This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chu, J. Articles by Luo, Z. Search for Related Content PubMed PubMed Citation Articles by Chu, J. Articles by Luo, Z.

Male Fertility Is Compatible with an Arg⁸⁴⁰Cys Substitution in the AR in a Large Chinese Family Affected with Divergent Phenotypes of AR Insensitivity Syndrome

Laboratory of Population and Quantitative Genetics, State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University (J.C., R.Z., W.Z., Y.H., Q.Q., H.Z., J.-C.W., S.T., X.L., Z.L.), Shanghai 200433, People’s Republic of China; Department of Surgery, First Affiliated Hospital of Guiyang Traditional Chinese Medicine College (Z.Z.), Guiyang 550001, People’s Republic of China; and University of Birmingham School of Biosciences (Z.L.), Edgbaston, United Kingdom B15 2TT

Address all correspondence and requests for reprints to: Dr. Zewei Luo, University of Birmingham School of Biosciences (Z.L.), Edgbaston, United Kingdom B15 2TT. or z.luo{at}bham.ac.uk

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Androgen insensitivity syndrome (AIS) is a disorder of male sexual development caused by an absent or dysfunctional AR. Fertile cases with mild AIS and slightly impaired AR activity had been reported in literature, and their external genitalia were documented to be usually normal or subnormal. We reported here an Arg⁸⁴⁰Cys substitution in the AR gene in a large Chinese pedigree affected with AIS. The mutant gene may result in infertility for some affected males with or without hypospadias. However, it was also observed that the mutation did not affect the fertility of the other patients. The gonadotropin levels for one of these patients were within the normal range. Thus, whether normal levels of the gonadotropins are necessary for the preserved fertility of patients affected with this genetic disorder remains to be elucidated.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
ANDROGEN INSENSITIVITY syndrome (AIS; MIM 300068) is an X-linked recessive disorder of male sexual development caused by an absent or dysfunctional AR (1). Patients with AIS have a spectrum of abnormalities ranging from a completely female phenotype to a less severe form of hypospadias and gynecomastia or a phenotypically normal, but infertile, male (2). Although the infertile but otherwise normal male used to be recognized as the mildest form of AIS (3), cases of fertile patients began to emerge in the literature during the last 2 decades (4, 5, 6, 7, 8, 9). These findings raise the possibility that impaired spermatogenesis is not a necessary consequence of androgen insensitivity and hence demonstrated wider phenotypic variation in AIS.

The fertile AIS patients reported to date have displayed a common feature of mildly impaired AR activities and slight undervirilization, such as gynecomastia and/or small penis. Some of the cases were identified to be the consequence of single base mutations in the AR gene (4, 5). None of AR gene defects that cause severe undermasculinization, such as hypospadias, was observed previously to be compatible with male fertility. In the present paper we reported an Arg⁸⁴⁰Cys substitution in a large Chinese family affected with a highly divergent clinical phenotype of AIS. In this family some affected males were infertile and showed gynecomastia and/or hypospadias, but some had fathered children normally.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Family

The pedigree is illustrated in Fig. 1. A family interview denied consanguineous marriages. There were 14 affected males in the family. The present study examined 22 individuals (III 13–16, 21, and 31–35; IV 23, 24, 26, 28, 36, 39, 40, 53, 54, 61, and 66; V 10), and their blood samples, after obtaining informed consent, were collected for genetic analysis. The research was conducted with official approval from the academic advisory board of Institute of Genetics of Fudan University.

View larger version (14K): [in this window] [in a new window]   Figure 1. Pedigree of a Chinese family affected with AIS. Individuals whose DNA was tested are indicated by an asterisk.

