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The genetics of polycystic ovary syndrome
Best Practice & Research Clinical Obstetrics and Gynaecology Vol. 18, No. 5, pp. 707–718, 2004 doi:10.1016/j.bpobgyn.2004.05.002 available online at http://www.sciencedirect.com
3 The genetics of polycystic ovary syndrome Paula Amato MD Joe Leigh Simpson*
MD
Department of Obstetrics and Gynecology, Baylor College of Medicine, 6550 Fannin, Suite 901A, Houston, TX 77030, USA
Polycystic ovary syndrome (PCOS) is a heterogeneous disorder. There is evidence for a genetic component in PCOS based on familial clustering of cases. The majority of the evidence supports an autosomal dominant form of inheritance. Steroidogenesis has been shown to be upregulated in PCOS theca cells, suggesting that the genetic abnormality in PCOS affects signal transduction pathways controlling the expression of a family of genes. Although a number of candidate genes have been proposed, the putative PCOS gene(s) has yet to be identified. Linkage and association studies implicate a region near the insulin receptor gene at chr 19p13.3. New genetic approaches, such as microarray technology, hold promise for elucidation of the pathophysiology underlying PCOS. Key words: genetics; hyperandrogenism; polycystic ovaries.
Polycystic ovary syndrome (PCOS) is a common endocrinopathy affecting 5 – 10% of women of reproductive age. The 2003 Rotterdam Consensus concluded that PCOS is a syndrome of ovarian dysfunction requiring two out of three of the following criteria for diagnosis: (i) oligo- or anovulation; (ii) hyperandrogenism and/or hyperandrogenemia; and (iii) polycystic ovary morphology. The exclusion of other etiologies, such as congenital adrenal hyperplasia, androgen-secreting tumors, and Cushing’s disease, is also necessary.1 The clinical manifestations of PCOS include menstrual irregularities, signs of hyperandrogenism, and obesity. Premature pubarche might be the earliest recognizable phenotype of PCOS.2 Hypersecretion of luteinizing hormone (LH) and insulin resistance are common features of PCOS, which is associated with an increased risk of type 2 diabetes mellitus (DM) and cardiovascular events.3 – 6 Some sections of this chapter inevitably reflect our previous reviews.7,8
PATHOPHYSIOLOGY Although the underlying pathophysiology of PCOS remains unknown, attention has centered upon primary defects in the hypothalamic –pituitary axis, ovarian function, * Tel.: þ 1-713-798-8360; Fax: þ1-713-798-8410. E-mail address: [email protected] (J.L. Simpson). 1521-6934/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.
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and insulin secretion and action. Hyperandrogenemia, LH hypersecretion, insulin resistance, and compensatory hyperinsulinemia are common biochemical features of PCOS. Hyperandrogenemia can result in the development of polycystic ovaries, as well as affect hypothalamic-pituitary function. LH hypersecretion is a common feature of PCOS. LH is believed to play a permissive role augmenting ovarian androgen production in PCOS. Insulin resistance and the associated compensatory hyperinsulinemia might be key factors in the pathophysiology of PCOS. Approximately 50% of PCOS women are insulin resistant when compared with age- and weight-matched controls.9 Insulin is postulated to augment the stimulatory action of LH on theca cell androgen biosynthesis through upregulation of genes encoding steroidogenic enzymes. Hyperinsulinemia stimulates ovarian androgen secretion directly and suppresses sex hormone binding globulin (SHBG) levels, resulting in an increase in bioavailable androgens. Androgens can in turn affect hypothalamic control of pituitary gonadotropin secretion, and also serve as precursors for peripheral aromatization to estrogen. Insulin-sensitizing drugs such as metformin and the thiazolinediones act by reducing insulin levels, which in turn reduces androgen levels. Freshly isolated theca cells collected from PCOS ovaries show enhanced production of dehydroepiandrosterone (DHEA), progesterone, 17-hydroxyprogesterone, and androstenedione.10 This increased steroidogenic activity is due to increased 3b-hydroxysteroid-dehydrogenase and 17a-hydroxylase/17,20-lyase activities.11,12 Northern blot analysis revealed that cytochrome P450 17-hydroxylase/17, 20desmolase (CYP17) and cytochrome P450 side-chain clearage enzyme (CYP11A) mRNAs were more abundant in PCOS theca cells than in normal theca cells. In addition, transient transfection experiments indicated that the CYP17 promoter is enhanced in PCOS theca cells compared to normal theca cells.13 The upregulation of steroidogenesis in PCOS theca cells might be the result of a genetic abnormality in these cells or a metabolic imprint received in vivo. Nelson et al11 showed that propagated PCOS thecal cells maintained in long-term cultures displayed enhanced steroidogenesis, suggesting that the upregulation of steroidogenesis is the result of a genetic abnormality, although a persistent metabolic imprint established in vivo cannot be excluded.
FAMILY STUDIES There appears to be evidence for a genetic component in PCOS based on familial clustering of cases. Hyperandrogenemia appears to be the strongest genetically inherited characteristic in familial cases. The investigation of the genetics of PCOS has been hampered by several factors. Because PCOS is associated with infertility, the availability of large pedigrees for linkage analysis is limited. Furthermore, because PCOS is a heterogeneous disorder1, family studies cannot be readily compared because of the use of different diagnostic criteria. In addition, a male phenotype for PCOS has not been clearly identified. These challenges notwithstanding, the majority of studies suggest a dominantly inherited trait of low penetrance and variable expressivity. This mode of inheritance would be consistent with the variable clinical findings in PCOS. However, polygenic, multifactorial inheritance or genetic heterogeneity cannot be excluded. Table 1 summarizes the familial studies. The first formal genetic study was by Cooper et al15, who studied 18 patients with Stein –Leventhal syndrome. Oligomenorrhea, hirsutism, and enlarged ovaries were
Table 1. Summary of studies assessing frequency of PCOS in female relatives.
