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Association of Cryptorchidism with a Specific Haplotype of the Estrogen Receptor Gene: Implication for the Susceptibility to Estrogenic Environmental Endocrine Disruptors

Department of Endocrinology and Metabolism, National Research Institute for Child Health and Development (R.Y., M.F., T.O.), Tokyo 157-8535, Japan; Department of Urology, Yamagata University School of Medicine and Yamagata Tokushukai Hospital (I.S.), Yamagata 990-9585, Japan; Department of Pediatrics, Keio University School of Medicine (R.Y., T.H.), Tokyo 160-8582, Japan; and Algorithm Team, Japan Biological Information Research Center, Japan Biological Informatics Consortium, and Division of Genomic Medicine, Department of Applied Biomedical Engineering and Science, Tokyo Women’s Medical University (N.K.), Tokyo 162-0054, Japan

Address all correspondence and requests for reprints to: Dr. Tsutomu Ogata, Department of Endocrinology and Metabolism, National Research Institute for Child Health and Development, 2-10-1 Ohkura, Setagaya, Tokyo 157-8535, Japan. E-mail: tomogata{at}nch.go.jp.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: The prevalence of cryptorchidism (CO) has increased during the past few decades in several countries, and this event has primarily been ascribed to the estrogenic effects of environmental endocrine disruptors (EEDs). Little is known, however, about the role of genetic susceptibility to EEDs in this phenomenon.

Objective: The objective of this study was to determine whether CO is associated with a specific haplotype of the gene for estrogen receptor (ESR1) that mediates the estrogenic effects of EEDs.

Design: This was a case-control study.

Setting: The study was performed at the National Research Institute and University Hospitals.

Subjects: Sixty-three cryptorchid males, aged 1–13 yr, and 47 control males, aged 4–12 yr, were studied.

Intervention: After genotyping 15 single nucleotide polymorphisms widely distributed in the greater than 300-kb genomic sequences of ESR1, haplotype analysis was performed.

Main Outcome Measure: Identification of a specific ESR1 haplotype associated with CO was the main outcome measure.

Results: A haplotype block was identified for an approximately 50-kb region encompassing single nucleotide polymorphisms 10–14 in the 3' region of ESR1 in both groups. The frequency of the estimated AGATA haplotype within the block was higher in the patients than in the control males (34.0% vs. 21.3%; P = 0.037), and the association of this haplotype with CO phenotype was significant in a recessive mode (P = 0.0060). The homozygosity for this haplotype was identified only in the patients, and the frequency of the homozygotes was significantly different between the two groups (10 of 63 vs. zero of 47; P = 0.0042).

Conclusions: The association of CO with homozygosity for the specific ESR1 haplotype suggests the relevance of genetic susceptibility to EEDs in the development of CO.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
CRYPTORCHIDISM (CO) IS a common ailment, defined as the failure of one or both testes to descend into the scrotum (1). It can take place as an isolated form or as a part of impaired male sex development or congenital malformation syndrome. Although CO accompanied by other genital and/or extragenital features often results from single gene abnormalities, apparently isolated CO, although it still can be caused by single gene abnormalities in rare cases, usually occurs as a multifactorial trait subject to various relatively minor genetic and environmental factors (1, 2).

The prevalence of CO appears to have increased during the last few decades, at least in several countries (3, 4). A similar tendency has been observed for hypospadias, defective spermatogenesis, and testicular cancer (4). Furthermore, deterioration of male genital and reproductive function has also been identified in many wildlife species (4). It has been hypothesized, therefore, that such adverse changes in males are interrelated events primarily caused by the deleterious effects of environmental endocrine disruptors (EEDs) (4, 5). In particular, estrogenic effects exerted by most EEDs may play a major role in the deterioration of male genital and reproductive health, because exposure to estrogenic agents is known to result in various genital and reproductive abnormalities, including CO (6).

The estrogenic effects of EEDs would depend on several factors, including the dosage of exposed EEDs, the developmental stage of EED exposure, and the genetic susceptibility to EEDs. Of these factors, the genetic susceptibility to EEDs may primarily be ascribed to variations in the genes for estrogen receptor (ER), because the estrogenic effects of EEDs are primarily mediated by ER (4, 5). In this regard, although estrogenic EEDs can bind to both ER encoded by ESR1 and ERß encoded by ESR2 with variable affinities (5), ER, rather than ERß, appears to play a critical role in male genital and reproductive health. A human male with mutant ESR1 and Esr1 knockout male mice have abnormal endocrine status and impaired spermatogenesis (7, 8), whereas a human male with mutant ESR2 has not been identified with a disease phenotype, and Esr2 knockout male mice show an apparently normal fertile phenotype (8). Thus, we performed ESR1 haplotype analysis in CO, as a first step in clarifying the role of genetic susceptibility to estrogenic EEDs that have been postulated as one of the major factors in the deterioration of male genital and reproductive health.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

