Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2006-0069
The Journal of Clinical Endocrinology & Metabolism Vol. 91, No. 7 2789-2791
Copyright © 2006 by The Endocrine Society
Epigenetic Modification of the X Chromosome Influences Susceptibility to Polycystic Ovary Syndrome
T. E. Hickey,
R. S. Legro and
R. J. Norman
Research Centre for Reproductive Health (T.E.H., R.J.N.), University of Adelaide, Department of Obstetrics and Gynecology, The Queen Elizabeth Hospital, Woodville, South Australia 5011, Australia; and Department of Obstetrics and Gynecology (R.S.L.), Penn State College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033
Address all correspondence and requests for reprints to: Theresa E. Hickey, Department of Obstetrics and Gynaecology, University of Adelaide, 1st Floor Maternity Building, The Queen Elizabeth Hospital, 28 Woodville Road, Woodville, South Australia 5011, Australia. E-mail: theresa.hickey{at}adelaide.edu.au.
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Abstract
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Context: The cause of polycystic ovary syndrome (PCOS) is unknown, although genetic and environmental influences are clearly implicated. Some genetic studies have suggested the involvement of X-linked genes in PCOS, but the influence of X chromosome inactivation (XCI) on manifestation of this disorder has not previously been examined.
Objective: The objective of the study was to test the null hypothesis that XCI has no influence on clinical presentation of PCOS.
Design: We examined patterns of XCI between sister pairs with the same genotype at a polymorphic locus on the X chromosome in families with PCOS.
Setting: The study was conducted at a private practice.
Participants: PCOS was defined as hyperandrogenemia with chronic anovulation. Forty families were studied in which DNA was obtained from at least one parent, the proband, and one sister that could be accurately diagnosed as being affected or unaffected.
Main Outcome Measure(s): Relative expression of two X-linked alleles was determined, and the ratio of one to the other represented the pattern of XCI.
Results: The statistical odds on a different clinical presentation between sisters was approximately 29 times higher in sister pairs with different patterns of XCI, compared with sister pairs with the same pattern of XCI (odds ratio 28.9; 95% confidence interval 4.0–206; P = 0.0008).
Conclusions: This study provides evidence to refute the null hypothesis and propose a closer inspection of X-linked genes in PCOS, one in which both genotype and epigenotype are considered. Environmental determinants of PCOS may alter clinical presentation via epigenetic modifications, which currently remain undetected in traditional genetic analyses.
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Introduction
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POLYCYSTIC OVARY SYNDROME (PCOS) is a complex endocrine disorder characterized by unexplained hyperandrogenism, chronic anovulation, and the occurrence of polycystic ovaries (1). Familial aggregation in this disorder implies underlying genetic influences (2), but extensive investigation into the identity of the gene(s) involved and their mode of transmission has yet to yield any valid candidates or clear inheritance patterns, due in part to the difficulties endemic to such an endeavor (3). Among the numerous genetic studies of PCOS, a few have suggested the involvement of genes located on the X chromosome. These include a study of women with mosaicism for Turner’s syndrome (4), two studies of family groups with PCOS (5, 6), and the only twin study of PCOS published to date (7). The latter study suggested that the symptomatic discordance observed in both mono- and dizygotic twins with PCOS could potentially result from unequal expression of X-linked genes. Women usually do not manifest X-linked diseases unless nonrandom inactivation of the normal X chromosome occurs, thereby increasing expression of the abnormal allele on the opposing chromosome (8). Significant differences in X chromosome inactivation (XCI) patterns between normal female monozygotic twins has been reported (9), and X-linked diseases can be differentially manifest in female twins due to variable patterns of XCI (10, 11). This suggests that differential XCI could occur in twin sisters discordant for PCOS and would confound traditional linkage studies involving genes on the X chromosome. To date, the only X-linked candidate gene for PCOS is the androgen receptor (AR), which Urbanek et al. (12) reported as having no significant linkage. Analyzing methylation status of sequences upstream of the highly polymorphic (CAG)n repeat region in the AR gene is the most common method used to determine XCI patterns in females (13). In the current study, we used this method to compare sisters in family groups with PCOS to determine whether XCI plays a role in the presentation of this disorder.
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Subjects and Methods
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Informed consent to participate in this study was sought from members of nuclear families previously recruited for genetic studies by one of the investigators (R.S.L.) in Hershey, PA, in which at least one sister had PCOS as defined by the 1990 National Institute of Child Health and Human Development criteria of hyperandrogenemia (HA) and chronic anovulation (14). A total of 40 families provided sufficient consent and genetic material to analyze at least one parent (to determine parental origin of alleles), the proband, and at least one sister. As described by Urbanek et al. (12), sisters were considered affected if given a diagnosis of PCOS or HA and unaffected if they had normal circulating androgen levels, were not taking any confounding medications (e.g. oral contraceptives), and had regular menstrual cycles (menses every 27–35 d). Sisters who were taking confounding medications or had irregular menstrual cycles in the absence of HA were considered to be of unknown status and were excluded from the study. The (CAG)n variable nucleotide repeat region in exon 1 of the human AR gene was used to genotype and determine XCI patterns between sisters, as previously described (15). This method allows determination of relative levels of inactivation of the two inherited X chromosomes, and by reciprocal association, the relative transcriptional activity expected from each one. Patterns of XCI were considered different between heterozygous sisters with the same genotype if: 1) opposing alleles were preferentially inactivated, or 2) one sister had a random pattern of XCI and the other had a nonrandom pattern (defined as 
60% inactivation of one allele). A logistic generalized estimating equation was used to estimate the odds ratio, comparing the odds of different clinical presentation in sister pairs with different patterns of XCI or the same pattern of XCI and adjusting for the fact that some sister pairs came from the same family (SAS 9.1; SAS Institute, Cary, NC). Significance was set at P < 0.05.