Author Cooper et al
Diagnostic criteria Oligomenorrhea, hirsutism, polycystic ovaries (culdoscopy, gynecography, or wedge resection)
Oligomenorrhea, hirsutism, and polycystic ovaries (exam and surgery)
Ferriman and Purdie
Hirsutism and/or oligomenorrhea (60% with polycystic ovaries by air contrast gynecography)
Lunde et al
Menstrual dysfunction, hyperandrogenism, obesity, infertility and polycystic ovaries (transabdominal ultrasound)
Hague et al
Menstrual irregularities, hirsutism, infertility, obesity and multicystic ovaries (wedge resection)
Carey et al
Polycystic ovaries by transabdominal ultrasound
Oligomenorrhea
Hirsutism Elevated 24-hour urinary 17-ketosteroids Enlarged ovaries Oligomenorrhea
Affected sisters (%)
Affected mothers (%)
9/19 (47%)
4/13 (31%)
14/24 (58%) 12/19 (63%)
4/13 (31%) 2/7 (29%)
10/19 (53%)
0/7 (0%) 16/67 (24%)
Hirsutism Hirsutism
30/337 (9%)
32/284 (5%)
Oligomenorrhea Hirsutism
32/337 (9%) 8/129 (6%)
24/284 (8%) 17/132 (13%)
Oligomenorrhea Hirsutism
19/129 (15%)
16/132 (12%)
Oligomenorrhea Polycystic ovaries (ultrasound) Elevated testosterone
Overall affected first-degree female relatives (%)
28/54 (52%)
28/107 (26%)
19/107 (18%) 37/50 (74%) 16/50 (32%) (continued on next page)
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Givens et al
Frequency of specific trait assessed
Author Norman et al
Legro et al Govind et al Kahsar-Miller et al
Diagnostic criteria Elevated androgens, decreased SHBG, polycystic ovaries (ultrasound)
Elevated testosterone and oligomenorrhea Polycystic ovaries (ultrasound) Oligomenorrhea and either hirsutism or
Frequency of specific trait assessed
Affected sisters (%)
Affected mothers (%)
11/15 (73%)
1/1 (100%)
Polycystic ovaries (ultrasound) Increased testosterone or androstenedione Hyperinsulinemia Hyperadrogenemia
13/15 (87%)
1/5 (20%)
10/15 (66%) 36/80 (45%)
5/5 (100%)
Polycystic ovaries PCOS as defined
35/53 (66%) 16/50 (32%)
15/29 (52%) 19/78 (24%)
Overall affected first-degree female relatives (%)
50/82 (61%)
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Table 1 (continued)
The genetics of polycystic ovary syndrome 711
much more common in sisters of cases than in sisters of controls. In addition, hirsutism or ‘pelosity’ was more prevalent in male relatives. In the 1970s, Givens and others at the University of Tennessee, Memphis, published reports indicating that PCOS could be inherited in an X-linked dominant fashion. Diagnostic criteria consisted of hirsutism and either polycystic or bilaterally enlarged ovaries. In the first report, Givens et al16 described two families in which multiple individuals in more than two generations were affected. In one kindred, affected females experienced myocardial infarction in their fifth decade; and acanthosis nigricans, insulin resistance, and hypertension were present in many family members. Most subjects were African – American. In a third kindred17, several males showed maturational arrest of spermatogenesis. Excluding index cases, Wilroy et al18 tabulated that 47% of female offspring of affected females were affected. Among the offspring of males with an elevated LH/follicle stimulating hormone (FSH) ratio, 89% of daughters were affected. That almost all daughters of affected males were affected is consistent with X-linked dominant inheritance. In the United Kingdom, Ferriman and Purdie19 studied 381 patients with hirsutism and/or oligomenorrhea and a control group of 179 women. Familial tendencies were greatest among hirsute women with enlarged ovaries. Of 188 patients with hirsutism and enlarged ovaries, 38 first-degree relatives had hirsutism, 30 oligomenorrhea, and 19 infertility. Of 96 patients with hirsutism but normal sized ovaries, the numbers of first-degree relatives with the above traits were 73, 15, and 10, respectively. In 179 controls, numbers were 7, 8, and 8. Baldness was significantly increased in male relatives of hirsute women. Later British studies provided additional data in support of heritability of PCOS, specifically autosomal dominant inheritance. Hague et al20 used ultrasound criteria and either hyperandrogenemia or LH hypersecretion to determine the frequency of PCOS in relatives of affected cases. PCOS was found in 45 of 52 (87%) of sisters of probands and in 24 of 36 (67%) of mothers. The frequency of affected relatives was dramatically higher than the 50% predicted for either autosomal dominant or X-linked dominant inheritance, raising doubts about specificity of diagnostic criteria. Non-Mendelian mechanisms would need to be invoked in order to account for such a distorted segregation ratio. More likely, criteria were overly sensitive, leading to diagnosis false positivity. In later British studies the frequency of affected relatives in PCOS was nearer the 50% expected for autosomal dominant inheritance. Genetic studies were performed by Carey et al.21 First-degree relatives of 10 probands were studied. Of 62 informative relatives, 58 were screened by ultrasound. Of the 58 female first-degree relatives, 51% were affected. Family studies were conducted in Norway by Lunde et al Using hirsutism and oligomenorrhea as inclusion criteria, Lunde et al22 found only 6 – 15% of first-degree relatives affected. Norman et al23 found that, in 15 probands, far more relatives were affected. Among sisters, 11 of 15 (73%) had polycystic ovaries by ultrasound, 13 of 15 (87%) had elevated testosterone (T), and 10 of 15 (66%) hyperinsulinemia. In the US (Pennsylvania), Legro et al24 studied 80 probands diagnosed on the basis of elevated testosterone associated with oligomenorrhea (, 6 menses/year); non-classical 21-hydroxylase deficiency was excluded. They found 36 of 80 (45%) sisters to be affected on the basis of hyperandrogenemia. Govind et al25 studied 29 probands and 10 control women. Diagnostic criteria consisted of polycystic ovaries on ultrasound with or without clinical or biochemical features of PCOS; 61% of female first-degree relatives were affected and 22% of male first-degree relatives had early onset (before age 30) male-pattern baldness. The prevalence was much
712 P. Amato and J. L. Simpson
higher than in the control families. Of a total of 71 sibs of PCOS probands, 39 (55%) were affected, which is consistent with autosomal dominant inheritance. Kahsar-Miller and co-workers26 considered the frequencies of oligomenorrhea and either hirsutism or elevated testosterone among first-degree female relatives within families of 93 probands with PCOS. A significantly higher rate of PCOS was observed among first-degree relatives than in the general population, suggesting genetic component in the disorder. Concordantly affected twins have been observed.27,28 However, discordance has been observed even for monozygotic twins.29 Using a large genealogy database to search for a founder effect and to evaluate the degree of heritability in PCOS, Ward et al30 showed that the degree of relatedness among a PCOS population was four-fold greater than the average degree of relatedness among a large random sampling of the same database.
