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The Journal of Clinical Endocrinology & Metabolism Vol. 86, No. 7 3207-3210
Copyright © 2001 by The Endocrine Society

Original Articles

Evidence That Longer Androgen Receptor Polyglutamine Repeats Are a Causal Factor for Genital Abnormalities

Han N. Lim1, Richard M. Nixon, Hong Chen, Ieuan A. Hughes and J. Ross Hawkins2

Department of Paediatrics (H.N.L., H.C., I.A.H., J.R.H.), University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 2QQ, United Kingdom; Medical Research Council Biostatistics Unit (R.M.N.), Institute of Public Health University Forvie Site, Cambridge CB2 2SR, United Kingdom; and Department of Paediatrics (H.C.), Pamela Youde Nethersole Hospital, Chai Wan, Hong Kong, China

Address correspondence and requests for reprints to: Han N. Lim, Ph.D., Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, United Kingdom. E-mail: hl215{at}mole.bio

Abstract

Moderate to severe undermasculinized genitalia was recently shown to be associated with longer polyglutamine repeats within the androgen receptor [AR(Gln)n]. However, it was unknown whether this was because longer AR(Gln)n contributed to the: 1) etiology; 2) severity; and/or 3) testicular maldescent. Therefore, AR(Gln)n length in 175 males with abnormal genitalia were analyzed according to etiology (known or unknown), severity (complete, severe, and moderate), or testis position (abdominal, inguinal, or scrotal). Etiology (P = 0.01) and severity (P = 0.02) but not testis position (P = 0.52) were associated with AR(Gln)n length. The association between the severity of the genital abnormalities and AR(Gln)n length was due to the close association of severity with the etiology (P < 0.0001). A highly selected group with moderate to severe genital abnormalities and multiple criteria to exclude known etiological factors had a greater AR(Gln)n length (mean, 25.33) than all other samples (mean, 23.11; P = 0.0004). The results suggest that AR(Gln)n length does not influence the severity of undermasculinization or testis descent but instead contributes to the causation of genital abnormalities in a subset of patients. These findings, together with a demonstrated relationship between severity and multifactorial etiology, are incorporated into a proposed model for the involvement of AR(Gln)n length in genital abnormalities.

THE ANDROGENS TESTOSTERONE and 5{alpha}-dihydrotestosterone are crucial to the differentiation of the male genital and reproductive system. Defects of androgen production caused by either aberrant testis development (e.g. Denys-Drash syndrome) or abnormal function (e.g. a sex steroid biosynthetic defect) can result in a wide variety of phenotypes including: completely female genitalia, ambiguous genitalia, isolated hypospadias, gynecomastia, and isolated male infertility (1, 2). Similar phenotypes can result from mutations in the androgen receptor (AR), which mediates the actions of the androgens. However, many patients with genital and reproductive defects that are consistent with insufficient androgen action have no detectable defect of androgen production and no AR mutation (3, 4).

In vitro AR transactivation studies have shown that AR function is reduced by increasing the length of the polymorphic polyglutamine repeat within the receptor [AR(Gln)n] (5, 6, 7). This seems to be physiologically and clinically important, because recent studies have shown that longer AR(Gln)n are associated with isolated male infertility and moderate to severe undermasculinized genitalia (8, 9, 10). However, it must be stressed that these studies had not demonstrated (as it was not possible with the described patient groups) that longer AR(Gln)n actually contributed to the cause. The association could have been due to longer AR(Gln)n increasing the severity of the undermasculinization in these disorders or alternatively contributing to testicular maldescent, which is commonly associated with male infertility and incomplete genital development (1, 4, 11).

The aim of this study was to examine a large heterogeneous population with abnormal male genital development that varied in the etiology, the severity of abnormal genitalia, and the level of testis descent, to determine which of these factors were associated with longer AR(Gln)n.

