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Risk Factors for Hypospadias in the Estrogen Receptor 2 Gene

Departments of Molecular Medicine and Surgery (A.B.-M., F.L., C.S., A.N.) and Clinical Neurosciences (I.K.), Karolinska Institutet, SE 171 76 Stockholm, Sweden; and Department of Women and Child Health (A.N.), Astrid Lindgren Children Hospital, Karolinska University Hospital, 171 76 Stockholm, Sweden

Address all correspondence and requests for reprints to: Ana Beleza-Meireles, Department of Molecular Medicine and Surgery, Building CMM 00, Karolinska University Hospital, Solna, SE 171 76 Stockholm, Sweden. E-mail: Ana.Beleza{at}ki.se; or Agneta.Nordenskjold{at}ki.se.

	Abstract

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Context: Hypospadias is a common inborn error of the male genitalia of complex, and still elusive, etiology. The presence of active estrogen receptors (ESRs) in the developing male urethra, predominantly the ESR2, has suggested a role of estrogens in the otherwise androgen-dependent male genital differetiation. Moreover, imbalances between these two steroid hormones have been suggested to disturb the external genital development. This has been supported by the association between longer (CA)n variants in the ESR2 gene with lower androgen levels as well as with hypospadias.

Objective: The aim of this study was to analyze the effect of ESR2 gene variants on the risk to hypospadias.

Design, Participants, and Methods: Four haplotype-tagging single nucleotide polymorphisms (rs2987983, rs1887994, rs1256040, and rs1256062), the (CA)n polymorphism, and two additional promoter single nucleotide polymorphisms (rs10483774 and rs1271572), mapping to a transcription factor binding region, were typed and analyzed in a Swedish cohort of 354 boys with nonsyndromic hypospadias and 380 healthy controls.

Results: Association was identified with longer variants of the (CA)n polymorphism in intron 6 and with a region of intense transcription factor binding, in the putative promoter region, mapping to rs2987983 and rs10483774. The two regions are in low-linkage disequilibrium, meaning that they are not necessarily inherited together as a haplotype; logistic regression analysis indicates that these two risk effects are not independent.

Conclusions: The present study evidences two nonindependent risk factors for hypospadias in the ESR2 gene. We discuss possible mechanisms that explain how these variants may affect male urethral development.

	Introduction

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THE DEVELOPMENT OF the external male genitalia is a complex process, involving genetic programming, cell differentiation, hormonal signaling, enzyme activity, and tissue remodeling (1). A disturbance in these processes might lead to hypospadias, a midline fusion defect of the male ventral urethra, defining a continuum, from a malformed urethral meatus at the subcoronal margin, or on the glans penis, to an intersex condition (1). Hypospadias is a common genital birth defect, affecting 0.3–7 in 1000 live male births worldwide (2). The reported incidence of hypospadias in Sweden was 1.53 in 1000 in 1982 (3). The etiology of hypospadias is considered multifactorial, with several genetic and environmental factors interacting, a model that yields the highest heritability indices (4, 5).

Familial clustering of hypospadias among first-degree relatives, as well as twin studies and segregation analysis have supported a strong heritable component in this disorder (5, 6). Androgens and the androgen receptor (AR) are essential for the development of the male external genital. Not surprisingly, disruption of the AR gene, as well as of genes involved in the androgen metabolism, including the 5-reductase type II and the 17-ß hydroxysteroid dehydrogenase, have been found in patients with hypospadias. However, these explain only a small subset of the cases of nonsyndromic hypospadias (7, 8, 9).

Environmental toxicants and xenoestrogens, acting during fetal life, have been partly implicated in an increasing incidence of hypospadias, as well as other reproductive tract abnormalities (10). Several lines of evidence have suggested that estrogens can modulate serum androgen levels (11, 12, 13). An additional concept explaining these disorders is of androgen-estrogen imbalances created by endocrine disruptors, affecting androgen production and action (13, 14). These effects could be influenced by gene variants in the estrogen or androgen pathways, an aspect that is plausible, given the ethnic differences in the response to environmental toxicants (15).

The importance of estrogens in male reproductive tract development has been increasingly acknowledged. Estrogens act through specific nuclear estrogen receptors (ESRs). Two forms of the human ESR, ESR1 and ESR2, occur, each with distinct tissue and cell patterns of expression and modest overall sequence identity (16). Functional ESRs were detected in differentiating male external genitalia, in which the ESR2 is the predominant form, localized at the same structures as the ARs, indicating interactions between effects exerted by the two steroid hormones (17, 18, 19). An excess of estrogens in the developing murine urethra results in an inhibition of the cell proliferation in a dose-dependent manner (20), and hypospadias has been induced in a mice model by maternal exposure to synthetic estrogens during pregnancy (21).