CANDIDATE GENES That a number of genes show altered patterns of expression suggests that the genetic abnormality in PCOS affects signal transduction pathways controlling the expression of a family of genes, rather than the abnormal expression of a single gene encoding a single steroidogenic enzyme. Consistent with this, cytogenetic studies have failed to identify common karyotypic abnormalities. A consistently observed aberration characterized by a specific breakpoint would indicate localization of a causative gene. Because abnormalities of steroidogenesis are a prominent feature of PCOS, investigators have long sought linkage or associations between PCOS and the various genes involved in the androgen biosynthetic pathway or metabolic pathways involved in insulin action. Linkage analysis is used to demonstrate the co-segregation of a genetic variant and a disease locus. Association studies assess whether a genetic variant is linked to a disease locus on a population scale. Family-based linkage disequilibrium test (TDT) determines whether parents heterozygous for a disease allele transmit that allele more often to their affected children than the non-disease allele. A number of linkage and association studies of candidate genes in PCOS have yielded negative results.31 – 39 Studies with positive and/or mixed results are discussed in more detail below. CYP17 (cytochrome P450 17-hydroxylase/17, 20-desmolase) Although initial studies suggested an association between cytochrome CYP17, which encodes 17-hydroxylase/17,20-lyase, and PCOS, subsequent studies failed to confirm this finding making this gene an unlikely PCOS gene candidate.40 – 42 CYP11A (cytochrome P450 side-chain cleavage enzyme) Gharani et al43 found evidence for a weak linkage between the CYP11A gene, which encodes the cholesterol side-chain cleavage enzyme, and hyperandrogenemia in PCOS women. An association study of 97 women with PCOS demonstrated a strong association between the CYP11A 50 UTR pentanucleotide repeat polymorphism with total serum testosterone levels. In addition to the pitfalls mentioned earlier, these early
The genetics of polycystic ovary syndrome 713
studies are limited because of failure to make appropriate statistical adjustments for multiple testing. In another study involving CYP11A, an association was found between a pentanucleotide repeat at 2 528 of the CYP11A gene and PCOS.44 However, other studies have failed to find a significant association between CYP11A and PCOS.34,45 Although perturbations in the CYP11A gene cannot easily account for altered expression of other steroidogenic enzymes, the locus remains a potential candidate gene. CYP21 (cytochrome P450 21-hydroxylase) CYP21 encodes 21-hydroxylase, the enzyme responsible for most cases of congenital adrenal hyperplasia (CAH). Recent studies have found a significant prevalence of CYP21 mutations in PCOS women with a normal 17-hydroxyprogesterone response to adrenocorticotropic hormone (ACTH) stimulation, calling into question the diagnostic distinction between PCOS and CAH.46,47 Androgen receptor Urbanek et al34 studied 150 families and failed to find evidence for association of the trinucleotide (CAG) repeat polymorphism in the X-linked androgen receptor gene and PCOS. However, this short CAG repeat length has been shown to be inversely associated with androgen levels.48 A study of 122 women with PCOS demonstrated a significantly greater frequency of longer CAG alleles and biallelic means (. 22 repeats) in exon 1 of the androgen receptor in women with PCOS compared to normal women.49 Iban˜ez et al50 found an association between the shorter androgen receptor gene CAG repeat polymorphism and precocious pubarche. Sex hormone binding globulin (SHBG) Hogenveen et al51 identified a polymorphism in the coding region of SHBG that encodes a missense mutation, P156L, in 4 of 482 women with PCOS, hirsutism, or ovarian dysfunction. Xita et al52 showed an association between the (TAAAA)n polymorphism in the SHBG and PCOS. Women with PCOS were found to have a significantly greater frequency of longer (TAAAA)n alleles (more than eight repeats) than normal women who had shorter alleles (less than eight repeats) in higher frequency. Insulin receptor A number of studies have examined the insulin receptor for gene sequence major mutations—with negative results.53 This suggests that if perturbed insulin action is integral to PCOS, the mechanism likely involves a target downstream of the insulin receptor. Dunaif et al54 found that , 50% of women with PCOS showed increased insulin receptor serine phosphorylation in skeletal muscle cells and fibroblasts. In a recent study, tyrosine autophosphorylation of the insulin receptor was also found to be decreased in ovaries of women with PCOS.55 Siegel et al56 showed an association
714 P. Amato and J. L. Simpson
between a C/T single nucleotide polymorphism at the tyrosine kinase domain of the insulin receptor gene and PCOS. Two separate studies have found linkage and association between a marker (D19S884 at chr 19p13.3) located near the insulin receptor gene and PCOS.34,57 The National Cooperative Program in Infertility Research conducted linkage and association studies using both sib-pair analysis and transmission/disequilibrium tests to assess a number of candidate genes in a cohort of Caucasian families. In this study, evidence for linkage and association with a region on chr 19p13.3 was found. The evidence for linkage was strongest at the marker, D19S884. The Heritage Family study found evidence for statistically significant linkage between a region at chr 19p13.3 and androgen levels in Caucasians, providing further evidence for an important role for the region at chr 19p13.3 in PCOS.58 Although the putative PCOS gene in this region remains to be identified, data suggest likely involvement in signal transduction mechanisms, leading to altered expression of a family of genes involved in steroidogenesis and/or insulin action. Insulin Waterworth et al59 found strong linkage and association between the class III allele at the insulin gene 50 VNTR (variable number tandem repeats) in the 50 region of the insulin gene and PCOS. However, in a larger study, Urbanek et