caucasians) was associated with hirsute PCOS patients and with higher serum testosterone levels (140). Diamanti-Kandarakis et al. (141), using NICHD criteria for the definition of PCOS, confirmed its association with absence of four-repeat alleles in Greek patients. In women from the United States, nine-repeat alleles were more frequent in PCOS patients, defined by oligomenorrhea and polycystic ovaries, and four- and six-repeat alleles were more frequent in controls, but these allelic differences did not influence CYP11A expression in theca cells (142).
Other studies failed to demonstrate linkage with the CYP11A locus in PCOS patients defined by NICHD criteria (132) or association of CYP11A VNTR alleles with functional hyperandrogenism (143). Moreover, in a small sample of hirsute hyperandrogenic patients from Spain (144), no consistent genomic abnormalities have been found in the entire CYP11A coding region, nor in the genes encoding the steroidogenic acute regulatory protein, steroidogenic factor-1, and dosage-sensitive sex reversal-adrenal hypoplasia gene on the X chromosome gene-1 (DAX-1), which are also involved in the first step of steroidogenesis and its regulation. The family-based study cited above failed also to demonstrate linkage or association with the steroidogenic acute regulatory protein (132). Finally, Gaasenbeek et al. (145) recently failed to confirm any influence of CYP11A VNTR alleles on polycystic ovaries and on serum testosterone levels in a series of experiments involving a large number of subjects, concluding that the existence of associations between CYP11A promoter variation and androgen-related phenotypes had been substantially overestimated in previous studies, including their own preliminary report cited above (140).
3. CYP21.
An exaggerated serum 17-hydroxyprogesterone response to ACTH stimulation is a common finding in women with PCOS or functional hyperandrogenism (121, 146). This finding prompted several groups to study CYP21, which encodes the 21-hydroxylase enzyme catalyzing the conversion of 17-hydroxyprogesterone into 11-deoxycortisol; increased serum 17-hydroxyprogesterone levels are the biochemical marker for 21-hydroxylase deficiency. This type of congenital adrenal hyperplasia is an autosomal recessive disease resulting from homozygosity or double heterozygosity for missense or nonsense mutations in CYP21 and is characterized by hyperandrogenism with or without mineralocorticoid deficiency.
However, whether or not heterozygous CYP21 mutations influence functional hyperandrogenism and PCOS is still matter of debate. As stated above, girls presenting with premature adrenarche and adolescent hyperandrogenism carry CYP21 mutations more frequently than nonhyperandrogenic controls (112). Clinically symptomatic heterozygotes for CYP21 mutations present with a phenotype that resembles that of PCOS (113), but it is unclear whether all carriers of CYP21 mutations have an increased risk of having PCOS. Moreover, in hyperandrogenic women carrying CYP21 mutations there is no clear concordance between the CYP21 genotype and the functional origin of androgen excess (147).
Finally, the presence of polycystic ovaries in ultrasound scans has been associated with increased frequency of DRW6 and decreased DR7 human leukocyte antigen haplotypes, whereas 21-hydroxylase deficiency is associated with the Bw47, B14, or DR1 haplotypes (148). Moreover, in families of patients with 21-hydroxylase deficiency, polycystic ovaries segregate independently from adrenal dysfunction (149).
4. HSD3B2.
This enzyme catalyzes the conversion of 5 steroids into 4 steroids in the adrenal and in the ovary. Mutations in HSD3B2 result in a rare form of classic congenital adrenal hyperplasia, causing various degrees of salt wasting in both sexes and incomplete masculinization of the external genitalia in genetic males because both adrenal and ovarian steroidogenesis are severely impaired (150). Biochemically, this disorder is characterized by a marked increase in serum 17-hydroxypregnenolone and dehydroepiandrosterone concentrations (150).
Mild increases in these steroid precursors, and in the ratio of 5 to 4 steroids, are not infrequent in hyperandrogenic patients, and hyperandrogenic patients presenting with increased 17-hydroxypregnenolone concentrations and/or increased 5 to 4 ratios after adrenal stimulation with cosyntropin were initially considered to have a nonclassic form of HSD3B2 deficiency (151, 152, 153). However, these mild increases in serum 17-hydroxypregnenolone concentrations and in the 5 to 4 ratios were interpreted by others as an exaggerated adrenal response to cosyntropin stimulation without a genetic origin (154). Molecular analysis of HSD3B2 of hyperandrogenic patients presenting with increased 5 steroids and increased 5 to 4 ratios revealed no abnormalities (155, 156), ruling out the existence of a nonclassic form of HSD3B2 deficiency in these women. In conceptual agreement, a marker close to the HSD3B2 locus was not in linkage or association with PCOS, defined by NICHD criteria, in the American family-based study cited above (132).
5. 17?-Hydroxysteroid dehydrogenases.
17?-Hydroxysteroid dehydrogenase type III is also known as 17-ketosteroid reductase. This enzyme catalyzes the conversion of androstenedione into testosterone in the testis. Up to 20 mutations in HSD17B3 have been identified as the cause of male pseudohermaphroditism because of testosterone deficiency (157). Pang et al. (158) hypothesized that genetic females with ovarian 17-ketosteroid reductase deficiency would probably have a normal female phenotype at birth and normal pubertal breast development but would present at puberty with virilization and menstrual disorders because of the sudden increase of the ovarian secretion of androstenedione. However, no mutations have been identified in HSD17B3 in Caucasian and African-American women presenting with PCOS defined by NICHD criteria (159). Also, no evidence for linkage or association with the type 1, type 2, and type 3 17?-hydroxysteroid dehydrogenase loci was found in the American family-based study cited previously (132).
6. Aromatase.
This enzyme, encoded by CYP19, is responsible for the conversion of C19 steroids (androgens) into C18 steroids (estrogens). Aromatase activity may be decreased in granulosa cells and follicles from women with PCOS, and the possible androgen excess resulting from this decreased activity might contribute to abnormal follicle development (160, 161). Although mutations in CYP19 have been associated with multicystic ovaries in a case report, the clinical picture was that of hypergonadotropic hypogonadism (162). Moreover, no evidence for linkage of CYP19 with PCOS was found in studies conducted in the United Kingdom (140) and in the United States (132).
7. 11?-Hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase.
These enzymes regulate the inactivation of cortisol into cortisone at the tissue level. 11?-Hydroxysteroid dehydrogenase type 1 converts cortisol into cortisone directly, and endoluminal hexose-6-phosphate dehydrogenase regenerates reduced nicotinamide adenine dinucleotide phosphate in the endoplasmic reticulum, thereby influencing the directionality of 11?-hydroxysteroid dehydrogenase type 1 activity. Mutations in the genes encoding 11?-hydroxysteroid dehydrogenase type 1 and hexose-6-phosphate dehydrogenase have been recently shown to cause cortisone reductase deficiency (163), and because the phenotypic features of affected women resemble those of PCOS, these genes should be considered candidates to explain the pathogenesis of PCOS. However, this possibility has not been explored in any series of patients with functional hyperandrogenism and PCOS to date.