Materials and Methods

Subjects

One hundred seventy-five patient samples with abnormal male genital development were selected from the Cambridge intersex database based on the availability of DNA and clinical details (4). The subjects were categorized as having both known and unknown causes for the genital abnormalities (Table 1Go). The known group contained subjects with a definitive diagnosis and those with features inconsistent with isolated genital undermasculinization. In the unknown group, 12 subjects had completely female genitalia and 76 subjects had moderate to severe undermasculinized genitalia [these were included in an earlier study comparing AR(Gln)n length between undermasculinized males and a normal population] (10). The Unknown group was defined using minimal criteria: a 46, XY karyotype; no other severe congenital abnormalities or abnormalities known to be associated with abnormal genitalia; no uterus present; no relationship to other samples in the study; and no known cause for the abnormalities. AR(Gln)n length was determined from the CAG repeat region (includes the CAA codon) within the AR gene by PCR amplification and genotyping on an ABI 373 (ABI, Foster City, CA) using Genescan software (see Refs. 10 and 12 for further details). Local ethics committee approval was obtained for the use of patient samples as part of a sexual development disorders research program.


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  		Table 1. Patient categorization based on clinical features and investigations

 
Statistical analysis

The relationship between AR(Gln)n length and the etiology of the genital abnormalities, the severity of genital abnormalities, and testis descent was assessed by univariate and multivariate ANOVA. AR(Gln)n length was treated as a continuous variable, and the other factors as categorical variables. An F-ratio was considered significant if the P value was less than 0.05. Post hoc pairwise comparison of the groups within each variable was performed using Fisher’s protected least significance difference, allowing for unequal sample numbers. The comparison of the severity of the genital abnormalities (complete, severe, and moderate) with the etiology (known and unknown) was performed using the {chi}2 test for association (http://faculty.vassar.edu/~lowry/newcs.html). Fisher’s exact test (http://home.clara.net/sisa/fisher.htm) was used to examine whether there was an association between moderate genital abnormalities (as opposed to complete and severe) and unknown etiology (as opposed to a known etiology). The mean AR(Gln)n length between the selected subgroup and that of all the other samples was compared using the unpaired t test. Unless otherwise stated, statistical analyses were performed using Statview (Abacus, Berkeley, CA).

Results

Etiology

The data set was categorized into two groups (Known and Unknown etiology) according to whether or not a cause for the genital abnormalities had been identified (Tables 1Go and 2Go). Because all possible androgen production defects and AR mutations were not excluded in the Unknown group, this will reduce the difference between this and the Known etiology group, which will favor the null hypothesis (i.e. no difference between the two groups). Despite this, the mean AR(Gln)n length of the Known etiology group (n = 87; mean, 22.85) was significantly less than the Unknown etiology group (n = 88; mean, 24.19; ANOVA, P = 0.01).


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  		Table 2. Mean AR(Gln)n length for each of the groups after sorting according to the etiology, severity of the genital abnormalities, and testis descent

 
Severity of the genital abnormalities

The data set was divided into three groups according to the severity of the genital abnormalities (Table 2Go): complete (completely female external genitalia; n = 60), severe (female genitalia with clitoromegaly or unfused scrotum with perineal hypospadias and micropenis; n = 61), and moderate (hypospadias and unfused scrotum or micropenis; n = 54). As a variable, severity was associated with AR(Gln)n length (ANOVA, P = 0.020). The group with moderate genital abnormalities had the longest AR(Gln)n, which was significantly greater than the completely female genitalia group (mean difference, 1.73; ANOVA, P = 0.0058).