al34 found no evidence for linkage of the insulin gene and PCOS and no association between the class III allele of the insulin VNTR and hyperandrogenemia. Vankova et al60 also found no association between the INS VNTR polymorphism and PCOS. Insulin receptor substrate proteins A study by El Mkadem et al61 revealed an association between the Gly972Arg IRS-1 variant and the Gly1057Asp IRS-2 variants and insulin resistance in women with PCOS. Ehrmann et al62 found no association of IRS-1 with PCOS. However, the IRS-2 Gly/ Gly polymorphism was associated with higher blood glucose levels in nondiabetic White and African –American women with PCOS. Iban˜ez et al63 found an increased frequency of the G972R variant of the IRS-1 gene among girls with a history of precocious puberty. Follistatin An initial study of 39 affected sibling pairs demonstrated statistically significant linkage to follistatin.33 However, subsequent larger studies and more detailed sequence analysis of the follistatin gene have not revealed significant linkage.64 Calpain-10 Calpain-10 is a cysteine protease that has been shown to be associated with susceptibility to type 2 diabetes. In a recent study, the 112/121-haplotype was associated with higher insulin levels in African – American women, and an increase risk of PCOS in both African – American and white women.65
The genetics of polycystic ovary syndrome 715
Gonzalez et al66,67 showed an association of the CAPN10 UCSNP-44 allele with PCOS in a Spanish population. Hadad et al68, however, found no association between CAPN10 gene variation and PCOS. Other Polymorphisms in the tumor necrosis factor receptor (TNF-R)69 and peroxisome proliferator-activated receptor-gamma (PPARg)70,71 have recently been associated with PCOS. In addition, microarray analysis identified genes that were overexpressed in PCOS theca cells compared to normal theca cells. These included aldehyde dehydrogenase 6, retinol dehydrogenase 2, and the transcription factor GATA6.72
SUMMARY There is clear evidence for an underlying genetic cause for PCOS based on familial clustering of cases. Most studies are consistent with an autosomal dominant pattern of inheritance. However, studies have been hampered by small sample sizes, errors in statistical analysis, and differences in diagnostic criteria, an inevitable consequence of PCOS being a heterogeneous disorder. Nonetheless, collectively, data are consistent with the concept that a gene or—more likely—several genes predispose to PCOS susceptibility. Data suggest that PCOS develops as the consequence of a primary genetic abnormality in ovarian androgen production, interacting with environmental factors or other factors causing hyperinsulinemia. Linkage and association studies in particular implicate a region on chromosome 19p13.3, near the insulin receptor gene. The putative PCOS gene in this region remains to be identified, but it is probably involved in signal transduction mechanisms leading to altered expression of a suite of genes affecting steroidogenesis and insulin action. Further studies utilizing molecular genetic approaches hold promise for elucidating the pathophysiology of PCOS.
Practice points † there is evidence for a genetic basis for PCOS; this is probably polygenic/ multifactorial † steroidogenesis is upregulated in PCOS theca cells † linkage and association studies implicate a region on chromosome 19p13.3
Research agenda † Identification of the putative PCOS gene(s) † the value of insulin-sensitizing medications as a treatment for PCOS † the effect of environmental factors on disease expression
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Human Molecular Genetics 1997; 6: 397–402. 44. Diamanti-Kandarakis E, Bartzis MI, Berfiele AT, et al. Microsatellite polymorphism (TTTTA)(n) at 2528 base pairs of gene cyp1alpha influences hyperandrogenemia in patients with polycystic ovary syndrome. Fertility and Sterility 2000; 73: 735 –741. 45. San Millan JL, Sancho J, Calvo RM & Escobar-Morreale HF. Role of the pentanucleotide (TTTTA)(n) polymorphism in the promoter of the cyp11a gene in the pathogenesis of hirsutism. Fertility and Sterility 2001; 75: 797– 802. 46. Escobar-Morreale HF, San Millan JL, Smith RR, et al. The presence of the 21-hydroxylase deficiency carrier status in hirsute women: phenotype-genotype correlations. Fertility and Sterility 1999; 72: 629–638. 47. Witchel SF & Aston CE. The role of heterozygosity for cyp21 in the polycystic ovary syndrome. Journal of Pediatric Endocrinology and Metabolism 2000; 13(supplement 5): 1315–1317. 48. Mifsud A, Ramirez S & Yong L. Androgen receptor gene CAG trinucleotide repeats in anovulatory infertility and polycystic ovaries. Journal of Clinical Endocrinology and Metabolism 2000; 85: 3484–3488. 49. Hickey T, Chandy A & Norman RJ. The androgen receptor CAG repeat polymorphism and X-chromosome inactivation in Australian Caucasian women with infertility related to polycystic ovary syndrome. Journal of Clinical Endocrinology and Metabolism 2002; 87: 161– 165.
718 P. Amato and J. L. Simpson 50. Iban˜ez L, Ong KK, Mongan N, Jaaskelainen J, et al. Androgen receptor gene CAG repeat polymorphism in the development of ovarian hyperandrogenism. Journal of Clinical Endocrinology and Metabolism 2003; 88: 3333–3338. 51. Hogeveen KN, Cousin P, Pugeat M, et al. Human sex hormone-binding globulin variants associated with hyperandrogenism and ovarian dysfunction. The Journal of Clinical Investigation 2002; 109: 973– 981. 52. Xita N, Tsatsoulis A, Chatzikyriakidou A & Georgiou I. Association of the (TAAAA)n repeat polymorphism in the sex hormone-binding globulin (SHBG) gene with polycystic ovary syndrome and relation to SHBG serum levels. Journal of Clinical Endocrinology and Metabolism 2003; 88: 5976–5980. 53. Talbot JA, Bicknell EJ, Rajkhowa M, et al. Molecular scanning of the insulin receptor gene in women with polycystic ovarian syndrome. Journal of Clinical Endocrinology and Metabolism 1996; 81: 1979–1983. 54. Dunaif A, Xia J, Book CV, et al. Excessive insulin receptor serine phosphorylation in cultured fibroblasts and in skeletal muscle. A potential mechanism for insulin resistance in the polycystic ovary syndrome. The Journal of Clinical Investigation 1995; 96: 801–810. 