8. Gonadotropins.
In as many as 40% of PCOS patients, LH hypersecretion is present (164). The gene encoding the ?-subunit of LH (LH?), which is responsible for LH specificity, has been explored in PCOS and hyperandrogenic women. Initially, two point mutations, Trp8Arg and Ile15Thr, were identified as the cause of an immunologically abnormal LH? molecule (165). These mutations induce structural changes in the mutant LH? molecule (166) and have a 15% prevalence worldwide, although with significant differences depending on the population studied (167). The resulting LH? molecule has an increased in vitro activity and a decreased in vivo half-life compared with the wild-type molecule (168).
This LH? variant has been associated with increased serum testosterone, estrogen, and SHBG levels in healthy women, but not in PCOS patients (169). Furthermore, the prevalence of this LH? variant is reduced in obese women with PCOS, suggesting a protective role against the development of hyperandrogenic symptoms in these women and a possible usefulness in determining the risk for PCOS in obese women (170, 171). However, no association with PCOS, hyperandrogenism, or serum androgen or estrogen concentrations has been found in other studies (172, 173).
Other variants in the LH? gene might influence hyperandrogenism and PCOS. A Gly102Ser mutation in exon 3 has been related to menstrual disorders in Chinese women (172, 174). Finally, in Japanese patients with ovulatory disorders, several SNPs in the promoter of the LH? gene (–894C/T, –1018G/C, –1036C/A, –1098C/T, and –1423C/T) are more frequent than in normal ovulatory women (175).
Polymorphisms in the FSH ?-subunit gene have also been reported to influence PCOS. In Chinese women, homozygosity for a thymine-cytosine substitution in exon 3 (codon 76, TAT to TAC) has been found more frequently in PCOS patients, as defined by oligomenorrhea and polycystic ovaries, compared with nonhyperandrogenic women, especially in obese patients, and correlating with higher serum androgen concentrations (176). In these women, two SNPs in the gene encoding the FSH receptor, Thr307Ala and Ser680Asn, showed similar allele distribution among PCOS patients and controls (177). Moreover, the search for mutations in exons 6, 7, 9, and 10 of the FSH receptor gene, which have been shown previously to inactivate the receptor, yielded negative results in Japanese patients with oligomenorrhea and polycystic ovaries (178). This locus was not in linkage or association with PCOS in a family-based study conducted in the United States (132). Finally, mutations in the gene encoding the GnRH receptor have not been found in PCOS patients, defined by NICHD criteria (179).
9. Dopamine receptor.
Given that dopamine is involved in the hypothalamic control of gonadotropin secretion, the association between polymorphisms in the dopamine receptor and disorders of ovulation and fertility has been studied. Legro et al. (180) reported that SNPs in exons 5 and 6 of the gene encoding the type 2 dopamine receptor influenced serum gonadotropin and prolactin levels, as well as parity and prevalence of miscarriage in women of Hispanic ancestry. In these women, a genomic variant in the type 3 dopamine receptor was associated with hyperandrogenic chronic anovulation and resistance to clomiphene citrate (181), but this association was not confirmed in a large series of non-Hispanic women (182).
10. SHBG.
SHBG regulates the access of testosterone and estradiol to target tissues. Decreased SHBG concentrations are characteristic of hyperandrogenic women, contributing to increased tissue androgen availability (183). Despite the fact that evidence for linkage or association was not found between a marker close to the SHBG locus and PCOS in a family-based study conducted in the United States (132), a missense Pro156Leu mutation in the SHBG gene, resulting in abnormal glycosylation and secretion of a SHGB mutant that retains binding capacity (184), has been detected in a women presenting with severe hyperandrogenism during pregnancy. However, it is seldom found in unselected hyperandrogenic women (184).
Recently, an association between a (TAAAA)n polymorphism in the promoter of the SHBG gene and PCOS has been reported (185). Longer alleles (more than eight repeats) were frequent in Greek PCOS patients, defined by NICHD criteria, whereas nonhyperandrogenic women presented with a higher frequency of shorter alleles (185). Furthermore, in the PCOS group, carriers of the longer allele genotypes had lower SHBG levels than in those with shorter alleles (185). Therefore, this polymorphism might be related to the low SHBG levels characteristic of PCOS, facilitating androgen availability to target tissues. Cousin et al. (186) studied the (TAAAA)n polymorphism in hirsute women and found strong disequilibrium linkage with an Asp327Asn SNP in exon 8 of SHBG, 327Asn alleles being associated with eight-repeat (TAAAA)n alleles, and resulting in increased serum SHBG levels when compared with subjects homozygous for 327Asp alleles. Moreover, longer (TAAAA)n alleles resulted in decreased serum SHBG levels when compared with six-repeat alleles (186), in conceptual agreement with the results in PCOS women described above (185).
11. Glucocorticoid receptor.
Mutations in the glucocorticoid receptor gene result in a compensatory increase in circulating ACTH, resulting in excess secretion of adrenal androgens. Therefore, the glucocorticoid receptor has been studied as a candidate gene for functional hyperandrogenism and PCOS. However, genomic abnormalities and polymorphisms in the glucocorticoid receptor gene do not appear to influence the pathogenesis of functional hyperandrogenism (187, 188) and PCOS (189).
12. Steroid 5-reductase (SRD5A).
This enzyme catalyzes the conversion of testosterone into the more potent androgen dihydrotestosterone. SRD5A1 and SRD5A2 are the two isoforms of the enzyme. Mutations in SRD5A2 result in male pseudohermaphroditism (190, 191). Because total SRD5A activity is increased in polycystic ovaries (192), the genes encoding the SRD5A isoforms can be considered candidate genes for functional hyperandrogenism and PCOS. To date, no data have been published regarding the possible involvement of molecular genetic variants in these genes in hyperandrogenic disorders.
13. AR.
The AR is a member of the superfamily of ligand-activated transcription factors that regulate many biological processes and is encoded by a gene located at Xq11–12 (193, 194). The AR has three functional domains responsible for transactivation, for ligand binding, and for binding to DNA. Exon 1 contains a VNTR polymorphism consisting of (CAG)n repeats, which encodes for a polyglutamine tract in the N-terminal transactivation domain of the AR protein (195).
The number of CAG repeats in the normal population varies between 11 and 31, with 20 repeats being the most frequent (196). The number of CAG repeats is inversely related to the transactivation of the AR and its activity (197). Thus, the decreased number of CAG repeats has been proposed to increase androgen activity at target tissues, favoring hirsutism (198), premature pubarche, and ovarian hyperandrogenism (116) in women, infertility in men (199), as well as androgen-dependent skin disorders in both men and women (200). A decreased number of AR gene CAG repeats has been suggested to explain the normal serum androgen levels found in some women with polycystic ovaries, infertility, and oligomenorrhea, in whom the hyperandrogenic symptoms would result from the intrinsic increase in the AR activity (201).
One of the two X chromosomes in every cell of a woman undergoes inactivation, a process that involves methylation of DNA and occurs in a random fashion. Skewed inactivation of X chromosome with the larger CAG repeat, favoring expression of the shorter allele, has been proposed to play a role for idiopathic hirsutism and PCOS (202, 203). However, we have shown that both the number of CAG repeats and the prevalence of skewed X chromosome inactivation were equally distributed in hirsute patients with or without hyperandrogenemia and in healthy women (204). Moreover, no evidence for linkage or association with PCOS was found with the AR locus in the family-based study conducted in the United States cited above (132).