Sixty-three Japanese patients with CO, aged 1–13 yr (median, 5.0 yr), were studied. All patients were born between 1986 and 2002 and were studied from 1995–2003. Forty-seven patients had unilateral CO, with the undescended testes being identified in the inguinal region in 39 patients and in the abdominal cavity in seven patients; the remaining patient had an apparently vanishing testis. Sixteen patients had bilateral CO, with both testes being found in the inguinal regions in 15 patients and in the abdominal cavity in the remaining patient. All 63 patients satisfied the following criteria: 1) lack of other discernible genital and extragenital features, including hypospadias; 2) 46,XY karyotype in 20 or more of the lymphocytes analyzed; 3) no demonstrable mutation of the genes for 5-reductase-2 (9), androgen receptor (AR) (10), and insulin-like 3 (INSL3) (Wada, Y., and T. Ogata, unpublished observations) that can cause CO (1, 2); 4) lack of significant difference in the allele and genotype frequencies of Val⁸⁹Leu polymorphism at exon 1 of SRD5A2 (Hasagawa, T., and T. Ogata, unpublished observations) that is known to reduce the 5-reductase-2 activity by about 30% (11); 5) no expansion of CAG repeat lengths at exon 1 of AR (12) that is known to constitute a susceptibility locus for undermasculinization (13); and 6) absence of a Yq microdeletion after examining 30 or more loci, including RBMY and DAZ (Sasagawa, I., and T. Ogata, unpublished observations), that could be relevant to the development of CO (14). Basal serum gonadotropin and testosterone values remained within the age-matched normal ranges, although provocation tests for the pituitary-gonadal axis were not performed in most patients.

For controls, 47 prepubertal boys with normal external genitalia, aged 4–12 yr (median, 7.5 yr), were similarly analyzed after obtaining their permission. They were seen because of short to low-normal stature (–1.5 to –3.0 SD) between 1993 and 2000 and were found to have no discernible abnormality by cytogenetic, skeletal, and endocrine studies. The median age was similar in the patients and the control males (P = 0.28, by Mann-Whitney U test).

All patients and control males came from the urban or suburban area of Tokyo or Yamagata City. They were free of particular residential environments, such as the vicinity of chemical factories or farms; specific dietary habits, such as vegetarianism or nearly pure meat or fish diet; and intake of drugs with hormonal effects.

Single nucleotide polymorphism (SNP) analysis

This study was approved by the institutional review board committees at National Center for Child Health and Development, Keio University Hospital, and Yamagata University Hospital. To identify informative SNPs that are associated with high minor allele frequencies and are widely distributed in the more than 300-kb genomic sequences of ESR1, we screened 30 SNPs reported in the National Center for Biotechnology Information Database (www.ncbi.nlm.nih.gov/). In brief, the mixture of an equal amount of leukocyte genomic DNA from 30 Japanese subjects was amplified by PCR with primers designed to amplify an approximately 300-bp region around each of the 30 SNPs, and the PCR product was subjected to direct sequencing on a CEQ 8000 autosequencer (Beckman Coulter, Fullerton, CA). When two predicted nucleotide peaks were clearly delineated on the autosequencer, the SNPs were regarded as having a certain degree of heterozygosity and were used in this study.

Genotyping was performed by the 5' nuclease assay (the TaqMan method) on an ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA), using leukocyte genomic DNA from each subject. The details of the TaqMan methods have been described previously (15).

Statistical significance of the differences in allele and genotype frequencies between the patients and the control males was analyzed by Pearson’s ² test using the R environment for statistical computing (www.r-project.org/). The odds ratio (OR) and the 95% confidence interval (CI) were calculated using the same environment.

Haplotype analysis

Haplotype analysis was performed to identify a specific haplotype associated with the CO phenotype. The haplotype is a list of alleles on the same chromosome, and the alleles at loci in a linkage disequilibrium (LD) status can be inherited as a unit (16). The degree of LD can be expressed as the pairwise |D’| value (the absolute value for the disequilibrium parameter) that ranges from 0 (complete absence of LD) to 1.0 (complete presence of LD) (17), and a chromosomal region associated with high |D'| values between different loci is called a haplotype block (or an LD block) (18). When a significant association is identified between a disease phenotype and a specific haplotype within the block, a susceptibility locus or loci can be mapped to the block region (19). Although haplotypes are usually not observed, the haplotypes present in a subject and the frequencies of the haplotypes in a population can be inferred using genotype data at separate loci (16). In this study, haplotype inference was performed by the maximum likelihood method using expectation maximization (EM) algorithm (20) implemented in the software LDSUPPORT (21), the pairwise |D'| values were estimated by the method of Terwilliger and Ott (16), and a haplotype block was determined by the method of Zhu et al. (22) using the software developed by Kamatani et al. (23).

