ANDROGEN RECEPTOR GENE ALTERATIONS ARE NOT ASSOCIATED WITH ISOLATED CRYPTORCHIDISM

0022-5347/96/1603-0663$03.00/0 Vol. 160,863-865,September 1998 Printed in U.S.A.

T H E JOURNAL OF UROLOGY

Copyright 0 1998 by AMERICAN UROL~CICAL ASSOCIATION, bc.

ANDROGEN RECEPTOR GENE ALTERATIONS ARE NOT ASSOCIATED WITH ISOLATED CRYPTORCHIDISM JOHN S. WIENER,* MARC0 MARCELLI, EDMOND T. GONZALES, JR.,DAVID R. ROTH DOLORES J. LAMB? From the Scott Department

of

AND

Urology, Department of Cell Biology and Department of Medicine, Division of Endocrinology, Baylor College of Medicine, and Texas Children’s Hospital, Houston, Texas

ABSTRACT

Purpose: Multiple theories of testicular descent exist but there is no consensus. Cryptorchidism is a component of the androgen insensitivity syndrome, suggesting that testicular descent may be a t least partially under the control of androgenic stimulation. To determine whether isolated cryptorchidism may be caused by androgen insensitivity, we screened a population of boys with isolated cryptorchidism for the presence of androgen receptor gene alterations. Materials and Methods: Deoxyribonucleic acid (DNA) was isolated from tissue collected from 21 patients with isolated cryptorchidism during orchiopexy. Patient selection was biased to maximize the likelihood of detection of a genetic etiology of cryptorchidism. The DNA was screened for androgen receptor gene alterations in exons 2 to 8 using single strand conformational polymorphism analysis. Results: No abnormalities in the androgen receptor gene were detected by single strand conformational polymorphism analysis in any patient. Conclusions: Mutations of the androgen receptor gene in the hormone and DNA binding domains of the protein appear to be an unlikely cause of isolated cryptorchidism. KEY WORDS:cryptorchism; receptors, androgen; testis; base sequence

The process of testicular descent in humans is not well understood. It is known that XY individuals with the complete androgen insensitivity syndrome have undescended testes, suggesting that the presence of androgens and a functioning androgen receptor are necessary for the testes to descend.1.2 Hutson studied inbred mice with the tfm mutation (conferring the phenotype of complete androgen insensitivity syndrome) and found that the testes were uniformly descended to the level of the internal inguinal ring but no further.3 This finding led him to conclude that the intraabdominal portion of testicular descent is not androgen mediated but the presence of functional androgens and androgen receptors is required for the testes to descend through the inguinal canal and into the scrotum. Further support for this hypothesis comes from the localization of androgen receptors in the rat gubernaculum and the spinal cord nucleus associated with the genitofemoral n e ~ e . ~However, ,s the applicability of the rodent model to human testicular descent is controversial.2 Androgen receptor localization studies in humans have demonstrated the presence of the receptor in the cremaster muscle but not in the gubernaculum.6 These studies were performed on postnatal tissues only, and it is not presently known whether the androgen receptor is expressed in the gubernaculum or cremaster a t the time of human fetal testicular descent. Cloning of the androgen receptor gene has allowed characterization of the genetic defects responsible for partial and complete androgen insensitivity syndrome.7 Most XY individuals with androgen receptor gene alterations have severe genital malformations and, thus, are easily recognizable. However, a minority of individuals may only exhibit isolated defects, such as infertility, gynecomastia or hypospadias, in

the absence of other abnormalities.’ We recently described an individual with isolated distal penile shaft hypospadias who had a missense mutation of exon 2 of the androgen receptor gene.* We hypothesized that individuals with isolated cryptorchidism likewise may harbor androgen receptor gene mutations, particularly if a family history of cryptorchidism is present. Therefore, we screened a cohort of 21 boys undergoing surgical management of cryptorchidism to detect androgen receptor gene alterations and determine whether such defects may be an etiology of isolated cryptorchidism. MATERIALS AND METHODS