14. Aldosterone synthetase.
The renin-angiotensin system may be hyperactive in women with PCOS (205, 206). Recently, a –344T/C SNP in the promoter of the gene encoding aldosterone synthetase, CYP11B2, has been proposed to influence the pathogenesis of PCOS, because C alleles are more frequent in these patients than in healthy controls (207). Interestingly, women homozygous for C alleles presented with increased plasma renin activity and increased serum angiotensin II, aldosterone, and testosterone levels compared with women homozygous for T alleles, suggesting that this polymorphism influences the activity of aldosterone synthetase and also contributes to androgen excess (207).
B. Genes involved in insulin resistance and associated disorders (Table 4)
1. Insulin receptor gene (INSR).
The presence of insulin resistance is common in PCOS (3) and is frequent in other hyperandrogenic patients (208, 209, 210). Therefore, the genes encoding the INSR and those encoding several molecules involved in postreceptor signaling have been studied in hyperandrogenic patients.
The INSR is a heterotetrameric glycoprotein consisting of two - and two ?-subunits, and it is encoded by a gene located at chromosome 19. A marker relatively close (1 cM) to INSR, D19S884, has been reported in association with PCOS, defined by NICHD criteria, using the transmission disequilibrium test in a family-based study (132), and in a case-control study (211), both conducted in the United States. However, this association was not confirmed in PCOS patients from Spain and Italy using a case-control design (212). In conceptual agreement with the latter, the 22 exons of INSR have been sequenced in PCOS patients with negative results (213). INSR contains several polymorphisms, yet most of them are silent polymorphisms or are located in intronic regions and are present with similar frequencies in patients with polycystic ovaries and hyperandrogenism and in controls (214).
Recently, Siegel et al. (215) have observed a C/T SNP at the tyrosine kinase domain of INSR associated with PCOS defined by NICHD criteria. This SNP could be a susceptible variant for PCOS, or it could be in linkage disequilibrium with another INSR polymorphism; such an association must be confirmed in future studies.
Studying cultured skin fibroblasts and muscle samples from PCOS patients defined by NICHD criteria, Dunaif et al. (216) found increased INSR serine phosphorylation, which decreases its protein tyrosine kinase activity, in as many as 50% of the cases. Subsequent studies in these patients confirmed in vivo the defect in insulin signaling, using serial skeletal muscle biopsies obtained during euglycemic glucose clamp studies (217). However, the search for mutation in the tyrosine kinase domain of the INSR gene did not show abnormalities other than polymorphisms in exon 17, which were not associated with insulin resistance (218). Therefore, the still unidentified factor responsible for the increased phosphorylation of the serine residues of the INSR appears to be extrinsic to the receptor and might also contribute to the increased serine phosphorylation of P450c17 also found in some PCOS patients (118).
2. IRS-1 and IRS-2.
After insulin binding, autophosphorylation of tyrosine residues results in the activation of the INSR, and tyrosine kinase activity phosphorylates intracellular substrates such as IRS-1 and IRS-2 (3). PCOS women present a defect in insulin receptor signaling characterized by a decreased IRS-1-associated phosphatidylinositol 3-kinase activity (217). Two common SNPs in the genes encoding insulin-receptor substrates, Gly972Arg in IRS-1 and Gly1057Asp in IRS-2, are susceptibility genes for type 2 diabetes mellitus (219, 220); they have been studied in PCOS patients, despite the fact that evidence for linkage or association with PCOS was not found with IRS-1 in a family-based study conducted in the United States (132).
Carriers of Arg972 IRS-1 alleles presented with increased fasting insulin levels compared with women homozygous for Gly972 alleles, whereas carriers of Asp1057 IRS-2 alleles presented with increased glucose and insulin levels 2 h after an oral glucose load and had an increased prevalence of glucose intolerance compared with subjects homozygous for Gly1057 alleles (102). However, a subsequent study in a larger series of PCOS patients showed only the effect of the Gly1057Asp polymorphism in IRS-2 on glucose tolerance, and no effect of the Gly972Arg polymorphism in IRS-1 (103). Surprisingly, the effect was just the opposite found previously, because the 2-h glucose values were actually increased in subjects homozygous for Gly1057 alleles when compared with carriers of Asp1057 alleles (103). Additional studies are needed to confirm the influence of these polymorphisms on glucose tolerance and insulin resistance in PCOS.
3. INS.
The presence of pancreatic ?-cell dysfunction in women presenting with PCOS appears to have a genetic origin (221). Therefore, INS has been studied in women with PCOS and functional hyperandrogenism. Waterworth et al. (222) found that women with menstrual disturbances and/or hirsutism and polycystic ovaries, who were homozygous for class III alleles, were more frequently anovulatory and had increased body mass index and fasting insulin compared with women homozygous for class I alleles. Moreover, class III alleles were associated with symptomatic women (222, 223). Paternal transmission of class III alleles from heterozygous fathers to anovulatory PCOS patients is more frequent than maternal transmission of the allele (222, 223, 224), and class III alleles predisposed these patients to both PCOS and type 2 diabetes mellitus. However, later case-control studies in European Caucasian women, conducted outside the United Kingdom, have failed to reproduce these results (225, 226), and the INS locus was not associated with PCOS in a linkage study in American PCOS patients (132).
4. IGF system.
IGFs, their receptors, binding proteins, and proteases are important for the normal development of the ovary (227). IGFs stimulate ovarian cellular mitosis and steroidogenesis, inhibit apoptosis, and might be related to the development of functional hyperandrogenism and PCOS (228).
No evidence for linkage with PCOS was found for markers close to the genes encoding IGF-I and IGF-binding proteins 1 and 3 in a family study conducted in the United States (132). San Millán et al. (229) recently found an association of PCOS with homozygosity for G alleles of the ApaI polymorphism in IGF-II, but not with a dinucleotide polymorphism in IGF-I, a trinucleotide polymorphism in the IGF-I receptor, or with an ACAA-insertion/deletion polymorphism at the 3' nontranslated region of the IGF-II receptor, previously described (230, 231, 232). G alleles of the ApaI polymorphism in IGF-II have been attributed to increased IGF-II mRNA in leukocytes compared with A alleles (233), and possibly result in increased liver IGF-II expression and secretion (234). Given that IGF-II stimulates adrenal (235, 236) and ovarian (237) androgen secretion, the increased frequency of homozygosity for these alleles might contribute to hyperandrogenism in some PCOS patients, provided we assume that G alleles may increase IGF-II expression at the ovary, as reported for other tissues. In the same case-control study involving PCOS patients cited above (229), we found that subjects homozygous for 90-bp alleles of a trinucleotide repeat polymorphism in the gene encoding IGF-I receptor had increased fasting glucose levels and fasting insulin resistance index compared with subjects carrying 93-bp alleles, but no association of any genotype with PCOS.
5. PPAR-2.
Activation of PPAR- by using the insulin sensitizer drugs, thiazolidinediones, has been one of the most important advances for the treatment of type 2 diabetes mellitus in past years. As described above, insulin resistance is a common finding in hyperandrogenic patients, and thiazolidinediones improve insulin sensitivity, hyperandrogenism, and ovulation in women with PCOS (238, 239, 240, 241, 242, 243).