55. Moran C, Huerta R, Conway-Myers BA, et al. Altered autophosphorylation of the insulin receptor in the ovary of a woman with polycystic ovary syndrome. Fertility and Sterility 2001; 75: 625–628. 56. Siegel S, Futterwet W, Davies TF, et al. A C/T single nucleotide polymorphism at the tyrosine kinase domain of the insulin receptor gene is associated with polycystic ovary syndrome. Fertility and Sterility 2002; 78: 1240– 1243. * 57. Tucci S, Futterweit W, Concepcion ES, et al. Evidence for association of polycystic ovary syndrome in Caucasian women with a marker at the insulin receptor gene locus. Journal of Clinical Endocrinology and Metabolism 2001; 86: 446 –449. 58. Ukkola O, Rankinen R, Gagno J, et al. A genome-wide linkage scan for steroids and SHBG levels in black and white families: the HERITAGE Family Study. Journal of Clinical Endocrinology and Metabolism 2002; 87: 3708–3720. 59. Waterworth DM, Bennett ST, Gharani N, et al. Linkage and association of insulin gene VNTR regulatory polymorphism with polycystic ovary syndrome. Lancet 1997; 349: 986– 990. 60. Vankova M, Vrbikova J, Hill M, et al. Association of insulin gene VNTR polymorphism with polycystic ovary syndrome. Annals of the New York Academy of Sciences 2002; 967: 558– 565. 61. El Mkadem SA, Lautier C, Lautier C, Macari F, et al. Role of allelic variants Gly972Arg of IRS-1 and Gly1057Asp of IRS-2 in moderate-to-severe insulin resistance of women with polycystic ovary syndrome. Diabetes 2001; 50: 2164–2168. 62. Ehrmann DA, Tang X, Yoshiuchi I, et al. Relationship of insulin receptor substrate-1 and -2 genotypes to phenotypic features of polycystic ovary syndrome. Journal of Clinical Endocrinology and Metabolism 2002; 87: 4297–4300. 63. Ibanez L, Marcos MV, Potau N, et al. Increased frequency of the G972R variant of the insulin receptor substrate-1 (Irs-1) gene among girls with a history of precocious pubarche. Fertility and Sterility 2002; 78: 1288–1293. 64. Urbanek M, Wu X, Vickery KR, et al. Allelic variants of the follistatin gene in polycystic ovary syndrome. Journal of Clinical Endocrinology and Metabolism 2000; 85: 4455–4461. 65. Ehrmann DA, Schwarz PE, Hara M, et al. Relationship of calpain-10 genotype to phenotypic features of polycystic ovary syndrome. Journal of Clinical Endocrinology and Metabolism 2002; 87: 1669–1673. 66. Gonzalez A, Abril E, Roca A, et al. Comment: CAPN10 alleles are associated with polycystic ovary syndrome. Journal of Clinical Endocrinology and Metabolism 2002; 87: 3971–3976. 67. Gonzalez A, Abril E, Roca A, et al. Specific CAPN10 gene haplotypes influence the clinical profile of polycystic ovary patients. Journal of Clinical Endocrinology and Metabolism 2003; 88: 5529–5536. 68. Haddad L, Evans JC, Gharani N, et al. Variation within the type 2 diabetes susceptibility gene calpain-10 and polycystic ovary syndrome. Journal of Clinical Endocrinology and Metabolism 2002; 87: 2606–2610. 69. Peral B, San Millan JL, Castello R, et al. Comment: the methionine 196 arginine polymorphism in exon 6 of the TNF receptor 2 gene (TNFRSF1B) is associated with the polycystic ovary syndrome and hyperandrogenism. Journal of Clinical Endocrinology and Metabolism 2002; 87: 3977–3983. 70. Orio F, Matarese G, Di Biase S, et al. Exon 6 and 2 peroxisome proliferator-activated receptor-gamma polymorphisms in polycystic ovary syndrome. Journal of Clinical Endocrinology and Metabolism 2003; 88: 5887–5892. 71. Korhonen S, Heinonen S, Hiltunen M, et al. Polymorphism in the peroxisome proliferator-activated receptor-gamma gene in women with polycystic ovary syndrome. Human Reproduction 2003; 18: 540–543. 72. Wood JR, Nelson VL, Ho C, et al. The molecular phenotype of polycystic ovary syndrome (PCOS) theca cells and new candidate PCOS genes defined by microarray analysis. Journal of Biological Chemistry 2003; 278: 26380–26390.
3 The genetics of polycystic ovary syndrome Paula Amato MD Joe Leigh Simpson*
MD
Department of Obstetrics and Gynecology, Baylor College of Medicine, 6550 Fannin, Suite 901A, Houston, TX 77030, USA
Polycystic ovary syndrome (PCOS) is a heterogeneous disorder. There is evidence for a genetic component in PCOS based on familial clustering of cases. The majority of the evidence supports an autosomal dominant form of inheritance. Steroidogenesis has been shown to be upregulated in PCOS theca cells, suggesting that the genetic abnormality in PCOS affects signal transduction pathways controlling the expression of a family of genes. Although a number of candidate genes have been proposed, the putative PCOS gene(s) has yet to be identified. Linkage and association studies implicate a region near the insulin receptor gene at chr 19p13.3. New genetic approaches, such as microarray technology, hold promise for elucidation of the pathophysiology underlying PCOS. Key words: genetics; hyperandrogenism; polycystic ovaries.
Polycystic ovary syndrome (PCOS) is a common endocrinopathy affecting 5 – 10% of women of reproductive age. The 2003 Rotterdam Consensus concluded that PCOS is a syndrome of ovarian dysfunction requiring two out of three of the following criteria for diagnosis: (i) oligo- or anovulation; (ii) hyperandrogenism and/or hyperandrogenemia; and (iii) polycystic ovary morphology. The exclusion of other etiologies, such as congenital adrenal hyperplasia, androgen-secreting tumors, and Cushing’s disease, is also necessary.1 The clinical manifestations of PCOS include menstrual irregularities, signs of hyperandrogenism, and obesity. Premature pubarche might be the earliest recognizable phenotype of PCOS.2 Hypersecretion of luteinizing hormone (LH) and insulin resistance are common features of PCOS, which is associated with an increased risk of type 2 diabetes mellitus (DM) and cardiovascular events.3 – 6 Some sections of this chapter inevitably reflect our previous reviews.7,8
PATHOPHYSIOLOGY Although the underlying pathophysiology of PCOS remains unknown, attention has centered upon primary defects in the hypothalamic –pituitary axis, ovarian function, * Tel.: þ 1-713-798-8360; Fax: þ1-713-798-8410. E-mail address: [email protected] (J.L. Simpson). 1521-6934/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.