The association between CO phenotype and estimated haplotypes was tested by the following methods. The difference in the frequencies of haplotypes between the patients and the control males was tested using the estimated population haplotype frequencies by Pearson’s ² test. The OR and the 95% CI were calculated using the R environment. The difference in the frequencies of diplotype configurations between the patients and the control males was tested using PENHAPLO software, in which EM algorithm was used to maximize the likelihood of the observed genotype and the qualitative phenotype (24). The diplotype configuration denotes a combination of two haplotypes in a subject, and this software tests the difference in the frequencies of diplotype configurations in a dominant mode (comparison the frequencies of subjects with the risk haplotype between cases and controls) and in a recessive mode (comparison of the frequencies of subjects with two risk haplotypes between cases and controls). The frequency of homozygotes for each haplotype was compared between the patients and the control males, using the R environment.

Sequence analysis of ESR1

All eight coding exons and their flanking regions (20–30 bp) were PCR-amplified with primers designed by us, using leukocyte genomic DNA. Subsequently, the PCR products were subjected to direct sequencing from both directions on the CEQ 8000 autosequencer.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
SNP analysis

Fifteen of the 30 SNPs were assessed as having a certain degree of heterozygosity by the screening method (SNPs 1–15 in Table 1 and Fig. 1). Subsequent genotyping showed that the minor allele frequencies of the 15 SNPs were 18–49% in the patients and 19–49% in the control males. The distance between neighboring SNPs ranged from approximately 6–38 kb in physical length. The genotyping results of the 15 SNPs were consistent with the Hardy-Weinberg equilibrium.

View larger version (32K): [in this window] [in a new window]   FIG. 1. Organization of ESR1 and the physical positions of 15 SNPs examined in the present study (25 ). The ESR1 gene consists of eight exons and extends over a distance of 300 kb in physical length. , Coding regions; , 5'- and the 3'-untranslated regions. The ESR1 cDNA is 6455 bp long and is translated into a 595-amino acid protein that is divided into six common series of structural regions, referred to as A–F. The functional domains are also shown. AF-1 (activation function 1) is a ligand-independent transactivation domain, and AF-2 (activation function 2) is a ligand-dependent transactivation domain.

Haplotype analysis

A haplotype block was identified for an approximately 50-kb region encompassing SNPs 10–14 in both the patients and the control males (Fig. 2). The |D'| value was greater than 0.9 between each pair of SNPs within the block. Although the |D'| value was greater than 0.9 between SNPs 1 and 2 and between SNPs 7 and 8 in the patients, these haplotype blocks were absent in the control males. Similarly, although the |D'| value was greater than 0.9 between SNPs 1 and 3 and between SNPs 8 and 9 in the control males, these haplotype block were absent in the patients.

View larger version (32K): [in this window] [in a new window]   FIG. 2. Pairwise linkage disequilibrium maps. A haplotype block encompassing SNPs 10–14 has been identified in both cryptorchid patients and control males.

Sequence analysis of ESR1

No mutation or variation was identified in the analyzed regions of the 10 patients homozygous for the AGATA haplotype and in 10 randomly examined control males.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The present study indicates that ESR1 harbors an approximately 50-kb haplotype block encompassing SNPs 10–14, and that the frequency of the AGATA haplotype is higher in the cryptorchid patients than in the control males. The results of haplotype frequencies would primarily be consistent with those of the allele and genotype frequencies. Although SNP 9 was not judged to reside within the block, the relatively high |D'| values between SNP 9 and SNPs 10–14 would imply the presence of a fairly strong LD between SNP 9 and the haplotype block. Furthermore, haplotype analysis revealed that the association of the AGATA haplotype with the CO phenotype was significant in a recessive mode, and that the homozygosity for the AGATA haplotype was exclusively identified in the patients and was significantly higher in prevalence in the patients than in the control males. These findings suggest that homozygosity for the AGATA haplotype markedly raises the susceptibility to the development of CO.

The A allele of SNP 12 was specific to the AGATA haplotype. It was exclusively present on the estimated AGATA haplotype in both the patients and the control males, and the 10 patients homozygotes for the A allele of SNP 12 were also homozygous for the AGATA haplotype. These findings imply that the A allele of SNP 12 is the key marker representing the AGATA haplotype, and that genotyping of SNP 12 can be used for screening of the AGATA haplotype.