The Pro12Ala SNP in PPAR-2 has been studied in women with PCOS, despite the fact that evidence for linkage or association with PCOS was not found for a marker close to the PPAR-2 gene, in a family-based study conducted in the United States (132). Ala12 alleles of the PPAR-2 gene favor weight gain in obese adults (244) and in obese hyperandrogenic girls and adolescents (110). Also, Ala12 alleles preserve insulin sensitivity in Caucasian men (109) and in Caucasian women presenting with PCOS defined by NICHD criteria (245). Recently, a marginally significant decrease in the frequency of the Ala12 allele has been reported in women with polycystic ovaries from Finland (246), but this result has not been confirmed in a small case-control study of PCOS patients conducted in Spain (229) or in a recently published study in Italian PCOS patients defined by NICHD criteria and ultrasonography (247). In the latter, a silent C to T substitution at position 142 in exon 6 was differentially distributed in PCOS patients and controls, T alleles being more frequent in women with PCOS (247). This silent polymorphism was not in linkage disequilibrium with the Pro12Ala polymorphism, but the possibility of an association with other unknown genomic variant in the PPAR-2 gene was not explored (247). Nevertheless, both Ala12 and T142 alleles are relatively uncommon (less than 20%) in normal and hyperandrogenic populations (229, 245, 246, 247), and therefore their putative influences on insulin sensitivity and/or hyperandrogenism would be restricted to a small number of PCOS patients.
6. Paraoxonase (PON1).
We have recently explored the –108C/T, Leu55Met, and Gln192Arg polymorphisms in the gene encoding serum PON1 in PCOS patients defined by NICHD criteria. The PON1 gene is expressed mainly in the liver and encodes for serum PON1, which is an antioxidant high-density lipoprotein-associated enzyme. Liver PON1 mRNA expression is influenced by genetic and environmental factors, and both androgens and proinflammatory mediators decrease liver PON1 expression (248). Interestingly, both androgen excess and proinflammatory genotypes contribute to the pathogenesis of PCOS (249, 250, 251).
Homozygosity for T alleles of the –108C/T polymorphism in PON1 was more frequent in patients compared with nonhyperandrogenic women (229). As expected from the association with PCOS, subjects homozygous for –108T alleles of PON1 presented with increased hirsutism scores, total testosterone, and free testosterone and androstenedione concentrations compared with carriers of –108C alleles (229). Moreover, in a logistic regression model, homozygosity for –108T alleles of PON1 was associated with a 7.1 odds ratio (95% confidence interval, 2.1–23.8) of having PCOS (229).
The –108C/T polymorphism is responsible of approximately 23% of PON1-expression levels in some cell systems, in which –108TT constructs showed reduced PON1 expression compared with –108CC constructs (252). We thus speculated that homozygosity for –108T alleles, hyperandrogenism, and proinflammatory genotypes might contribute to reduced PON1 expression, resulting in a higher oxidative stress in these women. The latter has been found in PCOS patients (253).
Oxidative stress may impair insulin action (254). Therefore, reduced serum PON1 activity might contribute to the insulin resistance of PCOS patients. This hypothesis is supported by the finding of reduced serum PON1 activity in other insulin-resistant disorders such as type 2 diabetes mellitus (255, 256) and cardiovascular atherosclerotic disease (257, 258). If confirmed in future studies, the association of homozygosity for –108T alleles of PON1 with PCOS might contribute to explaining the insulin resistance and the increased risk for atherosclerosis associated with this syndrome (259).
In our study (229), subjects homozygous for Met55 alleles presented with increased body mass index and indexes of insulin resistance compared with carriers of Leu55 alleles, further suggesting the involvement of PON1 in PCOS, despite the fact that the Leu55Met and Gln192Arg polymorphisms in PON1 were not associated with PCOS.
7. Human homolog for the sorbin and SH3-domain-containing-1 gene (SORBS1).
In addition to the studies in adolescents cited above (106), we have recently studied the Thr228Ala polymorphism in adult PCOS patients. Allele frequencies were similar in PCOS patients and nonhyperandrogenic women, but carriers of Ala228 alleles of SORBS1 presented with increased body mass index compared with subjects homozygous for 228T alleles (229), in conceptual agreement with a large study conducted in Europe (260).
8. Calpain-10.
This enzyme is a cysteine protease that plays a role in insulin secretion and action (261). The 112/121-haplotype combination of University of Chicago single nucleotide polymorphism (UCSNP)-43, UCSNP-19, and UCSNP-63 polymorphisms in the gene encoding calpain-10, located at 2q37.3, has been reported to increase the risk for diabetes (262). Ehrmann et al. (263) found no association between this haplotype and any of the phenotypic features of PCOS in Caucasian nondiabetic PCOS patients, defined by NICHD criteria, whereas the 112/121-haplotype was significantly associated with higher insulin levels in response to an oral glucose tolerance test in African-American, nondiabetic PCOS women. Moreover, when considering Caucasian and African-American, nondiabetic PCOS patients as a whole, the 112/121 haplotype was associated with a 2-fold increase in susceptibility to PCOS.
However, the association of calpain-10 SNPs with PCOS, as defined by polycystic ovaries, hyperandrogenism, and/or anovulation, was not confirmed by Haddad et al. (264) in 330 PCOS patients from the United Kingdom. We have studied three common polymorphisms in the calpain-10 gene in 97 Spanish hyperandrogenic patients and 37 controls, including UCSNP-43 (265). C alleles at the UCSNP-45 locus were associated with idiopathic hirsutism, but neither the UCSNP-43 nor the UCSNP-44 was associated with hyperandrogenism or PCOS (265). However, in a different population from the south of Spain, González et al. (266, 267) recently reported an association between PCOS and USCNP-44. Additional studies are needed to clarify this issue, especially because the physiological roles of calpain-10 remain mostly unknown.
9. Genes encoding for other molecules related to insulin resistance and associated disorders.
Among other genes tested, no association has been reported in PCOS with genomic variants in the genes encoding glycogen synthetase (268), resistin (269), leptin and its receptor (270), apoprotein E (271), or with variants in the genes of plasma cell differentiation antigen glycoprotein, protein tyrosine phosphatase 1B, and adiponectin (229).
C. Proinflammatory genotypes (Table 5)
Chronic inflammation is involved in the development of metabolic syndrome and cardiovascular disease (272, 273), and serum inflammatory markers cluster in patients with cardiovascular disease, suggesting a role in the pathogenesis of atherosclerosis (272, 273).
Inverse correlations have been reported between indexes of insulin sensitivity and inflammatory markers such as circulating levels of TNF- (274), soluble type 2 TNF receptor (TNFR2) (275), IL-6 (276), C-reactive protein (CRP) (276), and soluble intercellular cell adhesion molecule-1 (276).
Adipose tissue plays a central role in the relationship between cytokines and insulin resistance. The expression of TNF- and TNFR2 in adipose tissue is increased in obesity (277, 278). TNF- expression correlates with indexes of insulin resistance and decreases with weight loss in parallel with the improvement in insulin sensitivity (277). Similar results have been reported for IL-6 (279). Moreover, inflammatory cytokines may induce insulin resistance by direct actions on insulin-signaling postreceptor molecules (280) or by inducing central obesity through activation of the hypothalamic-pituitary-adrenal axis (281).