708 P. Amato and J. L. Simpson
and insulin secretion and action. Hyperandrogenemia, LH hypersecretion, insulin resistance, and compensatory hyperinsulinemia are common biochemical features of PCOS. Hyperandrogenemia can result in the development of polycystic ovaries, as well as affect hypothalamic-pituitary function. LH hypersecretion is a common feature of PCOS. LH is believed to play a permissive role augmenting ovarian androgen production in PCOS. Insulin resistance and the associated compensatory hyperinsulinemia might be key factors in the pathophysiology of PCOS. Approximately 50% of PCOS women are insulin resistant when compared with age- and weight-matched controls.9 Insulin is postulated to augment the stimulatory action of LH on theca cell androgen biosynthesis through upregulation of genes encoding steroidogenic enzymes. Hyperinsulinemia stimulates ovarian androgen secretion directly and suppresses sex hormone binding globulin (SHBG) levels, resulting in an increase in bioavailable androgens. Androgens can in turn affect hypothalamic control of pituitary gonadotropin secretion, and also serve as precursors for peripheral aromatization to estrogen. Insulin-sensitizing drugs such as metformin and the thiazolinediones act by reducing insulin levels, which in turn reduces androgen levels. Freshly isolated theca cells collected from PCOS ovaries show enhanced production of dehydroepiandrosterone (DHEA), progesterone, 17-hydroxyprogesterone, and androstenedione.10 This increased steroidogenic activity is due to increased 3b-hydroxysteroid-dehydrogenase and 17a-hydroxylase/17,20-lyase activities.11,12 Northern blot analysis revealed that cytochrome P450 17-hydroxylase/17, 20desmolase (CYP17) and cytochrome P450 side-chain clearage enzyme (CYP11A) mRNAs were more abundant in PCOS theca cells than in normal theca cells. In addition, transient transfection experiments indicated that the CYP17 promoter is enhanced in PCOS theca cells compared to normal theca cells.13 The upregulation of steroidogenesis in PCOS theca cells might be the result of a genetic abnormality in these cells or a metabolic imprint received in vivo. Nelson et al11 showed that propagated PCOS thecal cells maintained in long-term cultures displayed enhanced steroidogenesis, suggesting that the upregulation of steroidogenesis is the result of a genetic abnormality, although a persistent metabolic imprint established in vivo cannot be excluded.
FAMILY STUDIES There appears to be evidence for a genetic component in PCOS based on familial clustering of cases. Hyperandrogenemia appears to be the strongest genetically inherited characteristic in familial cases. The investigation of the genetics of PCOS has been hampered by several factors. Because PCOS is associated with infertility, the availability of large pedigrees for linkage analysis is limited. Furthermore, because PCOS is a heterogeneous disorder1, family studies cannot be readily compared because of the use of different diagnostic criteria. In addition, a male phenotype for PCOS has not been clearly identified. These challenges notwithstanding, the majority of studies suggest a dominantly inherited trait of low penetrance and variable expressivity. This mode of inheritance would be consistent with the variable clinical findings in PCOS. However, polygenic, multifactorial inheritance or genetic heterogeneity cannot be excluded. Table 1 summarizes the familial studies. The first formal genetic study was by Cooper et al15, who studied 18 patients with Stein –Leventhal syndrome. Oligomenorrhea, hirsutism, and enlarged ovaries were
Table 1. Summary of studies assessing frequency of PCOS in female relatives.
Author Cooper et al
Diagnostic criteria Oligomenorrhea, hirsutism, polycystic ovaries (culdoscopy, gynecography, or wedge resection)
Oligomenorrhea, hirsutism, and polycystic ovaries (exam and surgery)
Ferriman and Purdie
Hirsutism and/or oligomenorrhea (60% with polycystic ovaries by air contrast gynecography)
Lunde et al
Menstrual dysfunction, hyperandrogenism, obesity, infertility and polycystic ovaries (transabdominal ultrasound)
Hague et al
Menstrual irregularities, hirsutism, infertility, obesity and multicystic ovaries (wedge resection)
Carey et al
Polycystic ovaries by transabdominal ultrasound
Oligomenorrhea
Hirsutism Elevated 24-hour urinary 17-ketosteroids Enlarged ovaries Oligomenorrhea
Affected sisters (%)
Affected mothers (%)
9/19 (47%)
4/13 (31%)
14/24 (58%) 12/19 (63%)
4/13 (31%) 2/7 (29%)
10/19 (53%)
0/7 (0%) 16/67 (24%)
Hirsutism Hirsutism
30/337 (9%)
32/284 (5%)
Oligomenorrhea Hirsutism
32/337 (9%) 8/129 (6%)
24/284 (8%) 17/132 (13%)
Oligomenorrhea Hirsutism
19/129 (15%)
16/132 (12%)
Oligomenorrhea Polycystic ovaries (ultrasound) Elevated testosterone
Overall affected first-degree female relatives (%)
28/54 (52%)
28/107 (26%)
19/107 (18%) 37/50 (74%) 16/50 (32%) (continued on next page)
The genetics of polycystic ovary syndrome 709
Givens et al
Frequency of specific trait assessed
Author Norman et al
Legro et al Govind et al Kahsar-Miller et al
Diagnostic criteria Elevated androgens, decreased SHBG, polycystic ovaries (ultrasound)
Elevated testosterone and oligomenorrhea Polycystic ovaries (ultrasound) Oligomenorrhea and either hirsutism or
Frequency of specific trait assessed
Affected sisters (%)
Affected mothers (%)
11/15 (73%)
1/1 (100%)
Polycystic ovaries (ultrasound) Increased testosterone or androstenedione Hyperinsulinemia Hyperadrogenemia
13/15 (87%)
1/5 (20%)
10/15 (66%) 36/80 (45%)
5/5 (100%)
Polycystic ovaries PCOS as defined
35/53 (66%) 16/50 (32%)
15/29 (52%) 19/78 (24%)
Overall affected first-degree female relatives (%)
50/82 (61%)
710 P. Amato and J. L. Simpson
Table 1 (continued)
The genetics of polycystic ovary syndrome 711
much more common in sisters of cases than in sisters of controls. In addition, hirsutism or ‘pelosity’ was more prevalent in male relatives. In the 1970s, Givens and others at the University of Tennessee, Memphis, published reports indicating that PCOS could be inherited in an X-linked dominant fashion. Diagnostic criteria consisted of hirsutism and either polycystic or bilaterally enlarged ovaries. In the first report, Givens et al16 described two families in which multiple individuals in more than two generations were affected. In one kindred, affected females experienced myocardial infarction in their fifth decade; and acanthosis nigricans, insulin resistance, and hypertension were present in many family members. Most subjects were African – American. In a third kindred17, several males showed maturational arrest of spermatogenesis. Excluding index cases, Wilroy et al18 tabulated that 47% of female offspring of affected females were affected. Among the offspring of males with an elevated LH/follicle stimulating hormone (FSH) ratio, 89% of daughters were affected. That almost