The proband (IV 54) was born with penile hypospadias. Bilateral gynecomastia occurred at his age of 17 yr. He had normal karyotype (46,XY). Both his hypospadias and bilateral gynecomastia were surgically corrected. At the age of 27 yr, he fathered a healthy boy. The proband had nine affected cousins (IV 8, 9, 23, 28, 31, 36, 39, 61, and 64; photograph of IV 23 in Fig. 2). Two of them (IV 8 and 9) were abandoned at birth because of ambiguous external genitalia. Morphological phenotypes of the proband and his five affected cousins (IV: 23, 28, 31, 39 and 61) are summarized in Table 1. It can be seen that the affected individuals displayed varied degrees of undermasculinization.

View larger version (178K): [in this window] [in a new window]   Figure 2. Photograph of patient IV 23 at 13 yr of age, showing left cryptorchidism and gynecomastia.

Hormone, semen test, and testicular biopsy

Plasma T, LH, and FSH levels were measured by RIA for individuals IV 39, 54, and 61. The hormone measures of the proband (IV 54) were scored in three blood samples collected on different days. Semen was collected from IV 31, 39, 54, and 61 after 3 d of abstinence. For the proband, repeated measures were obtained on three ejaculations. Testicular biopsies were performed in IV 39 and 61.

Paternity test

Genomic DNA was extracted from peripheral lymphocytes of the 22 members who donated the blood samples according to standard procedures (10). Four highly polymorphic and randomly selected microsatellite markers (D8S1119, D10S1239, D14S250, and D15S655) were genotyped on an ABI PRISM 377 DNA sequencer (PE Applied Biosystems, Inc., Foster City, CA) to confirm the genetic relationship among these individuals.

Sequencing of the AR gene

Mutation detection. The whole coding sequence of the AR gene was amplified by PCR in one nuclear family (III 13 and 14; IV 23 and 24) using the primers listed in Table 2. All but 1 segment were amplified by 40 cycles of denaturation at 95 C for 45 sec, annealing at 60-68 C for 30 sed, and extension at 72 C for 1 min, using Taq DNA polymerase (AmpliTaq DNA polymerase, Perkin-Elmer Corp., Norwalk, CT) on a GeneAmp PCR system 9600. For segment flanked by primers A7 and A8, a final concentration of 10% dimethylsulfoxide was added, and the denaturation temperature was adjusted to 96 C. Amplified fragments were purified using the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) and sequenced from both directions on an ABI PRISM 377 DNA sequencer with the BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Inc.).

Linkage analysis

Genotypes of the mutant locus in all 22 family members were assayed by PvuI digestion of the PCR fragments containing coding sequence of exon 7. The LOD score was calculated using the computer program LINKAGE (12).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Hormone and semen analyses

Hormone and semen data for individuals IV 31, 39, 54, and 61 are summarized in Table 3. The semen measurements for the proband (IV 54) were within the normal range, and this was in agreement with his proven fertility. However, the other three cousins of the proband had abnormal semen. Moreover, the LH and FSH levels of the proband were also normal, whereas his affected cousins were abnormal for the corresponding measures. All of these four patients had T levels above normal. Testicular biopsy performed on IV 39 discovered dysplasia of the seminiferous tubule, with only a few primary spermatocytes. For individual IV 61, primary spermatocytes were normal, but his second spermatocytes were scarce, indicating arrest of spermatogenesis.

Genotypes of the 22 individuals at the 4 microsatellite marker loci showed that the segregation pattern at the marker loci was consistent with the blood relationship among members of the pedigree under study (Fig. 4).

View larger version (26K): [in this window] [in a new window]   Figure 4. Genotypes of a nuclear family under examination at four microsatellite marker loci and at the AR gene mutant site. Individuals whose DNA was tested are indicated by an asterisk. The proband is indicated with an arrow. A, The genotypes of four randomly selected markers (D8S1119, D10S1239, D14S250, and D15S655) are listed below each corresponding individual. B, Digestion of a 325-bp PCR fragment covering exon 7 of the AR gene with PvuI yields subfragments of 198 and 127 bp, as in normal individuals. The recognition site of PvuI is absent at the Arg840Cys substitution. The affected individuals show only one undigested fragment. In female carriers, both the two digested subfragments and the undigested mutant fragment are seen. Due to incomplete digestion, a weak 325-bp band is seen in some of the normal individuals. However, that will be not confused with the female carriers whose 325-bp band is significantly brighter than the 198- and 127-bp ones.