Because obesity and insulin resistance are common findings in hyperandrogenic women (3), chronic inflammation might be involved in the pathogenesis of functional hyperandrogenism and PCOS. In animal models in which polycystic ovaries were induced by neonatal administration of estradiol, the production of TNF- and IL-6 was increased in ovaries and in peritoneal macrophages (282). The concentrations of IL-12 were decreased, and production of IL-13 and the number of activated lymphocytes were increased in follicular fluid of women with PCOS (283).
The study of serum inflammatory markers in PCOS has resulted in conflicting reports. Increased CRP levels have been reported in PCOS patients defined by NICHD criteria (284, 285). Similarly, increased serum IL-6 (286) and TNF- (287, 288, 289) concentrations have been reported in women with PCOS or functional hyperandrogenism.
However, we have recently reported that obesity, and not PCOS, appears to be the major determinant of the increase in serum CRP and IL-6 in premenopausal women, and this and other inflammatory markers such as serum TNF-, soluble TNFR2, or soluble intercellular cell adhesion molecule-1 are not increased by PCOS when controlling for confounding factors such as smoking and obesity (290). Similar findings have been published recently by Mohlig et al. (291). These results cast doubt upon the usefulness of these serum inflammatory molecules as markers of the inflammatory process associated with hyperandrogenism. On the contrary, another novel inflammatory marker of cardiovascular risk, IL-18, is increased in serum both by obesity and by PCOS, suggesting that this molecule may be a useful marker of inflammation in PCOS patients (292).
Given that proinflammatory genotypes influence obesity, type 2 diabetes mellitus, and insulin resistance-related disorders (273), over the past years our group has studied genomic variants in the genes encoding several inflammatory mediators and their receptors. Some of these variants are associated with functional hyperandrogenism and PCOS.
1. TNF-.
TNF- induces reproductive changes that closely resemble those found in patients with PCOS and functional hyperandrogenism. TNF- facilitates the effects of insulin and IGF-I on the ovary in a dose-dependent and additive fashion (293), stimulating proliferation and steroidogenesis in rat theca cells in vitro (293, 294). Moreover, TNF- may be involved in apoptosis and anovulation in the rat ovary (295).
We have recently studied serum TNF- levels and nine common polymorphisms (–1196C/T, –1125G/C, –1031T/C, –863C/A, –857C/T, –316G/A, –308G/A, –238G/A, and –163G/A) in the TNF- gene in 60 hyperandrogenic women and 27 healthy controls matched for body mass index (288). As a group, hyperandrogenic patients presented with increased serum TNF- levels, but this increase was only present in lean patients when compared with lean controls, and not in obese patients (288).
No differences between patients and controls were found in the allele frequencies of any of the polymorphisms studied (288). In conceptual agreement, –308G/A alleles were equally distributed between patients with polycystic ovaries and hyperandrogenic symptoms and controls in other studies (296, 297), and similar results were reported for the –805C/T polymorphism in the TNF- gene (298). However, when considering patients and controls as a whole in our series, carriers of –308A alleles presented with increased serum androgen and 17-hydroxyprogesterone levels before and after stimulation with the GnRH analog leuprolide (288). Therefore, polymorphisms in the TNF- gene do not appear to play a major role in the pathogenesis of functional hyperandrogenism and PCOS but might be a modifying factor for phenotypic traits associated with these disorders.
2. TNFR2 gene (TNFRSF1B).
TNFR2 mediates most of the metabolic effects of TNF- (299). We have recently studied serum soluble TNFR2 levels and several polymorphisms in the TNFRSF1B in women with functional hyperandrogenism, including PCOS defined by NICHD criteria (250).
TNFRSF1B has been studied in several metabolic disorders. The 1690T/C variant in exon 10 has been described to influence body mass index and insulin resistance (300). The CA-repeat polymorphism in intron 4 and the Met196Arg polymorphism in exon 6, which are in strong linkage disequilibrium, influence serum lipid levels (301, 302, 303) and diastolic blood pressure (302); linkage studies suggest that the TNFRSF1B locus is associated with hypertension (302) and familial combined hyperlipidemia (301). Moreover, the CA-repeat polymorphism has been recently proposed as a contributing factor to coronary artery disease (304).
In our series, the uncommon 196Arg allele of the Met196Arg (676T/G) polymorphism in exon 6 of TNFRSF1B was more frequent in patients with PCOS compared with healthy controls (250). When the study was extended to include Italian subjects, this variant was more frequent not only in PCOS patients but also in women with hyperandrogenic hirsutism and regular menstrual cycles (250). However, the Met196Arg polymorphisms did not influence any phenotypic trait associated with hyperandrogenism, insulin resistance, or obesity when studying patients and controls separately (250).
We also studied three SNPs in the 3'-untranslated region of TNFRSF1B in exon 10, 1663G/A, 1668T/G, and 1690T/C, which were not associated with hyperandrogenism (250). Serum soluble TNFR2 levels were not increased in hyperandrogenic women compared with controls, but they were influenced by the interaction between the 1663G/A and 1668T/G variants. We hypothesized that the Met196Arg variant in TNFRSF1B might contribute to PCOS by modulating TNF- actions at target tissues.
3. IL-6.
Among cytokines, IL-6 circulates in plasma and acts in distant tissues (305). TNF- stimulates IL-6 secretion by adipocytes, and mounting evidence suggests that IL-6 is also implicated in insulin resistance and associated syndromes (273, 306, 307, 308). Although serum IL-6 levels are not increased in women presenting with functional hyperandrogenism and PCOS (249, 290), IL-6 concentrations are increased in peritoneal fluid in clomiphene-resistant, anovulatory PCOS patients, suggesting a role in the pathogenesis of hyperandrogenic disorders (309).
We recently studied four common polymorphisms in the promoter of the IL-6 gene (–597G/A, –572G/C, –373A(n)T(n), and –174G/C) in 85 hyperandrogenic patients and 25 healthy women (249). The –597G/A and –174G/C variants were in strong disequilibrium linkage. When considering the three biallelic SNPs, five haplotypes were found (relative frequencies in parentheses): GGG (0.505), AGC (0.377), GGC (0.059), GCG (0.055), and GCC (0.005). The frequency of the GGG haplotype was increased in patients (0.559) compared with controls (0.320), and conversely, the frequency of the AGC haplotype was reduced in patients (0.318) compared with controls (0.580) (P < 0.02). Homozygosity and heterozygosity for –597G and –174G alleles were more frequent in controls, and controls carrying these alleles presented with increased serum IL-6, cortisol, 11-deoxycortisol, and 17-hydroxyprogesterone levels, and a tendency toward increased serum total testosterone levels compared with controls homozygous for –597A and –174C alleles. These findings suggest a protective role for the latter against IL-6 excess, adrenal hyperactivity, and hyperandrogenism. The –572G/C and –373A(n)T(n) were not associated with hyperandrogenism or with any androgen-related phenotypic trait (249). In conceptual agreement, Mohlig et al. (291) recently reported that the heterozygous –174G/C genotype in PCOS patients was associated with lower serum androstenedione levels.
4. IL-6 receptor.
This is a heterodimeric receptor consisting of two membrane-bound glycoproteins: an 80-kDa IL-6 binding unit and a 130-kDa IL-6 signal transducer (IL-6 receptor ? or gp130). gp130 is a transducer chain shared by other cytokines and is responsible for signal transduction of the chain-ligand complex through the Janus kinase/signal transducer and activator of the transcription pathway (310).