all daughters of affected males were affected is consistent with X-linked dominant inheritance. In the United Kingdom, Ferriman and Purdie19 studied 381 patients with hirsutism and/or oligomenorrhea and a control group of 179 women. Familial tendencies were greatest among hirsute women with enlarged ovaries. Of 188 patients with hirsutism and enlarged ovaries, 38 first-degree relatives had hirsutism, 30 oligomenorrhea, and 19 infertility. Of 96 patients with hirsutism but normal sized ovaries, the numbers of first-degree relatives with the above traits were 73, 15, and 10, respectively. In 179 controls, numbers were 7, 8, and 8. Baldness was significantly increased in male relatives of hirsute women. Later British studies provided additional data in support of heritability of PCOS, specifically autosomal dominant inheritance. Hague et al20 used ultrasound criteria and either hyperandrogenemia or LH hypersecretion to determine the frequency of PCOS in relatives of affected cases. PCOS was found in 45 of 52 (87%) of sisters of probands and in 24 of 36 (67%) of mothers. The frequency of affected relatives was dramatically higher than the 50% predicted for either autosomal dominant or X-linked dominant inheritance, raising doubts about specificity of diagnostic criteria. Non-Mendelian mechanisms would need to be invoked in order to account for such a distorted segregation ratio. More likely, criteria were overly sensitive, leading to diagnosis false positivity. In later British studies the frequency of affected relatives in PCOS was nearer the 50% expected for autosomal dominant inheritance. Genetic studies were performed by Carey et al.21 First-degree relatives of 10 probands were studied. Of 62 informative relatives, 58 were screened by ultrasound. Of the 58 female first-degree relatives, 51% were affected. Family studies were conducted in Norway by Lunde et al Using hirsutism and oligomenorrhea as inclusion criteria, Lunde et al22 found only 6 – 15% of first-degree relatives affected. Norman et al23 found that, in 15 probands, far more relatives were affected. Among sisters, 11 of 15 (73%) had polycystic ovaries by ultrasound, 13 of 15 (87%) had elevated testosterone (T), and 10 of 15 (66%) hyperinsulinemia. In the US (Pennsylvania), Legro et al24 studied 80 probands diagnosed on the basis of elevated testosterone associated with oligomenorrhea (, 6 menses/year); non-classical 21-hydroxylase deficiency was excluded. They found 36 of 80 (45%) sisters to be affected on the basis of hyperandrogenemia. Govind et al25 studied 29 probands and 10 control women. Diagnostic criteria consisted of polycystic ovaries on ultrasound with or without clinical or biochemical features of PCOS; 61% of female first-degree relatives were affected and 22% of male first-degree relatives had early onset (before age 30) male-pattern baldness. The prevalence was much
712 P. Amato and J. L. Simpson
higher than in the control families. Of a total of 71 sibs of PCOS probands, 39 (55%) were affected, which is consistent with autosomal dominant inheritance. Kahsar-Miller and co-workers26 considered the frequencies of oligomenorrhea and either hirsutism or elevated testosterone among first-degree female relatives within families of 93 probands with PCOS. A significantly higher rate of PCOS was observed among first-degree relatives than in the general population, suggesting genetic component in the disorder. Concordantly affected twins have been observed.27,28 However, discordance has been observed even for monozygotic twins.29 Using a large genealogy database to search for a founder effect and to evaluate the degree of heritability in PCOS, Ward et al30 showed that the degree of relatedness among a PCOS population was four-fold greater than the average degree of relatedness among a large random sampling of the same database.
CANDIDATE GENES That a number of genes show altered patterns of expression suggests that the genetic abnormality in PCOS affects signal transduction pathways controlling the expression of a family of genes, rather than the abnormal expression of a single gene encoding a single steroidogenic enzyme. Consistent with this, cytogenetic studies have failed to identify common karyotypic abnormalities. A consistently observed aberration characterized by a specific breakpoint would indicate localization of a causative gene. Because abnormalities of steroidogenesis are a prominent feature of PCOS, investigators have long sought linkage or associations between PCOS and the various genes involved in the androgen biosynthetic pathway or metabolic pathways involved in insulin action. Linkage analysis is used to demonstrate the co-segregation of a genetic variant and a disease locus. Association studies assess whether a genetic variant is linked to a disease locus on a population scale. Family-based linkage disequilibrium test (TDT) determines whether parents heterozygous for a disease allele transmit that allele more often to their affected children than the non-disease allele. A number of linkage and association studies of candidate genes in PCOS have yielded negative results.31 – 39 Studies with positive and/or mixed results are discussed in more detail below. CYP17 (cytochrome P450 17-hydroxylase/17, 20-desmolase) Although initial studies suggested an association between cytochrome CYP17, which encodes 17-hydroxylase/17,20-lyase, and PCOS, subsequent studies failed to confirm this finding making this gene an unlikely PCOS gene candidate.40 – 42 CYP11A (cytochrome P450 side-chain cleavage enzyme) Gharani et al43 found evidence for a weak linkage between the CYP11A gene, which encodes the cholesterol side-chain cleavage enzyme, and hyperandrogenemia in PCOS women. An association study of 97 women with PCOS demonstrated a strong association between the CYP11A 50 UTR pentanucleotide repeat polymorphism with total serum testosterone levels. In addition to the pitfalls mentioned earlier, these early
The genetics of polycystic ovary syndrome 713