Patient accrual. Study patients were accrued from the operative case loads of 2 pediatric urologists (D. R. R. and E.T.G.).The sole inclusion criterion was the presence of isolated cryptorchidism, that is in the absence of any other known genitourinary abnormalities. These cases were not consecutive to maximize the number of patients with bilateral cryptorchidism, intra-abdominal testes and a positive family history of genital malformations. Informed consent was obtained from parents under an approved protocol of the Institutional Review Boards of Texas Children’s Hospital and Baylor College of Medicine. Tissue collection. Tissue samples were collected in the operating room at orchiopexy and snap frozen to -70C. In accordance with the approved protocol, only discarded tissue was used. Usually this tissue consisted of the hernia sac excised at orchiopexy but skin was obtained if a circumcision was performed simultaneously. Genomic deoxyribonucleic acid (DNA) preparation. The specimens were first digested for 2 hours at 37C in 250 p1. of *pted for ublication April 9, 1998. Current adflress: P.O.Box 3831, Duke University Medical Cen- a collagenase solution consisting of 0.5 mg./ml. of collageter, Durham, North Carolina 27710. nase, 10 mM. tris hydrochloric acid, pH 8.0 and 25 a. ?Requests for reprints: Laboratory for Male Reproductive Research, Baylor College of Medicine, One Baylor Plaza,Room 440E, ethylenediaminetetraaceticacid. A second digestion was performed using 1 mgJml. proteinase K in 250 p1. of an ad&Houston, Texas 77030. 863

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ANDROGEN RECEPTOR GENE MUTATIONS ARE NOT ASSOCIATED WITH CRYPTORCHIDISM

tional lysis buffer consisting of 200 mM. sodium chloride and 1%sodium dodecyl sulfate for 3 hours at 55C. The proteinase K digestion step was repeated if complete digestion did not occur. ARer proteolysis DNA was purified by standard extraction techniques in phenoYchlorofodisoamy1 alcohol (25: 24:l) and precipitation in cold ethanol. Single strand conformationul polymorphism (SSCP).8. The coding regions of exons 2 to 8 of the androgen receptor were amplified individually by polymerase chain reaction (PCR) using specific oligonucleotide primers. Each specimen was analyzed at each exon using 1 pg. DNA. During each amplification reaction a positive control and a negative control were included to verify the reaction and to exclude contamination. 32phosphorusdeoxy~dinetriphosphate was used as a radiolabel in each PCR reaction for imaging the reaction products. SSCP analysis of the PCR products was performed by electrophoresis in a denaturing 5.4% acrylamide gel (10 ml. 1OX tO.9 M. tris, 0.9 M. boric acid and 20 mM. ethylenediaminetetraacetic acid], 10 ml. glycerol, 12 ml. 45% acrylamide, 3 ml. 1.6% ammonium persulfate and 59 ml. water). Before loading 5 pl. DNA samples were denatured in 20 pl. formamide and 2.1 p1.10 mM. sodium hydroxide at lOOC for 10 minutes and then iced. Electrophoresis was performed at 400 v. for 14 to 24 hours. After drying, the gel was exposed to standard x-ray film overnight. Genetic alterations could then be detected as band shifts on the gel because PCR products containing mutant DNA demonstrate a Merent migration pattern in comparison to wild type DNA. RESULTS

Of the 21 boys included in the genetic analysis 9 had bilateral undescended testes and 12 had a unilateral undescended testis (see table). Considering the highest location of either testis in a single patient, 5 had at least 1 intraabdominal testis, 13had testes within the inguinal canal and 3 had maldescended testes at or below the external inguinal ring. Two boys had a family history of cryptorchidism and 2 had a family history of hypospadias. One boy also had congenital heart disease and another had multiple dysmorphisms but no identifiable chromosomal anomaly. Sufficient amounts of DNA were extracted from tissue from all 21 patients and were of adequate quality for SSCP analysis of each of the androgen receptor gene exons 2 to 8. No abnormal SSCP band patterns were noted for any sample in any of the 7 androgen receptor gene exons studied. Positive controls consisting of DNA with known mutations for each exon demonstrated appropriate band shifts, verifying the validity of the analysis. These findings of wild type SSCP band patterns exclusivelyin all patients in the coding regions of exons 2 to 8 strongly suggest that isolated cryptorchidism is not associated with androgen receptor gene alterations. DISCUSSION

Androgen receptor gene alterations have been well characterized in humans and are usually associated with partial or complete androgen insensitivity syndrome, a phenotype that

Patient characteristics

No.Pta. No. pts. Laterality: Unilat. Bilat. Testicular loeation: Abdominal Inguinal Superficialinguinal pouch Pos. family history: Cryptorchidism Hmsuadias