Mutation detection. A single base substitution of C to T at site 3851 was identified in exon 7 of the AR gene (Fig. 3). This mutation causes an Arg⁸⁴⁰Cys substitution (number according to Ref.13) and disruption of a PvuI site. No other mutation was found in the coding sequence. IV 23 carried a single copy of the mutant allele. His mother and sister (III 14 and IV 24) were heterozygotes of the mutant allele, and his father (III 13) carried only the normal allele. The mutation was also confirmed in another nuclear family (III 33 and 34; IV 61 and 66). To statistically exclude abundance of the mutant gene in natural population, 50 randomly selected chromosomes were assayed based on the PvuI digestion, and no such substitution was found in these chromosomes.

View larger version (36K): [in this window] [in a new window]   Figure 3. Detection of Arg840Cys substitution in exon 7 of AR gene by sequencing. In an affected individual, a C at base 3851 was replaced by T, causing an Arg840Cys substitution. In female carriers, both nucleotides are present.

Linkage analysis

The mutant allele consistently cosegregated with the disease phenotype (Fig. 4). Linkage analysis revealed a complete linkage ( = 0) between disease locus and the polymorphic site detected (LOD_max = 3.61) when the population frequency of the disease gene and the frequency of the mutant allele were assumed to be 0.1% (14).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Defects in male sexual differentiation and development may be explained genetically by a wide spectrum of mutations in the AR gene (15). The present study reports a complete linkage (LOD_max = 3.61 at = 0) between an Arg⁸⁴⁰Cys substitution in AR and AIS in a large Chinese pedigree.

Although the Arg⁸⁴⁰Cys substitution in the AR gene had been demonstrated to be associated with male infertility and severe undervirilization (16, 17, 18), a total of five fertile cases were reported in the present study to be compatible with the amino acid substitution. One of the major features of the family is the high phenotypic variation in undermasculinization, undervirilization, and/or fertility among the patients. The proband (IV 54) of the affected pedigree, one of the five fertile patients, was genetically analyzed and tested for semen and hormone data. The other four fertile patients (II 7 and 11; III 9 and 11) were not tested due to unavailability of their blood samples. However, the carrier status of the mutant gene for individuals II 7 and 11 can be readily and precisely inferred. In fact, marriages of both II 7 and 11 were not consanguineous. This makes it unlikely that the disease gene was transmitted from their wives (II 8 and 12). In addition, all five male children of II 7 and 11 were normal, and all three female children were heterozygotes. It can be anticipated from the pedigree structure that the disease gene carried by individuals IV 36, 39, 54, and 61 was paternally inherited from II 7 and 11.

Among the fertile cases with impaired AR activity reported to date, the most severe morphological phenotype is the slightly diminished penis size (4, 5, 6, 7, 8, 9). However, in the pedigree reported here, the patient who showed hypospadias and gynecomastia (IV 54) was fertile. At the other extreme case, the patient (IV 31) showed slight undervirilization, but had azoospermia. This indicates that androgen action may influence spermatogenesis and genitalia differentiation through different pathways and that spermatogenesis may be normally accomplished under a wide range of defects in the AR.

Semen and hormone analyses were carried out for some of the affected members. Patient IV 54 showed hypospadias and gynecomastia, but had normal semen density and motility and normal levels of LH and FSH. The normal semen and hormone data of the patient are paralleled by his preserved male fertility. The normal LH level of this patient may reveal that the function of AR is partially rescued. Sharing the same phenotype with patient IV 54, however, infertile patients IV 31, 39, and 61 had azoospermia, and the last two individuals had abnormally high hormone levels. An elevated LH level is recognized as one of the basic features of AIS and reflects the impaired androgen action in regulating gonadotropin secretion. It will be intriguing to clarify whether normal LH and FSH levels in IV 54 are prerequisite for the individual to maintain his fertility or whether the elevated LH and FSH levels in IV 39 and 61 have a toxic effect on spermatogenesis and, in turn, impair fertility. The arguments are supported by the previous observation that nearly all fertile subjects with AIS had normal LH and FSH levels (4, 5, 6, 7, 8, 9).