studies are limited because of failure to make appropriate statistical adjustments for multiple testing. In another study involving CYP11A, an association was found between a pentanucleotide repeat at 2 528 of the CYP11A gene and PCOS.44 However, other studies have failed to find a significant association between CYP11A and PCOS.34,45 Although perturbations in the CYP11A gene cannot easily account for altered expression of other steroidogenic enzymes, the locus remains a potential candidate gene. CYP21 (cytochrome P450 21-hydroxylase) CYP21 encodes 21-hydroxylase, the enzyme responsible for most cases of congenital adrenal hyperplasia (CAH). Recent studies have found a significant prevalence of CYP21 mutations in PCOS women with a normal 17-hydroxyprogesterone response to adrenocorticotropic hormone (ACTH) stimulation, calling into question the diagnostic distinction between PCOS and CAH.46,47 Androgen receptor Urbanek et al34 studied 150 families and failed to find evidence for association of the trinucleotide (CAG) repeat polymorphism in the X-linked androgen receptor gene and PCOS. However, this short CAG repeat length has been shown to be inversely associated with androgen levels.48 A study of 122 women with PCOS demonstrated a significantly greater frequency of longer CAG alleles and biallelic means (. 22 repeats) in exon 1 of the androgen receptor in women with PCOS compared to normal women.49 Iban˜ez et al50 found an association between the shorter androgen receptor gene CAG repeat polymorphism and precocious pubarche. Sex hormone binding globulin (SHBG) Hogenveen et al51 identified a polymorphism in the coding region of SHBG that encodes a missense mutation, P156L, in 4 of 482 women with PCOS, hirsutism, or ovarian dysfunction. Xita et al52 showed an association between the (TAAAA)n polymorphism in the SHBG and PCOS. Women with PCOS were found to have a significantly greater frequency of longer (TAAAA)n alleles (more than eight repeats) than normal women who had shorter alleles (less than eight repeats) in higher frequency. Insulin receptor A number of studies have examined the insulin receptor for gene sequence major mutations—with negative results.53 This suggests that if perturbed insulin action is integral to PCOS, the mechanism likely involves a target downstream of the insulin receptor. Dunaif et al54 found that , 50% of women with PCOS showed increased insulin receptor serine phosphorylation in skeletal muscle cells and fibroblasts. In a recent study, tyrosine autophosphorylation of the insulin receptor was also found to be decreased in ovaries of women with PCOS.55 Siegel et al56 showed an association
714 P. Amato and J. L. Simpson
between a C/T single nucleotide polymorphism at the tyrosine kinase domain of the insulin receptor gene and PCOS. Two separate studies have found linkage and association between a marker (D19S884 at chr 19p13.3) located near the insulin receptor gene and PCOS.34,57 The National Cooperative Program in Infertility Research conducted linkage and association studies using both sib-pair analysis and transmission/disequilibrium tests to assess a number of candidate genes in a cohort of Caucasian families. In this study, evidence for linkage and association with a region on chr 19p13.3 was found. The evidence for linkage was strongest at the marker, D19S884. The Heritage Family study found evidence for statistically significant linkage between a region at chr 19p13.3 and androgen levels in Caucasians, providing further evidence for an important role for the region at chr 19p13.3 in PCOS.58 Although the putative PCOS gene in this region remains to be identified, data suggest likely involvement in signal transduction mechanisms, leading to altered expression of a family of genes involved in steroidogenesis and/or insulin action. Insulin Waterworth et al59 found strong linkage and association between the class III allele at the insulin gene 50 VNTR (variable number tandem repeats) in the 50 region of the insulin gene and PCOS. However, in a larger study, Urbanek et al34 found no evidence for linkage of the insulin gene and PCOS and no association between the class III allele of the insulin VNTR and hyperandrogenemia. Vankova et al60 also found no association between the INS VNTR polymorphism and PCOS. Insulin receptor substrate proteins A study by El Mkadem et al61 revealed an association between the Gly972Arg IRS-1 variant and the Gly1057Asp IRS-2 variants and insulin resistance in women with PCOS. Ehrmann et al62 found no association of IRS-1 with PCOS. However, the IRS-2 Gly/ Gly polymorphism was associated with higher blood glucose levels in nondiabetic White and African –American women with PCOS. Iban˜ez et al63 found an increased frequency of the G972R variant of the IRS-1 gene among girls with a history of precocious puberty. Follistatin An initial study of 39 affected sibling pairs demonstrated statistically significant linkage to follistatin.33 However, subsequent larger studies and more detailed sequence analysis of the follistatin gene have not revealed significant linkage.64 Calpain-10 Calpain-10 is a cysteine protease that has been shown to be associated with susceptibility to type 2 diabetes. In a recent study, the 112/121-haplotype was associated with higher insulin levels in African – American women, and an increase risk of PCOS in both African – American and white women.65
The genetics of polycystic ovary syndrome 715
Gonzalez et al66,67 showed an association of the CAPN10 UCSNP-44 allele with PCOS in a Spanish population. Hadad et al68, however, found no association between CAPN10 gene variation and PCOS. Other Polymorphisms in the tumor necrosis factor receptor (TNF-R)69 and peroxisome proliferator-activated receptor-gamma (PPARg)70,71 have recently been associated with PCOS. In addition, microarray analysis identified genes that were overexpressed in PCOS theca cells compared to normal theca cells. These included aldehyde dehydrogenase 6, retinol dehydrogenase 2, and the transcription factor GATA6.72
SUMMARY There is clear evidence for an underlying genetic cause for PCOS based on familial clustering of cases. Most studies are consistent with an autosomal dominant pattern of inheritance. However, studies have been hampered by small sample sizes, errors in statistical analysis, and differences in diagnostic criteria, an inevitable consequence of PCOS being a heterogeneous disorder. Nonetheless, collectively, data are consistent with the concept that a gene or—more likely—several genes predispose to PCOS susceptibility. Data suggest that PCOS develops as the consequence of a primary genetic abnormality in ovarian androgen production, interacting with environmental factors or other factors causing hyperinsulinemia. Linkage and association studies in particular implicate a region on chromosome 19p13.3, near the insulin receptor gene. The putative PCOS gene in this region remains to be identified, but it is probably involved in signal transduction mechanisms leading to altered expression of a suite of genes affecting steroidogenesis and insulin action. Further studies utilizing molecular genetic approaches hold promise for elucidating the pathophysiology of PCOS.
Practice points † there is evidence for a genetic basis for PCOS; this is probably polygenic/ multifactorial † steroidogenesis is upregulated in PCOS theca cells † linkage and association studies implicate a region on chromosome 19p13.3
Research agenda † Identification of the putative PCOS gene(s) † the value of insulin-sensitizing medications as a treatment for PCOS † the effect of environmental factors on disease expression
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