The mean AR(Gln)n length increased as the severity of the genital abnormalities decreased: complete (mean, 22.70), severe (mean, 23.54), and moderate (mean, 24.43) (Table 1Go). The subdivision of these three subgroups into those with a known and an unknown etiology demonstrated that this trend was related to an increasing proportion of samples with an unknown etiology as severity decreased: complete, 12 of 60 (20%); severe, 35 of 61 (57%); moderate, 41 of 54 (76%). In subjects with an unknown etiology, an increase in mean AR(Gln)n was associated (ANOVA, P = 0.041) with a decrease in severity (complete, 22.33; severe, 23.89; moderate, 24.93; Fig. 1Go). In contrast, the mean AR(Gln)n length of subjects with a known etiology did not show a significant change with severity: complete (mean, 22.79), severe (mean, 23.08), and moderate (mean, 22.85) (ANOVA P = 0.94). The relationship between the severity and the etiology of the genital abnormalities was significant ({chi}2 = 32.8, P < 0.0001), therefore, adjustments (in a multivariate ANOVA) could not be made for both of these factors together, or for each other.



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  		Figure 1. Comparison of the severity of the genital abnormalities and mean AR(Gln)n length in all samples, the Known and Unknown etiology groups and the Selected group.

 
Testis descent

Subjects were divided into three groups (Table 2Go): an abdominal testis group (at least one intra-abdominal testis; n = 52), an inguinal testis group (at least one inguinal testis but excluding those with one intra-abdominal testis; n = 68), and a bilateral scrotal testes group (n = 55). AR(Gln)n length was not different between the three testis descent groups (P = 0.52). Testis descent was also not associated with the AR(Gln)n when adjusted for differences in either severity of undermasculinization or the etiology of the genital abnormalities (multivariate ANOVA, P = 0.97 and P = 0.77, respectively).

Using multiple parameters to define a subgroup with longer AR(Gln)n length

A subset of patients was selected that had both less severe genital abnormalities and the exclusion of known causes for the abnormal genitalia, to determine whether combining these two factors further increased the mean AR(Gln)n length. The criteria for selection were: moderate or severe abnormal genitalia; no uterus and no severe congenital malformation; a normal 46, XY karyotype; a normal AR binding study; normal testosterone levels; a positive response to human CG stimulation; and a normal urinary steroid profile or testosterone to 5{alpha}-dihydrotestosterone ratio (details of the normal reference ranges used for the hormonal and AR binding studies have previously been described; Ref. 10). This highly Selected group had an average AR(Gln)n length (n = 33; mean, 25.33) that was greater than all other samples (n = 142; mean, 23.11; P = 0.0004). In addition, the Selected group had a trend of increasing AR(Gln)n with decreasing severity [severe (mean, 24.68) and moderate (mean, 26.21); Fig. 1Go]. The Selected subgroup with moderate genital abnormalities (n = 14) also had an AR(Gln)n length that was greater than all other samples (n = 161, P = 0.0014).

Discussion

The complexity of the multistep process involved in male reproductive development is reflected by the many factors that, when disrupted, can lead to genital abnormalities. The disruption of a single factor is sometimes sufficient to cause abnormal genital development, irrespective of other genetic [e.g. AR(Gln)n length] and environmental factors (2). The clinical investigation of abnormal genitalia currently focuses on the identification of these single factor causes. As expected, the group with an identifiable cause for the genital abnormalities (Known etiology group) was not associated with longer AR polyglutamine repeats. Conversely, the Unknown etiology group is more likely to contain samples that are due to multiple interacting factors, none of which can alone cause abnormal genitalia (i.e. a multifactorial cause). The higher mean AR(Gln)n within the Unknown etiology group indicates that this polymorphism may be one of the factors contributing to abnormal genitalia.

Within the Known etiology group there was no increase in AR(Gln)n length as the severity increased. In contrast, there was a trend of an increasing AR(Gln)n length with decreasing severity of the genital abnormalities within the Unknown etiology group. This is the opposite to what would be expected if longer AR(Gln)n worsened the severity. It was also observed that the proportion of subjects with an Unknown etiology increased as the severity of the genital abnormalities decreased. Together these findings suggest that as the severity decreases, the relatively subtle effect of longer AR(Gln)n within the normal range contributes to the multifactorial cause (rather than the severity) of a greater proportion of samples (refer to the model in Fig. 2Go). Conversely, the greater the severity of the genital abnormalities, the greater the likelihood that it is caused by a single factor. Therefore, many of the subjects in the Unknown group with completely female genitalia should be examined using more rigorous methods than were required for this study, to identify mutations in candidate genes. The inclusion of such investigations into future studies is predicted to demonstrate an even greater difference in mean AR(Gln)n length between the multifactorial (Unknown) and predominantly single factor (Known) etiology groups.