21

12 9

5

13 3 2

2

often includes cryptorchidism.1 Due to our recent finding of an androgen receptor gene mutation in a patient with isolated penile hypospadias: we hypothesized that isolated cryptorchidism may also be associated with androgen receptor gene alterations. Our molecular genetic analysis of 21 patients with isolated cryptorchidism revealed no abnormalities of the coding sequences of exons 2 to 8 of the androgen receptor gene. Based on these findings, we conclude that androgen receptor gene alterations are unlikely to be an etiology of isolated cryptorchidism. Our series of 21 patients undergoing surgical correction of cryptorchidism was selected to create a bias for the potential detection of a genetic etiology of cryptorchidism. We assumed that patients with a positive family history of genital abnormalities would be more likely to harbor androgen receptor gene alterations. Therefore, we included 2 patients in our cohort with a positive family history of cryptorchidism and 2 with a positive family history of hypospadias. One patient with cryptorchidism was a cousin of the patient with penile hypospadias in whom we had previously characterized a mutation of exon 2 of the androgen receptor: and the mother of the boy with cryptorchidism was a carrier of the same mutation (unpublished data). We also included 9 cases of bilateral cryptorchidism (43%)intentionally to overrepresent this population of patients. Cryptorchidism typically has been reported to be bilateral in 15% of cases in unbiased series.10 We hypothesized that if an endocrinopathy existed, such as an androgen receptor defect, a global effect would be seen and bilateral cryptorchidism might result. Five patients in our study had an intra-abdominal testis or testes, a condition found in the more severe degrees of the androgen insensitivity syndrome. No patient had any known syndromes associated with cryptorchidism to avoid potentially confounding etiologies. Despite this select population of cryptorchid patients, no androgen receptor gene alterations were noted in our study. It is possible that our techniques could have missed an androgen receptor gene alteration in a patient. SSCP is a widely accepted technique and has been shown in mixing studies to be sensitive enough to detect more than 90% of genetic alterations (Lamb and DeMayo, unpublished data). In our prior experience SSCP analysis using the same sets of primers was successful in detecting alterations of the androgen receptor gene.8.9 More sensitive methods, such as direct sequencing, are prohibitively labor-intensive to screen for mutations of genes of large size in numerous patients. We limited our analysis to exons 2 to 8 of the androgen receptor gene and excluded exon 1 and the noncoding regions of the gene. We cannot rule out the possibility that patients with isolated cryptorchidism may harbor androgen receptor gene mutations in exon 1 or in noncoding regions but we believe it highly unlikely. Of the more than 40 known mutations of the gene that have been associated with partial androgen insensitivity syndrome phenotypes none has been within exon 1or noncoding regions.11 Additionally, exon 1is 5 to 13-fold larger than exons 2 t o 8, making SSCP analysis much more difficult. Our analysis was also limited by the amount of DNA available from the small tissue quantity obtained in the hernia sac but additional sample acquisition by biopsy or phlebotomy was prohibited by the institutional review boards. However, we were able to analyze completely all of the coding regions of exons 2 to 8 in all patients with the available DNA. Genetic etiologies of cryptorchidism seem to be present in at least a minority of cases. Cryptorchidism is a common component of syndromes associated with chromosomal aberrations12but no chromosomal defects have been consistently associated with isolated cryptorchidism.13.14 A number of case reports of familial occurrence of cryptorchidism have demonstrated that a heritable component does exist,15-17 and epidemiological studies have shown a consistent pattern of

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ANDROGEN RECEPTOR GENE MUTATIONS ARE NOT ASSOCIATED WITH CRYPTORCHIDISM

inheritance for isolated cryptorchidism. Of boys with undescended testes 3.9 to 4.0% of their fathers and 6.2 to 9.8%of their brothers also have undescended testes.18-19This familial incidence vastly exceeds the incidence of cryptorchidism in the general population (0.8%).12 The presence of cryptorchidism in the androgen insensitivity syndrome is strong evidence for at least a partial role for androgen mediated mechanisms in testicular descent. Our inability to demonstrate androgen receptor gene alterations in a select cohort of 21 patients with undescended testes suggests that these genetic defects are an unlikely cause of isolated cryptorchidism. However, the biology of the androgen receptor in developing male genitalia is poorly understood at this time and, undoubtedly, the timing and amount of androgen receptor expression in target tissues are important in testicular development and descent. Expression of the androgen receptor in the rat gubernaculum declines during progression from fetal to postnatal life.20 In addition, the presence of androgens have been demonstrated to up regulate the expression of its receptor in the urogenital tract of the rat before but not aRer birth.21,22Thus, testicular descent likely requires more than presence of a normal (wild type) androgen receptor gene. The androgen receptor and its substrate (androgens) may need to be expressed at a critical time in the critical tissue and in a sufficient amount for testicular descent to occur properly. Elucidation of the complex hormonal interactions required for testicular descent will require further research into these pathways in rodents and man. CONCLUSIONS