Furthermore, variants of the ESR2 influence serum levels of testosterone. Westberg et al. (22) have shown that individuals with relatively longer lengths of the (CA)n repeat polymorphism in the intron 6 of ESR2 displayed lower androgen levels. Recently, we reported an association of hypospadias with longer (CA)n repeat lengths in ESR2 in a small cohort (23), which we speculated being due to lower levels of androgens (22, 23, 24). Because no coding or splicing mutations were found in this gene (23), one could either explain these results by a direct effect of the longer (CA)n polymorphisms (25) or by another sequence variation in linkage disequilibrium (LD) with that polymorphism. Association with polymorphisms in the ESR2 gene has also been documented in other male reproductive tract disorders, such as male infertility (26) and prostate cancer risk (27) in Caucasians.

These observations prompted us to examine the (CA)n polymorphism as well as haplotype-tagging single nucleotide polymorphisms (SNPs) (27) in a population extended material of nonsyndromic hypospadias cases and healthy controls.

	Patients and Methods

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Patients and controls

In our study, 354 boys with nonsyndromic hypospadias were selected through medical records in Sweden. As a control group, we used DNA from an anonymous sample constituted of 380 healthy voluntary blood donors at Karolinska University Hospital, Sweden. The two groups have comparable ethnical backgrounds. Genomic DNA was extracted from blood using a standard phenol/chloroform protocol.

The protocol for the research project has been approved by a suitably constituted Ethics Committee of the institution within which the work was undertaken, and it conforms to the provisions of the Declaration of Helsinki in 1995 (as revised in Edinburgh 2000). Subject, and/or their parents, gave informed consent, and patient anonymity has been preserved.

Selection of ESR2 polymorphisms

Four haplotype-tagging SNPs (htSNP:rs2987983, rs1887994, rs1256040, and rs1256062) in ESR2 have been chosen because they capture 99.6% the haplotype variation in the Swedish population (27). Two additional SNPs (rs10483774 and rs1271572) were added because they map to a region of predicted intense transcription factor (TF) binding in the putative promoter region. The (CA)n repeat polymorphism was also included in this study (Table 1).

SNP typing was performed using a 5'-nuclease TaqMan assay together with fluorescently labeled probes using standard protocols (Applied Biosystems, Foster City, CA). The samples were analyzed on an ABI 7900HT (Applied Biosystems). The lengths of the (CA)n repeat were analyzed in an ABI 3730 DNA analyzer (Applied Biosystems). The lengths of the (CA)n containing fragments were ascertained by two different PCRs. The length of the fragment containing the (CA)n (Fig. 1) ranges from 143–171 bp.

View larger version (24K): [in this window] [in a new window]   FIG. 1. Distribution on (CA)n length across the patients and controls. The fragment lengths range from 143–171 bp. The y-axis is presented in percentage of all observations (obs). Shorter repeats are underrepresented in the patients’ group (P < 0.002).

To identify potentially associated polymorphisms, we performed global tests of allele and genotype frequency differences among the cases and controls. We initially performed ² and Fisher exact tests. Odds ratios (ORs) and odds ratio’s 95% confidence intervals (OR₉₅s) were estimated. These analyses were conducted using Statistica 7.0 (StatSoft, Inc., Tulsa, OK). Furthermore, single locus and multi-marker haplotype association tests were performed using two different algorithms, Haploview version 3.32 (28) and Unphased version 3.0.3 (29), thus providing complementary validity to our results. The calculations were performed both with and without missing data estimation. The threshold for rare haplotypes frequency identification was 0.005. The P values were corrected for multiple testing by performing permutations as follows. The trait values were randomly shuffled between subjects 1000 times. In each permutation, the minimum P value is compared with the minimum P value observed in all the analyses in the original data set. Pair-wise LD between polymorphisms was measured by estimating D' (the normalized disequilibrium coefficient, which ranges from 0–1) and the squared correlation coefficient r². Hardy-Weinberg equilibrium of the genotype frequencies at each SNP in cases and controls was accessed with Haploview version 3.32 (28).

To determine whether the associated risk gene variants were independent, logistic regression analysis was performed using the SAS computer program (SAS Institute Inc., Cary, NC), in which the "proc logistic" command was used. This resulted in a standard logistic regression model in which the regression coefficients were logarithms of the odd ratios. Models were compared using the log likelihood ratio test, which approximates to a ² distribution with 1 df (the difference in number of factors in the models). Initially, different genetic models for each marker against each other were tested (dominant, recessive, and codominant models). The analysis presented in Table 5 only includes data for the best model (codominant). To get OR estimates for the different genotypes, variables were recoded into design variables.