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  		Figure 2. A model for the involvement of AR(Gln)n length in genital abnormalities. Longer AR(Gln)n cause a relatively small reduction in AR function and, thus, are likely to be most important when there are other "weak" causal factors (i.e. those factors that cannot by themselves cause genital abnormalities). Therefore, in cases with less severe genital abnormalities where a greater proportion of cases have a multifactorial cause, longer AR(Gln)n are more important.

 
Testis descent, particularly through the inguinal canal down to the scrotum, is generally believed to be an androgen-dependent process because in patients with AR mutations the testes are most frequently found in the inguinal region (Table 2Go). It is, therefore, surprising that testicular maldescent showed no association with AR(Gln)n length in this and another study (13). This might be because testis descent, unlike genital development, is relatively insensitive to small changes in AR function caused by variation in AR(Gln)n length. This explanation is consistent with the findings from other studies, which have identified AR mutations in cases with genital abnormalities and no testicular maldescent but not in cases with isolated testicular maldescent (4, 14).

The presence of the highest mean AR(Gln)n length in the Selected group demonstrates the importance of clinical features and investigations in defining the subgroups where longer AR(Gln)n play a major role. The selection of different patient subgroups with variable involvement of AR(Gln)n length may explain the conflicting results generated by association studies for other disorders with a heterogeneous cause such as male infertility, prostate cancer, and breast cancer (8, 9, 12, 15, 16, 17, 18, 19). Therefore, it is essential that studies of multifactorial disorders have sufficient clinical data and subjects to detect an association within the patient group, between AR(Gln)n length (or any other polymorphism) and a plausible biological or clinical parameter such as age of onset or severity.

Subjects with moderate to severe undermasculinized genitalia had previously been shown to have a greater mean AR(Gln)n length than controls from the normal population (10). In this study, the analysis of a heterogeneous group with abnormal genitalia demonstrates that this association is related to the etiology and not the severity of the genital abnormalities or testis descent. Longer AR(Gln)n seem to contribute to the etiology of the genital abnormalities in a subset without an identifiable single factor cause (i.e. a multifactorial etiology). Furthermore, multifactorial etiology and longer AR(Gln)n were shown to be associated with decreasing severity. These findings are a step toward defining the role of AR(Gln)n length and its incorporation into the diagnosis and management of abnormal genitalia.

Acknowledgments

We thank all the clinicians who sent samples and details to the Cambridge intersex database. We are also grateful to Alison Dunning and Simon McBride for sharing reagents and the ABI373 sequencer.

Footnotes

1 Supported by the Birth Defects Foundation and an Overseas Research Student’s award. Back

2 Present address: Incyte Genomics Ltd., 214 Science Park, Cambridge CB4 0WA, United Kingdom. Back

Received January 8, 2001.

Revised March 2, 2001.

Accepted March 23, 2001.