We have recently studied common polymorphisms in both subunits of the IL-6 receptor in a series of 145 hyperandrogenic women and 45 controls from Spain (251). The uncommon Arg148 allele of the Gly148Arg polymorphism in the gp130 gene was more frequent in controls compared with hyperandrogenic patients, and controls carrying Arg148 alleles had lower 11-deoxycortisol and 17-hydroxyprogesterone concentrations, a lower response of androstenedione to 1–24 adrenocorticotropin, and an almost statistically significant decrease in free testosterone levels, suggesting that, as occurred for the IL-6 polymorphisms described above, Arg148 alleles in the gp130 gene have a protective effect against androgen excess and adrenal hyperactivity (251). When considering patients and controls as a whole, a microsatellite CA-repeat polymorphism in the 80-kDa IL-6 binding unit locus was associated with obesity. The frequency of the common 149-bp allele was markedly increased in obese women compared with controls, further supporting the involvement of inflammatory genotypes in obesity and related syndromes (251).
Overall, our studies over the past years suggest that chronic inflammation underlies the pathogenesis of functional hyperandrogenism and PCOS, as has been proposed for other disorders associated with insulin resistance, and that proinflammatory genotypes may be involved.
D. Other candidate genes
1. Follistatin.
This protein binds to activin, inhibiting its action both in vivo and in vitro (311). Activin and follistatin are expressed in multiple tissues, including pituitary, ovary, adrenal, and pancreas (312, 313). In the ovary, activin promotes follicular development and inhibits androgen secretion by theca cells (314). Conversely, transgenic mice overexpressing follistatin present with follicular arrest and infertility (315), characteristics frequently found in hyperandrogenic women.
However, the expression of human inhibin/activin subunit, follistatin, type 2 activin receptor mRNAs, and their encoded proteins in ovarian follicles from PCOS patients suggested that an increase in the availability of activin, relative to inhibin, was actually present in the arrested follicles in PCOS patients (316). Moreover, the location of the mRNAs of the follistatin subunits is not altered in PCOS (317).
Initial molecular genetic studies in humans by Urbanek et al. (132) suggested evidence for linkage between the follistatin locus and PCOS, as cited above. Subsequent studies by these authors (318) have failed to confirm the involvement of the follistatin gene with PCOS in a large multiethnic study. The coding regions and some introns of the follistatin gene were sequenced, disclosing at least 17 polymorphisms; however, 16 of them were rare, making a significant contribution of these variants to the pathogenesis of hyperandrogenism unlikely (318). Moreover, the only common polymorphism found, located in exon 6 but not translated, was not associated with PCOS when correcting for multiple testing, and the authors concluded that contributions to the etiology of PCOS from the follistatin gene, if any, are probably small (318).
In conceptual agreement, no mutations in the follistatin gene have been found in Chinese PCOS patients defined by menstrual dysfunction, hyperandrogenism, and polycystic ovaries (319), and the only mutation found in our series of patients from Spain, a silent G951A variant, was equally distributed in PCOS patients and in healthy women (320).
2. Thrombophilic factors.
Women with PCOS have increased miscarriage rates, as happens in women with inherited thrombophilic conditions. The secretion of plasminogen activator inhibitor-1 (PAI-1) in adipose tissue is enhanced by inflammatory cytokines and by insulin (321, 322, 323). In agreement, the increased circulating PAI-1 levels found in PCOS patients decrease after treatment with insulin sensitizers (324). Homozygosity for 4G alleles of the –675 4G/5G polymorphism in the gene encoding PAI-1, which modulates PAI-1 activity, have been reported in association with obesity (325) and PCOS (326). We have found a similar trend in our series on Spanish women, although the difference in allele frequencies did not reach statistical significance, possibly because of small sample size (229).
Other genetic abnormalities associated with thrombophilia have been studied in PCOS. Tsanadis et al. (327) recently reported that the prevalence of antithrombin III, protein S and protein C deficiencies, factor V Leiden, prothrombin G20210A factor, and methylene tetrahydrofolate reductase 677C/T mutations is not increased in Greek patients with polycystic ovaries, menstrual dysfunction, and hyperandrogenism when compared with nonhyperandrogenic controls. Similar results have been found for the methylene tetrahydrofolate reductase variant in Italian women (328).
3. Microsomal epoxide hydrolase.
Two SNPs, Tyr113His and His139Arg, in the gene encoding the detoxifying enzyme microsomal epoxide hydrolase have been studied in women with PCOS, defined by the presence of polycystic ovaries and hyperandrogenic symptoms (329). Although none of the polymorphisms was associated with PCOS, the presence of the His113-Arg139 haplotype was associated with an odds ratio for PCOS of 2.28 and 95% confidence interval of 1.1–4.8 (329). However, it is unclear how changes in the activity of this enzyme might relate to functional hyperandrogenism and PCOS, and therefore, this result should be considered with caution unless confirmed in future studies.
4. Bone morphogenetic proteins.
The intraovarian bone morphogenetic protein system is involved in the control of granulosa cell proliferation and cytodifferentiation and plays a role in oocyte development (330). The genes encoding the growth differentiation factor 9 and bone morphogenetic protein 15 have been studied in Japanese women with polycystic ovaries, but no missense mutations have been found in these women (331).
VI. Hyperandrogenism, PCOS, and Survival Advantage
The increasing prevalence of complex metabolic disorders in developed countries raised the possibility that, from an evolutionary perspective, the pathogenetic mechanisms underlying these disorders might have provided survival advantages (332). However, these previously beneficial mechanisms may lead to disease with prolonged life expectancy or when these subjects are exposed to the present lifestyle in Western countries.
As suggested by Witchel et al. (333) for congenital adrenal hyperplasia, which is one of the most common inherited disorders with carrier frequencies of approximately 10% in all world populations studied to date and a relatively common cause of hyperandrogenism in children and adults, the presence of hyperandrogenism in women might have provided survival advantage for these women and their children.
The rapid maturation of the reproductive axis found in these subjects, together with the increase in assertive behavior resulting from increased androgen secretion, might be advantageous during times of environmental stress (333, 334, 335). Moreover, the relative infertility of these women could increase the interval between pregnancies, decreasing the birth rate and favoring maternal and infant survival (333). In addition to the lower rate of pregnancies secondary to oligoovulation, pregnancies may also occur at an older age, favoring the survival of these women. In agreement, an early beginning of fecundity is associated with higher mortality rates in animal models (335).
Yet survival advantage may also contribute to explaining the association of hyperandrogenism with other disorders related to insulin resistance. Human metabolism may be genetically adapted to the dominant conditions that have predominated for ages: near-continuous physical activity, a diet rich in carbohydrates and proteins yet poor in fat, and long periods of famine or food shortage (336, 337, 338). Survival was therefore favored by a combination of thrifty genotypes and phenotypes, in which insulin resistance played a central role (Fig. 2).