Although cryptorchidism may result from androgen insensitivity in patients with the androgen insensitivity syndrome, androgen receptor gene alterations have not been associated with isolated cryptorchidism. Our genetic analysis of 21 patients with isolated cryptorchidism failed to demonstrate any alterations of the coding regions of exons 2 to 8 of the androgen receptor gene. Since these are the regions of the gene that have been associated with androgen insensitivity, we conclude from these findings that androgen receptor gene alterations are an unlikely cause of isolated cryptorchidism. REFERENCES

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Expression of androgen protein within the lumbar spinal cord during ontologic development and following antiandrogen induced cryptorchidism. J. Urol., 152 766, 1994. 6. Johansen, T. E. and Klein, H.: Evidence of androgen receptivity in the pathway of testicular descent in humans. A postnatal study. Eur. Urol., 2 3 466, 1993. 7. Lubahn, D. B., Brown, T. R., Simental, J. A., Higgs, H. N., Migeon, C. J., Wilson, E. M. and French, F. S.: Sequence of the introdexon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity. Proc. Natl. Acad. Sci., 86.9534, 1989. 8. Sutherland, R. W., Wiener, J . S., Hicks, J. P., Marcelli, M., Gonzales, E. T., Jr., Roth, D. R. and Lamb, D. J.: Androgen receptor gene mutations are rarely associated with isolated penile hypospadias. J . Urol., 156: 828,1996. 9. Marcelli, M., Tilley, W. D., Wilson, C. M., Grif6n, J. E., Wilson, J. D. and McPhaul, M. J.: Definition of the human androgen receptor gene structure permits the identification of mutations that cause androgen resistance: premature termination of the receptor protein at amino acid residue 588 causes complete androgen resistance. Mol. Endocr., 4 1105, 1990. 10. Cendron, M., Huff, D. S., G at i n g , M. A., Synder, H. M., 111and Duckett, J. W.: Anatomical morphological and volumetic analysis: a review of 759 cases of testicular maldescent. J . Urol., 149 570, 1993. 11. Quigley, C. A., De Bellis, A., Marschke, K. B., el-Awady, M. K., Wilson, E. M. and French, F. S.: Androgen receptor defects: historical, clinical, and molecular perspectives. Endocr. Rev., 1 6 271, 1995. 12. Kogan, S.:Cryptorchidism. In: Clinical Pediatric Urology. Edited by P. P. Kelalis, L. R. King and A. B. Belman. Philadelphia: W. B. Saunders Co., vol. 2,pp. 1050-1083,1992. 13. Dewald, G. W., Kelalis, P. P. and Gordon, H.: Chromosomal studies in cryptorchidism. J . Urol., 117: 110,1977. 14. Bartone, F. F. and Schmidt, M. A.: Cryptorchidism; incidence of chromosomal anomalies in 50 cases. J. Urol., 127: 1105,1982. 15. Perrett, L. J. and O'Rourke, D. A,: Hereditary cryptorchidism. Med. J . Aust., 2 5 1289, 1969. 16. Savion, M., Nissenkorn, I., Servadio, C. and Dickerman, 2.: Familial occurrence of undescended testes. Urology, 2 3 355, 1984. 17. Carragher, A. M. and McLean, R. D.: Familial bilateral cryptorchidism. Brit. J. Clin. Pract., 44:688, 1990. 18. Czeizel, A., Erijdi, E. and Toth, J.: Genetics of undescended testes. J. Urol., 126 528, 1981. 19. Jones, I. R. G. and Young, I. D.: Familial incidence of cryptorchidism. J. Urol., 127: 508, 1982. 20. Bentvelsen, F. M. and George, F. W.: The fetal rat gubernaculum contains higher levels of androgen receptor than does the postnatal gubernaculum. J. Urol., 160 1564,1993. 21. Bentvelsen, F. M., McPhaul, M. J., Wilson, J. D. and George, F. W.: The androgen receptor of the urogenital tract of the fetal rat is regulated by androgen. Mol. Cell. Endocrin., 105 21, 1994. 22. Husmann, D. A., McPhaul, M. J . and Wilson, J . D.: Androgen receptor expression in the developing rat prostate is not altered by castration, flutamide, or suppression of the adrenal axis. Endocrinology, 128 1902,1991.