Although functional studies have not been carried out, it may be possible that the specific haplotype enhances ER signaling, facilitating the development of CO. Indeed, the enhanced estrogenic signaling could cause CO because of two major factors. First, excessive estrogenic effects may decrease the expression of INSL3 in Leydig cells, affecting the development of gubernaculum that is indispensable for the transabdominal testicular descent and may play a certain role in the transinguinal testicular descent (1, 2). Impaired Insl3 expression and the resultant occurrence of intraabdominal CO have been demonstrated in mice exposed to nonsteroidal as well as steroidal estrogenic agents (26, 27). Furthermore, targeted homozygous disruption of Insl3 has resulted in intraabdominal CO in mice (28, 29), and heterozygous mutations of INSL3 have been identified in patients with intraabdominal or inguinal CO (30). Second, excessive estrogenic effects may suppress gonadotropin production in pituitary gonadotropes and steroidogenic enzyme activity in Leydig cells, resulting in a relative deficiency of androgens that contribute to the transabdominal testicular descent by regressing the cranial suspensory ligament and is required for transinguinal testicular descent (1, 2). Administration of estrogens is known to attenuate gonadotropin secretion in men and testicular CYP17A1 activity in rodents (8, 31), and various disorders with primary and secondary androgen deficiency are frequently associated with CO (1). In support of the relevance of the two factors, rodent Esr1 is expressed in Leydig cells and pituitary gonadotropes, and human ESR1 is expressed in pituitary gonadotropes, although the expression of human ESR1 in Leydig cells has not been confirmed (8, 25). In this regard, because excessive estrogenic effects could affect both transabdominal and transinguinal testicular descent, this would explain why patients homozygous for the AGATA haplotype had various types of CO.

If the AGATA haplotype can indeed facilitate the effects of estrogenic EEDs, it remains to be clarified how the AGATA haplotype enhances ER signaling. However, because no variation was identified for the eight exons and their flanking sequences in the 10 patients homozygous for the haplotype as well as in 10 control males, this would exclude the relevance of a variation in such regions to the enhanced ER signaling. In this context, it has been demonstrated that several intronic SNPs can enhance the transcription activities of the corresponding genes (32, 33, 34). It is possible, therefore, that ESR1 also carries a functional intronic SNP(s) that is capable of facilitating ESR1 transcription and is tightly linked to the AGATA haplotype.

In this study, age-matched prepubertal boys were used as the control males. This was primarily to match the dosage of exposed EEDs between the patients and the control males, because the amount of EEDs is predicted to increase with time. Actually, we analyzed DNA samples obtained from boys with short to low-normal stature, because it was impossible to collect such samples from healthy boys. In this regard, it is known that patients with ER or aromatase deficiency have normal stature in childhood in the absence of estrogen effects, although they exhibit tall stature from the pubertal period of normal children (7, 35). It is also known that patients with hyperestrogenism show normal to relatively tall stature in childhood in the presence of exaggerated estrogen effects, although they become short with pubertal development (36). It is unlikely, therefore, that the identified haplotype is relevant to the short-stature phenotype in the control males, rather than to the CO in the patients.

Several points should be made with respect to the present study. First, some patients may have some unidentified pathological cause(s) of CO, such as single gene disorders. Second, there may be some unknown genetic and environmental differences between the patients and the control males. Third, it remains to be determined whether similar results can be reproduced in other, presumably EED-relate, undermasculinized conditions and in other countries with an increased prevalence of undermasculinization. Indeed, although multiple traits have been examined for the possible association with SNPs or haplotypes in the 5' region of ESR1, such as the XbaI site SNP (SNP 1 in this study) and the PvuII site SNP at intron 1, which is only approximately 50 bp away from the XbaI site (37, 38, 39), there has been no study dealing with undermasculinization or analyzing SNPs or haplotypes in the 3' region of ESR1.

Despite the above caveats, the present study provides a useful clue to clarify the relevance of an individual’s genetic susceptibility to the development of presumably EED-related disorders. In summary, we propose that homozygosity for the specific ESR1 haplotype increases susceptibility to the development of CO in response to estrogenic EEDs.

	Acknowledgments

 
We thank Toshikazu Ito and Shiori Furihata (Algorithm Team, Japan Biological Information Research Center, Japan Biological Informatics Consortium) for statistical advice.

	Footnotes

 
This work was supported by a grant for Child Health and Development from the Ministry of Health, Labor, and Welfare (14C-1) and a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture (16086215).

First Published Online May 17, 2005

Abbreviations: AR, Androgen receptor; CI, confidence interval; CO, cryptorchidism; EED, environmental endocrine disruptor; EM, expectation maximization; ER, estrogen receptor; ESR1, gene for estrogen receptor ; ESR2, gene for estrogen receptor ß; INSL3, insulin-like 3; LD, linkage disequilibrium; OR, odds ratio; SNP, single nucleotide polymorphism.

Received February 2, 2005.

Accepted May 11, 2005.

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