Insulin resistance increases glucose availability for brain metabolism. It also increases salt and water retention and sympathic tone and induces endothelial dysfunction, favoring an increase in blood pressure, obviously beneficial when trauma occurs. Similarly, the increased coagulability and decreased fibrinolysis associated with insulin resistance are defensive mechanisms against bleeding. But more important is that insulin resistance favors obesity, protecting against starvation, and obesity contributes to a proinflammatory state through the secretion of several cytokines, contributing to the defense against infection, and possibly to the development of functional hyperandrogenism and PCOS.
When the environmental conditions change and access to food is not restricted, significant trauma and epidemics seldom occur, and life expectancy increases markedly, these defensive mechanisms are no longer beneficial, and the price to pay is atherosclerosis and cardiovascular disease (Fig. 2).
For the reasons outlined above, it is not surprising that the genomic variants associated with obesity, type 2 diabetes mellitus, and other disorders in which insulin resistance plays a major role are frequently associated with functional hyperandrogenism and PCOS. However, genomic variants-associated insulin resistance may contribute to hyperandrogenism only indirectly, by inducing insulin resistance and hyperinsulinemia and/or by direct actions at the adrenal or the ovary. For example, TNF- induces insulin resistance by interfering with IRS-1-mediated insulin signaling (280) and also has reproductive actions that closely resemble those found in patients with PCOS and functional hyperandrogenism, as summarized above (293, 294).
Therefore, the precise elucidation of the mechanisms underlying these associations requires studies of the functional consequences of the genomic variants associated with hyperandrogenism in different target tissues, especially when some of the genomic variants appear to facilitate insulin resistance and hyperandrogenism, whereas others may protect against these disorders.
At present, the emerging picture for the molecular genetic mechanisms leading to functional hyperandrogenism and PCOS is that of a complex interaction between predisposing and protective genomic variants, and the strong impact of modifying environmental factors including diet, exercise, and lifestyle (Fig. 3).
VII. Explanations for the Lack of Reproducible Association of Hyperandrogenism and PCOS with Molecular Genetic Abnormalities and Genomic Variants
To date, most of the associations between genomic variants and functional hyperandrogenism or PCOS failed to replicate when studied in different populations. An immediate consideration is that most studies conducted to date have been modest with regard to the number of subjects included. A small sample size, leading to a lack of statistical power to detect the modest effects that genomic variants possibly play in the pathogenesis of a complex disorder such as functional hyperandrogenism, might explain the negative results found in many cases when efforts have been made to confirm previous studies. Although at present there are no doubts about the need of studies with large sample sizes, lack of consistency of the reported findings is not limited to hyperandrogenism and is a recurrent problem in the study of many complex metabolic disorders (339). Therefore, other factors might also contribute to the discrepancies observed when studying candidate genes for hyperandrogenism in different populations, summarized below.
A. Ascertainment issues
One of the essential requirements for the effectiveness of molecular genetic studies is a clear definition of the phenotype under study. This has not been the case for functional hyperandrogenism and PCOS. As an example, the linkage studies cited above (140, 222) of CYP11A and INS in women from the United Kingdom relied mostly on the use of ultrasonography for the diagnosis of PCOS, and this series included "ovulatory" PCOS patients. On the contrary, a linkage study conducted in the United States relied on the NICHD criteria (132), in which chronic anovulation is strictly required for the diagnosis of PCOS (9). Given the considerable differences in the criteria used to define the phenotype, it is unlikely that the genes associated with the disease would be the same in both studies.
Moreover, the spectrum of disorders covered by the criteria derived from the Rotterdam Consensus Workshop (11, 12) is even broader than those included with the NICHD definition: PCOS would be diagnosed not only in women presenting with hirsutism, hyperandrogenemia, and anovulation, but also in patients presenting with anovulation and polycystic ovaries but no clinical and/or biochemical evidence of hyperandrogenism. Therefore, these revised criteria are unlikely to be of significant advantage over previous ones with respect to molecular genetic studies. And to further complicate the search for PCOS-related genomic abnormalities in family-based studies, the male PCOS phenotype is still uncertain.
Because of the heterogeneous and syndromic nature of PCOS, it would be more helpful for molecular genetic studies to use criteria oriented toward the identification of specific clinical, reproductive, and metabolic traits associated with hyperandrogenism. This would require an important effort to identify and especially to standardize the methods used to identify these traits in large populations such as those needed for family-based or case-control designs.
Instead of trying to reach a consensus on how to diagnose precisely a disorder that is heterogeneous by definition, the practical issue would be to standardize the definitions and methods used to identify specific traits such as insulin resistance, overweight, visceral adiposity, dyslipidemia, hirsutism, acne, oligoovulation, and many others that characterize or are frequently found in hyperandrogenic women.
Given the apparently multigenic etiology for functional hyperandrogenism and PCOS, this approach would permit a more precise definition of the kind of hyperandrogenic patients included in the different molecular genetic studies. It would facilitate well-sized multicollaborative studies and thereby increase the probability of success in identifying genomic variants and abnormalities related to these particular traits that might be used as diagnostic and/or therapeutic targets in the future. It is hoped that scientific societies that address the study of hyperandrogenism, such as the recently created Androgen Excess Society (http://www.androgenexcesssociety.org), will serve as an adequate forum where these efforts in standardization could be made.
In addition to the problems of defining the PCOS phenotype, sampling issues also should be considered. For family-based studies, a common sampling procedure is to collect from an affected proband with at least one or two affected relatives, instead of the ideal, but rarely performed, procedure of selecting families from the population at random (340). Nonrandom ascertainment makes it more likely that families with multiple PCOS members enter the study than families with no affected relative, resulting in biased heritability estimates (340, 341). Another common source of error is that the affected status in relatives of the proband is typically determined on the basis of his or her report because of constraints in time and resources (340). Moreover, affected subjects may be more likely to be aware of the diagnosis in their relatives than nonaffected subjects (340), or more likely to misinterpret the symptoms of their relatives (340), such as considering the presence or absence of hirsutism as the equivalent of having or not having PCOS.
B. Involvement of environmental factors
The interactions between genetic and environmental factors are essential for the comprehension of the pathogenesis of common complex disorders (342). The precise knowledge of these interactions requires long-term studies analyzing the impact of different environmental factors in specific subgroups of patients, because controlling all the confounding environmental variables is extremely difficult (342).
Environmental factors such as weight gain may trigger the development of PCOS in predisposed women, as occurs in other complex metabolic disorders in which insulin resistance plays a major role. The identification and precise delimitation of the contribution of the environmental influences triggering the development of functional hyperandrogenism and PCOS may direct the search for the specific proteins and/or genomic variants involved to the metabolic pathways influenced by these environmental factors.
However, the environmental factors contributing to complex metabolic disorders may change depending on the population studied, because diet, exercise, and lifestyle have wide ethnic variations. Therefore, it should not be surprising that the genomic abnormalities contributing to these disorders may also change depending on the environmental conditions (i.e., the genes contributing to PCOS in obese sedentary women from a Western country are probably different from those involved in the PCOS phenotype of lean women from the Mediterranean area or from Asia). Finally, to further complicate the study of complex metabolic disorders, the phenotype changes during the life of the affected subjects as age advances. Therefore, different phenotypic traits may not be present when these women were phenotyped, but became apparent later in life, constituting one more confounding factor that is especially difficult to control in molecular genetic studies.