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Results
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In total, 88 sister pairs were formed from the 40 family groups, in which seven families provided multiple sister pairs and 33 families provided a single sister pair. Group distribution, percent of sister pairs with the same genotype, and number of heterozygous pairs with the same genotype suitable for XCI analysis are presented in Table 1
. For comparison of patterns of XCI, the 18 affected-affected sister pairs were subdivided into two groups based on whether they had the same clinical presentation (PCOS-PCOS; n = 9 or HA-HA; n = 2) or a different clinical presentation (PCOS-HA; n = 7). Analysis of patterns of XCI among all groups (Table 2) shows that in the majority (84%) of cases in which sister pairs had the same genotype but a different clinical presentation, a different pattern of XCI was evident, which implies a nonequivalent dose of gene products from a particular X chromosome. In contrast, this occurred in the minority (15%) of cases in which sister pairs had the same genotype and the same clinical presentation. Statistically, the odds on a different clinical presentation were approximately 29 times higher in sister pairs with different patterns of XCI, compared with sister pairs with the same pattern of XCI (odds ratio 28.9; 95% confidence interval 4.0–206; P = 0.0008). This suggests that the odds of having a different clinical presentation are dependent on the pattern of XCI. Overall, XCI analysis was determined in a total of 28 unaffected sisters and 54 affected sisters. There was no statistical difference (P = 0.07) in distribution of XCI patterns between these two groups as a whole, indicating that affected sisters do not have a higher prevalence of nonrandom XCI per se. Therefore, the impact of XCI had to be considered on a sister-pair basis to implicate a particular X chromosome in the disease process. An exemplary family group with multiple participating siblings is illustrated in Fig. 1.
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FIG. 1. Example of X chromosome segregation and activation among sisters in a family group with PCOS. Diagnosis for the mother could not be determined (ND). The three affected sisters all inherited the same maternal X chromosome, whereas their unaffected (UA) sisters inherited the alternate maternal X chromosome. The graph depicts the degree to which each X chromosome is active, as inferred by measuring inactivation of the opposing X chromosome. Among affected sisters there is a clear difference in XCI patterns between the sister with PCOS, in whom the paternal X is nonrandomly inactivated, and the two sisters with HA who display random XCI. Theoretically, this provides the PCOS sister with a greater dose of gene products transcribed from the maternal X inherited by all affected sisters. The two unaffected sisters also substantially inactivate the paternal X chromosome, thereby accentuating the effect of inheriting different maternal X chromosomes by diminishing the influence of the common paternal X chromosome. Although a particular maternal X chromosome was implicated in this particular family, we did not observe a parent-of-origin effect of implicated X chromosomes in the study as a whole.
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Discussion
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PCOS is clearly a disorder conditioned by both genetic and environmental influences (16), but to date, there have been no studies that examine how environmental influences could have an impact at the genetic level in this disorder. Epigenetic modification of DNA could be a means by which an environmental influence directly affects the behavior of genes without altering the genetic code itself, and new evidence comparing epigenetic differences in monozygotic twins indicates that such differences are likely to be involved in the etiology of complex diseases (17). We provide the first evidence that in families affected by PCOS, differential XCI occurs in the majority of cases in which phenotype does not segregate with genotype and in which heterogeneous symptoms occur among affected sisters with the same genotype. This suggests that, in addition to genetic linkage, inactivation of the X chromosome is an important contributor to manifestation of the PCOS phenotype.
Mounting evidence suggests that the origins of PCOS occur in utero or early life, a concept powerfully supported by prenatally androgenized animal models (18). Timing of gestational androgen excess is important because different PCOS-associated characteristics arise when the hormonal insult is administered at various stages of fetal organogenesis (18). Clonal selection of cells that are dependent on X-linked genes for growth and differentiation during development is the most likely cause of nonrandom X-inactivation patterns in adult tissue (8), although mutations of genes involved in the X-inactivation process can cause complete inactivation of a particular X chromosome (19, 20). Naumova et al. (21) report a strong sister-sister correlation in degree of XCI, consistent with our observation that sister pairs with the same genotype and the same clinical presentation had similar XCI patterns. Divergence from this norm in sister pairs with a different clinical presentation for PCOS suggests a developmental clonal selection process, perhaps one influenced by a hyperandrogenic environment. Our findings accord with the concept of an early origin for PCOS and offer a possible mechanism through which environmental conditions during gestation or early life influence gene expression patterns later in life. Variable familial XCI of an X-linked gene could also explain why the migration of PCOS in family groups tends to behave in a manner suggestive of a dominant gene effect with incomplete penetrance (22).
Whereas our study further substantiates candidature of the AR, our results implicate any gene on the X chromosome subject to inactivation. Our analysis was performed on DNA from peripheral blood leukocytes. Examination of other cell types corresponding to specific target tissues likely to be involved with PCOS is desirable, although ovarian tissue from family members would be extremely difficult to obtain and represents a challenge for future investigations.
We believe our findings warrant a closer inspection of X-linked genes in PCOS, one in which both genotype and epigenotype are considered. Examination of epigenetic modifications to the genetic code may be necessary to unravel some of the confounding complexities that have so far hindered the identification of causal genes for this disorder.
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Footnotes
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Disclosure statement: T.E.H., R.S.L., and R.J.N. have nothing to declare.
First Published Online April 24, 2006
Abbreviations: AR, Androgen receptor; HA, hyperandrogenemia; PCOS, polycystic ovary syndrome; XCI, X chromosome inactivation.
Received January 12, 2006.
Accepted April 18, 2006.
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Abstract
Introduction