The present study demonstrates that the single nucleotide substitution polymorphism explains the segregation of the AIS symptom in a large AIS pedigree, but the molecular mechanisms of the high variation in disease phenotype among the patients is still unclear (19).

Polymorphisms at the (Gln)_n and (Gly)_n regions within the AR gene have been found to be associated with the trans-activational function of AR (11). The three affected individuals (IV 23, 36, and 54), who showed various phenotypes, had an identical genotype of (Gln)₂₁. The allele is inherited from the founder carrier at generation 1 and retained a stable transmission from generation to generation in the pedigree. In addition, all individuals shared the same (Gly)₂₃. These data thus exclude the explanation that the varied disease phenotypes were due to the varying repeat numbers of the amino acid residuals.

Nuclear receptor coregulators may play roles in modulating transcriptional regulation of androgen through their interaction with AR (20). The effect of AR gene mutation on its trans-activational function could have been attenuated by increased activities of some coactivators. Varied attenuation effects in different patients may result from varied individual coregulator activities or their different combinations and thus result in the phenotypic diversity.

In addition, the variation in phenotype among the different patients may be explained by fluctuation in androgen secretion during certain periods of sexual development. Indeed, The activity of the AR with the Arg⁸⁴⁰Cys substitution has been shown to be reduced under the normal physiological level of androgen, but the mutant AR showed an improved activity when the androgen level was high (17). Variable androgen levels even within its physiological range might be sufficient during the highly sensitive period of genitalia differentiation or spermatogenesis in some cases, but not in others.

Finally, the different expression levels of the AR gene and 5-reductase activities might have contributed to the phenotypic variation. To investigate the molecular mechanisms that underlie the phenotypic diversity in this pedigree, we have established genital skin fibroblast cell lines from some normal members and patients of the pedigree. The future research will be focused on the differentiation in 1) transcriptional and translational levels, 2) ligand binding ability, and 3) trans-activational function of the AR gene among genital skin fibroblast cell lines from different patients.

	Acknowledgments

 
We are grateful for two anonymous reviewers for their constructive criticisms and comments that have been very helpful in improving the manuscript. We thank the family members who attended the study.

	Footnotes

 
This work was supported by China’s Basic Research Project (Grant G199805100) and the National Natural Science Foundation of China (Grant 39993420).

¹ J.C. and R.Z. contributed equally to this work.

Abbreviations: AIS, Androgen insensitivity syndrome.

Received March 7, 2001.