C. Possible polygenic etiology for functional hyperandrogenism and PCOS
It has been suggested that the phenotypic heterogeneity observed in PCOS patients, even within the same family, could be attributed to the interaction of a small number of genes with one another and with environmental factors (18, 22, 33). Furthermore, given the large number of genomic variants found associated with functional hyperandrogenism and PCOS to date, the emerging picture may be that of a complex metabolic disorder resulting from small predisposing or protecting effects arising from the interaction of multiple genomic variants and several environmental factors.
Even if a more precise definition for hyperandrogenic phenotypes was used, and that has not been the case, the classic requisite of replication of linkage or association of a genomic abnormality with monogenic diseases may not be applicable for complex disorders because the functional consequences of the genomic variant may be only apparent in certain populations or when a particular environmental factor is present (339). However, it is important that internal replication of findings is provided in larger series, because this might reveal false-positive associations reported in preliminary studies when limited sample sizes were studied.
Nowadays, it is technically feasible to genotype large series of individuals for multiple genomic variants, allowing whole-genome scans and high-throughput candidate gene analysis. These approaches will disclose reliable information of the relative contribution of these genomic variants to functional hyperandrogenism and PCOS and will facilitate the study of gene to gene and gene-environment interactions that probably contribute to the development of these prevalent disorders.
Finally, to avoid spurious associations from being considered causative of any disease-associated trait, every effort should be made to demonstrate in vivo or in vitro a functional consequence of the associated genomic variant that might reasonably account for the contribution of the variant to the disorder or its associated traits.
D. Limitations of the genetic techniques used to date
As discussed previously, the molecular genetic studies regarding PCOS and functional hyperandrogenism conducted to date had important limitations, especially because adequately sized whole-genome scans and large case-control association studies are still lacking, and therefore associations with genomic variants that have small effects on hyperandrogenism might have been missed by these studies.
Another source of confusion is the fact that quality control of genetic data has not been as strict as that applied to other methods used in clinical research or even in routine clinical practice. Genetic data are usually obtained from a single measure, and reliability and reproducibility of these analyses might be a problem, because genotyping errors may severely bias the estimates of genetic studies (339, 343). Although departure from the Hardy-Weinberg equilibrium may be useful for the detection of these genotyping errors (344), and although modern methods incorporate models of typing error (343, 345), efforts in standardization of these techniques should be made in the future (339).
VIII. Future Perspective: Functional Hyperandrogenism and PCOS in the Age of "Omics"
The suffix "omics" is being applied to recent technologies that are exponentially increasing our knowledge of human biology. Perhaps at present the most developed one is genomics.
Genomics aims to map, sequence, and analyze all the genes and their products in the genome, with the final intention of providing the complete and accurate DNA sequence of an organism (56, 346). The number of genes in the human genome is in the order of 35,000, and because of exon shuffling, alternative splicing, and posttranslational modifications, as many as 100,000 different proteins may be encoded in the human genome. Therefore, identification of all the genes and proteins of the human organism and their interactions requires an enormous effort.
Genomics is in constant evolution, and more specific subspecialties are being developed, such as functional genomics. Its aim is the identification of the biological functions of genes and their products, and how they interact with the environment in health and disease (347). To date, functional genomics has contributed to unravel the mechanisms of many diseases (348, 349, 350), and the genomic approach might be especially adequate for the study for complex polygenic disorders, given that traditional molecular genetic approaches have not been successful to date (351).
Genomic techniques, such as differential gene expression analyzed by DNA microarrays, allow the identification of genes that are differentially overexpressed or suppressed in patients compared with controls. This approach has the advantage over molecular genetic analyses in that the result integrates the presence of molecular genetic abnormalities with both gene-gene and gene-environment interactions. Therefore, the genes identified can be considered potential candidates to explain the disorder and also potential diagnostic and therapeutic targets. Recently, comparison of gene expression in cultured theca cells from PCOS patients and controls using DNA microarrays identified the genes encoding aldehyde dehydrogenase 6 and retinol dehydrogenase 2 as candidate genes for PCOS (352). These factors play a role in all-trans-retinoic acid biosynthesis and the transcription factor GATA6, which increase the expression of 17-hydroxylase (352), a characteristic of PCOS theca cells (137). In the near future, undergoing studies using DNA microarrays to compare the expression profiles of tissues such as adipose tissue and muscle, which are essential for the development of insulin resistance, will undoubtedly contribute to the identification of candidate genes in hyperandrogenic women.
But given that gene expression and protein concentration and activity are poorly correlated (353), modern techniques have been developed to study the "proteome" of a cell, tissue, or organism. Proteomics allows large-scale analysis of proteins, including their relative abundance, distribution, posttranslational modifications, functions, and interactions with other molecules (347). For example, it is possible to examine the expression of more than 1000 proteins by coupling mass spectrometry technology with several separation methods. Proteomic analysis of tissues involved in the pathogenesis of functional hyperandrogenism and PCOS, such as ovarian theca and granulosa cells, adrenal cortex, sc and omental adipose tissue, and muscle, are essential for our understanding of the complex interactions between the genome and the environment that underlie these disorders.
Ultimately, the identification of genes and proteins related to functional hyperandrogenism and PCOS, and their interactions with the environment, will be an essential step for the development of more precise diagnostic techniques, the identification of new therapeutic targets, and the identification of particular individuals that, because of their genetic background, may be predisposed to certain complications of the syndrome or respond differently to available treatments.
IX. Summary
Functional hyperandrogenism and PCOS appear to be complex multigenic disorders, arising from the interaction of predisposing and protective genetic variants that might have been selected during evolution because of a previous survival advantage, with environmental influences that play an important role in the expression of the hyperandrogenic phenotype.
Among others, genomic variants in genes pertaining to the regulation of androgen biosynthesis, insulin resistance, metabolic syndrome, and proinflammatory genotypes are involved in the genetic predisposition to functional hyperandrogenism and PCOS. Progress in this area requires adequately sized multicenter collaborative studies in which modifying environmental factors such as ethnicity, diet, and lifestyle are identified with precision.
In the future, classic molecular genetic techniques such as linkage analysis in the form of a whole-genome scan and large case-control studies, as well as modern genomic and proteomic approaches, will hopefully provide new insights into the pathogenesis of functional hyperandrogenism and PCOS, with the ultimate aim of improving the prevention, diagnosis, and treatment of these prevalent disorders.
Acknowledgments
This work was supported by Grants FIS 02/0741 and RGDM G03/212 from the Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, and by Grant CAM 08.6/0010/2001 from the Consejería de Educación, Comunidad de Madrid, Spain.
Footnotes
First Published Online November 23, 2004
Abbreviations: AR, Androgen receptor; CRP, C-reactive protein; CYP, cytochrome P450; gp130, gp130 subunit of IL-6 receptor; HSD, hydroxysteroid dehydrogenase; INS, insulin gene; INSR, insulin receptor gene; IRS, insulin receptor substrate; LH?, ?-subunit of LH; PAI-1, plasminogen activator inhibitor-1; PCOS, polycystic ovary syndrome; PON1, paraoxonase; PPAR-2, peroxisome proliferator-activated receptor-2; SORBS1, human homolog for the sorbin and SH3-domain-containing 1 gene; SNP, single nucleotide polymorphism; SRD5A, steroid 5-reductase; TNFR2, type 2 TNF receptor; VNTR, variable number of tandem repeats.
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