A search for putative regulatory genetic variation in the ESR2 gene was performed by RAVEN-Regulatory Analysis of Variation in Enhancers (http://www.cisreg.ca/cgi-bin/RAVEN/a). The putative regulatory function of SNPs is scored using phylogenetic footprinting and TF binding site analysis. The selection criteria included a view range of 2000 bp upstream the transcription start, a TF score of 90%, a conservation sliding window of 50 base pairs, a conservation cutoff of 0.8, and a minimum SNP-caused score difference of 1.5 (29, 30).

	Results

Top Abstract Introduction Patients and Methods Results Discussion References

 
Genotyping of the four haplotype tagging SNPs, rs2987983, rs1887994, rs1256040, and rs1256062 (27), and of the (CA)n repeat polymorphism (23), yielded a success rate of more than 95%. All loci were in Hardy-Weinberg equilibrium. Single marker analysis revealed significant association with the SNP rs2987983 (P < 0.01), in the putative promoter region, and to the (CA)n repeat polymorphism (P < 0.02), in intron 6 of ESR2 (Table 2). The estimated P value after correcting for multiple comparisons by performing 1000 permutations remains <0.05.

Comparing the length of the fragment containing the (CA)n polymorphism in patients and controls, with a T test assuming equal variances, confirmed that the (CA)n polymorphism is longer in patients (Figs. 1 and 2), yielding a P value < 0.002. Using the fragment length of 159 bp (22) as threshold above which a fragment is considered long (fragments < 159 bp S = short; fragments 159 bp L = long), the L allele is more common among patients than in controls (Table 2), with an OR of 1.43 (OR₉₅ 1.1–1.8; P < 0.004). The comparison of SS vs. SL vs. LL genotype frequencies of the (CA)n polymorphism in patients and controls reveals that the LL genotype is more common among patients than controls with an OR for the LL genotype of 1.48 (OR₉₅ 1.1–2.0; P < 0.02).

View larger version (17K): [in this window] [in a new window]   FIG. 2. Comparison between the length of the fragment containing the (CA)n polymorphism (box and whiskers plot). (CA)n is longer in patients (P) than in controls (C). T test assuming equal variances: t value = –3; P < 0.002. Mean length patients = 159.7; mean length controls = 158.9. SD patients = 4.6; SD controls = 5.2.

Global D' for all tested polymorphisms, the normalized disequilibrium coefficient, is 0.846. This high value is mostly due to a high LD between the SNPs rs2987983 and rs1256040, defining an approximately 1700-bp long haplotype block (Fig. 3); LD is much lower to and between the remaining SNPs. D' between rs2987983 and (CA)n is low (0.35), indicating that these two polymorphisms in ESR2 gene are in low LD, meaning that they are not necessarily inherited together as a haplotype.

View larger version (70K): [in this window] [in a new window]   FIG. 3. LD plot drawn with Haploview version 3.32: high D' values between the SNPs 1 and 2. LD is lower to the remaining SNPs. D' calculated by Unphased version 3.0.3: global D' 0.85. However, D' between rs2987983 and (CA)n, the two associated markers, is low (0.35).

Three models were tested, one with only rs2987983, one with only the CA microsatellite, and one with both polymorphisms. The model containing only the CA microsatellite had the lowest P value (P < 0.003), followed by the one with both polymorphisms (P < 0.004); the worst fit was observed for the model containing only rs2987983 (P < 0.03). The SNP rs2987983 is not independently associated when the CA microsatellite is included in the model (Table 5). The significance of a model containing rs2987983 and the CA repeat is significantly better (P < 0.02) than the significance of a model only containing rs2987983, whereas a model containing the CA repeat is not improved significantly by adding the rs2987983 marker.

The results suggest that the two polymorphisms are not independently associated; of the studied polymorphisms, the primary risk factors for hypospadias in the ESR2 sequence is longer lengths of the (CA)n polymorphism.

	Discussion

Top Abstract Introduction Patients and Methods Results Discussion References

 
A previously unsuspected role of estrogens in the male reproductive tract changed the idea that estrogen reproductive functions were confined to females. The present study evidences two risk factors for hypospadias in the ESR2 sequence: association was found with longer variants of the (CA)n polymorphism in intron 6, and with gene variants in the putative promoter. The combination of the two risk genotypes is also overrepresented in patients. These two polymorphisms are not necessarily inherited together as a haplotype, as demonstrated by a low LD. Their effects are, though, not independent, with the (CA)n polymorphism as the primary risk factor for hypospadias in the ESR2 sequence. The general low ORs reveal that the effects of these gene variants are low, as expected from a complex etiology.