References

  1. Quigley CA, De Bellis A, Marschke KB, El-Awady MK, Wilson EM, French FS. 1995 Androgen receptor defects: historical clinical and molecular perspectives. Endocr Rev. 16:271–321.[CrossRef][Medline]
  2. Migeon CJ, Wisniewski AB. 1998 Sexual differentiation: from genes to gender. Horm Res. 50:245–251.[CrossRef][Medline]
  3. Batch JA, Williams DM, Davies HR, et al. 1992 Androgen receptor gene mutations identified by SSCP in fourteen subjects with androgen insensitivity syndrome. Hum Mol Genet. 1:497–503.[Abstract/Free Full Text]
  4. Ahmed SF, Cheng A, Dovey L, et al. 2000 Phenotypic features androgen receptor binding and mutational analysis in 278 clinical cases reported as androgen insensitivity syndrome. J Clin Endocrinol Metab. 85:658–665.[Abstract/Free Full Text]
  5. Kazemi-Esfarjani P, Trifiro MA, Pinsky L. 1995 Evidence for a repressive function of the long polyglutamine tract in the human androgen receptor: possible pathogenetic relevance for the CAGn-expanded neuropathies. Hum Mol Genet. 4:523–527.[Abstract/Free Full Text]
  6. Chamberlain NL, Driver ED, Miesfeld RL. 1994 The length and location of CAG trinucleotide repeats in the androgen receptor N-terminal domain affect transactivation function. Nucleic Acids Res. 22:3181–3186.[Abstract/Free Full Text]
  7. Choong CS, Wilson EM. 1998 Trinucleotide repeats in the human androgen receptor: a molecular basis for disease. J Mol Endocrinol. 21:235–257.[CrossRef][Medline]
  8. Tut TG, Ghadessy FJ, Trifiro MA, Pinsky L, Young EL. 1997 Long polyglutamine tracts in the androgen receptor are associated with reduced trans-activation impaired sperm production and male infertility. J Clin Endocrinol Metab. 82:3777–3782.[Abstract/Free Full Text]
  9. Dowsing AT, Yong EL, Clark M, McLachlan RI, Kretser DM, Trounson AO. 1999 Linkage between male infertility and trinucleotide repeat expansion in the androgen receptor gene. Lancet. 354:640–643.[CrossRef][Medline]
  10. Lim HN, Chen H, McBride S, et al. 2000 Longer polyglutamine repeats in the androgen receptor are associated with moderate to severe undermasculinised genitalia in XY males. Hum Mol Genet. 9:829–834.[Abstract/Free Full Text]
  11. Hutson JM. 1986 Testicular feminization—a model for testicular descent in mice and men. J Pediatr Surg. 21:195–198.[Medline]
  12. Dunning M, McBride S, Gregory J, et al. 1999 No association between androgen or vitamin D receptor gene polymorphisms and risk of breast cancer. Carcinogenesis. 20:2131–2135.[Abstract/Free Full Text]
  13. Sasagawa I, Suzuki Y, Tateno T, Nakada T, Muroya K, Ogata T. 2000 CAG repeat length of the androgen receptor gene in Japanese males with cryptorchidism. Mol Hum Reprod. 6:973–975.[Abstract/Free Full Text]
  14. Weiner JS, Marcelli M, Gonzales ET, Roth DR, Lamb DJ. 1998 Androgen receptor gene alterations are not associated with isolated cryptorchidism. J Urol: 160:863–865.
  15. Legius E, Vandershueren D, Spiessens C, D’Hooghe TD, Matthijs G. 1999 Association between CAG repeat number in the androgen receptor and male infertility in a Belgian study. Clin Genet. 56:166–167.[CrossRef][Medline]
  16. Rebbeck TR, Kantoff PW, Krithivas K, et al. 1999 Modification of BRCA1-associated breast cancer risk by the polymorphic androgen-receptor CAG repeat. Am J Hum Genet. 64:1371–1377.[CrossRef][Medline]
  17. Stanford JL, Just JJ, Gibbs M, et al. 1997 Polymorphic repeats in the androgen receptor gene: molecular markers of prostate cancer risk. Cancer Res. 57:1194–1198.[Abstract/Free Full Text]
  18. Bratt O, Borg A, Kristoffersson U, Lundgren R, Zhang QX, Olsson H. 1999 CAG repeat length in the androgen receptor gene is related to age at diagnosis of prostate cancer and response to endocrine therapy but not to prostate cancer risk. Br J Cancer. 81:672–676.[CrossRef][Medline]
  19. Correa-Cerro L, Wohr G, Haussler J, et al. 1999 CAGn CAA and GGN repeats in the human androgen receptor gene are not associated with prostate cancer in a French-German population. Eur J Hum Genet. 7:357–362.[CrossRef][Medline]



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