The associated promoter polymorphisms map to a region of intense TF binding. We propose that these variants may alter the binding and responsiveness to transcription regulators. Indeed, as a steroid receptor, ESR2 is subjected to a complex regulation by coregulators and general TFs, which modulate the transcription of ESR2-target gene expression. Promoter polymorphisms have been associated with the risk for several diseases (31, 32), and the SNP rs2987983 has previously been associated with prostate cancer risk in a Swedish cohort, underlining a possible functional consequence of this variant in the male reproductive tract (27). To date, no functional studies on this polymorphism have been performed.

With regard to the (CA)n polymorphism, Westberg et al. (22) have suggested that longer (CA)n lengths in ESR2 may affect the hormonal production of androgens in the adrenal glands, explaining the associated lower levels of testosterone. Moreover, it has been demonstrated that intronic microsatellite repeats may lead to altered gene transcription, mRNA splicing, export to cytoplasm, induce heterochromatin- mediated-like gene silencing, or interactions with coregulators (25), which could underlie other possible mechanisms.

Because ESR2 is the predominant ESR in the developing male urethra (18, 19), it is possible that sequence variants of the ESR2 gene may affect the male genital development, which may be due to direct effects of this receptor on urethral development; or by an indirect effect, modulating the androgen pathway (22, 24) and affecting the balance between estrogen and androgen effects, which appears to be essential for correct sex development (17).

Interactions between estrogens and androgens seem a very likely mechanism to explain how the risk to hypospadias can be influenced by polymorphisms in ESR2. Not only do estrogens modulate androgen levels, but also ARs and ESRs, both ligand-dependent nuclear factors, interact at multiple levels to regulate target gene expression and, consequently, cell function. The physiological interplay between androgens and estrogens include: 1) negative feedback of estradiol on the hypothalamus-pituitary-gonadal axis (24, 33); 2) binding to and control of steroid hormone binding globulin (34); 3) interactions between AR and ESR through specific domains affecting their transactivational properties (35, 36); 4) estrogen-induced down-regulation of AR by proteosome degradation pathway (37); and 5) inhibition of androgen action by 7-estradiol via both ESR1 and ESR2 (38).

Furthermore, AR-ESR interactions could occur at the DNA and protein level. At the DNA level, these receptors may compete for the same binding sites or affect protein-DNA interactions. Interactions at the protein level may happen directly, or by squelching effect, to compete with common transcriptional factors or coregulators (36). It appears that the consensus DNA binding elements of AR and ESRs are distinct, and that ESRs do not bind to consensus androgen response elements and vice versa (39). However, it is not clear whether ESR can bind to the functional androgen responsive elements of an androgen-target gene.

Direct effects of the ESRs may also underline the disorders in the male reproductive tract. Sequence variants may interfere with some of the proposed functions of ESR2, such as antiproliferative action, regulation of apoptosis, and control of antioxidant gene expression, which may directly affect urethral development (40). Moreover, direct interaction of estrogenic chemicals and phytoestrogens with ESR ß has been documented (41). Xenoestrogens create changes in the ESR2 mRNA expression profile, as well as in the binding response, of this steroid receptor.

The incidence of disorders of the male reproductive tract is increasing in the last decades, partially due to an increased exposure to environmental toxicants (10, 14). Because hypospadias is a disorder with complex etiology, including both genetic and environmental factors, we speculate that variants in ESR2 could influence responses to environmental endocrine disrupters, which would also explain the ethnic differences in the susceptibility to these agents (15). Interactions between estrogens and androgens in the modulation of hormonal actions increase the level of complexity of the hormonal-regulated sexual differentiation and susceptibility to endocrine disruption.

In summary, we provide evidence for the involvement of variants in the ESR2 gene in hypospadias. We also support the concept that estrogens and their receptors are important for the developing male urethra, and suggest that polymorphisms in ERS2 may interact with environmental toxicants in influencing the risk for hypospadias.

	Acknowledgments

 
The authors thank Jodie Painter, who helped edit the text.

	Footnotes

 
This work was supported by the Portuguese Fundação para a Ciência e Tecnologia, which finances A.B.-M., and the Swedish society for medical research, which supports C.S. This work was also supported by grants from Her Royal Highness Crown Princess Lovisa Foundation, the Swedish Research Council, Karolinska Institutet, Erik Rönnberg donationer (Riksbankens jubileumsfond), and the Swedish Society of Medicine.

Disclosure Statement: The authors have no conflicts of interest.

First Published Online June 19, 2007

Abbreviations: AR, Androgen receptor; ESR, estrogen receptor; LD, linkage disequilibrium; OR, odds ratio; OR₉₅, odds ratio’s 95% confidence interval; SNP, single nucleotide polymorphism; TF, transcription factor.

Received March 9, 2007.

Accepted June 12, 2007.

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