Accepted October 9, 2001.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

1. Griffin JE, Leshin M, Wilson JD 1982 Androgen resistance syndromes. Am J Physiol 6:E81–E87
2. Quigley C, De Bellis A, Marschke K, El-Awady M, Wilson EM, French FS 1995 Androgen receptor defects: historical, clinical, and molecular perspectives. Endocr Rev 16:271–321[CrossRef][Medline]
3. Aiman J, Griffin JE, Gazak JM, Wilson JD, MacDonald PC 1979 Androgen insensitivity as a cause of infertility in otherwise normal men. N Engl J Med 1:223–227
4. Giwercman A, Kledal T, Schwartz M, Giwercman YL, Leffers H, Zazzi H, Wedell A, Skakkebaek NE 2000 Preserved male fertility despite decreased androgen sensitivity caused by a mutation in the ligand-binding domain of the androgen receptor gene. J Clin Endocrinol Metab 85:2253–2259[Abstract/Free Full Text]
5. Tsukada T, Inoue M, Tachibana S, Nakai Y, Takebe H 1994 An androgen receptor mutation causing androgen resistance in undervirilized male syndrome. J Clin Endocrinol Metab 79:1202–1207[Abstract]
6. Grino PB, Griffin JE, Cushard WG, Wilson JD 1988 A mutation of the androgen receptor associated with partial androgen resistance, familial gynecomastia, and fertility. J Clin Endocrinol Metab 66:754–761[Abstract]
7. Gottlieb B, Pinsky L, Beitel LK, Trifiro M 1999 Androgen insensitivity. Am J Med Genet 89:210–217[CrossRef][Medline]
8. Pinsky L, Kaufman M, Killinger DW 1989 Impaired spermatogenesis is not an obligate expression of receptor-defective androgen resistance. Am J Med Genet 32:100–104[CrossRef][Medline]
9. Larrea F, Benavides G, Scaglia H, Kofman-Alfaro S, Ferrusca E, Medina M, Perez-Palacios G 1978 Gynecomastia as a familial incomplete male pseudohermaphroditism type 1: a limited androgen resistance syndrome. J Clin Endocrinol Metab 46:961–970[Abstract]
10. Sambrook J, Fritsch EF, Maniatis T 1989 Molecular cloning: a laboratory manual, 2nd Ed. Cold Spring Harbor: Cold Spring Harbor Laboratory
11. Chamberlain NL, Driver ED, Miesfeld RL 1994 The length and location of CAG tri-nucleotide repeats in the androgen receptor N-terminal domain affect transactivation function. Nucleic Acids Res 22:3181–3186[Abstract/Free Full Text]
12. Terwilliger JD, Ott J 1994 Handbook of human linkage analysis. Baltimore: Johns Hopkins University Press
13. Lubahn DB, Brown TR, Simental JA, Higgs HN, Migeon CJ, Wilson EM, French FS 1989 Sequence of the intron/exon junction 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 USA 86:9534–9838[Abstract/Free Full Text]
14. Wieacker P, Griffin JE, Wienker T, Lopez JM, Wilson JD, Breckwoldt M 1987 Linkage analysis with RFLPs in families with androgen resistance syndromes: evidence for close linkage between the androgen receptor locus and the DXS1 segment. Hum Genet 76:248–252[CrossRef][Medline]
15. The Lady Davis Institute for Medical Research 2001 The androgen receptor gene mutation database. http://ww2.mcgill.ca/androgendb
16. Beitel LK, Kazemi-Esfarjani P, Kaufman M, Lumbroso R, Digeorge M, Killinger DW, Trifiro MA, Pinsky L 1994 Substitution of arginine-839 by cysteine or histidine in the androgen receptor causes different receptor phenotypes in cultured cells and coordinate degrees of clinical androgen resistance. J Clin Invest 94:546–554
17. Bevan CL, Brown BB, Davies HR, Evans BAJ, Hughes LA, Patterson MN 1996 Functional analysis of six androgen receptor mutations identified in patients with partial androgen insensitivity syndrome. Hum Mol Genet. 5: 265–273
18. McPhaul MJ, Marcelli M, Zoppi S, Wilson CM, Griffin JE, Wilson JD 1992 Mutations in the ligand-binding domain of the androgen receptor gene cluster in two regions of the gene. J Clin Invest 90:2097–2101
19. Rodien P, Mebarki F, Mowszouicz I, Chaussain JL, Young J, Morel Y, Schaison G 1996 Different phenotypes in a family with androgen insensitivity caused by the same M780I point mutation in the androgen receptor gene. J Clin Endocrinol Metab 81:2994–2997[Abstract]
20. McKenna NJ, Lanz RB, O’Malley BW 1999 Nuclear receptor coregulators: cellular and molecular biology. Endocr Rev 20:321–344[Abstract/Free Full Text]

This article has been cited by other articles:

				C. Bouvattier, B. Mignot, H. Lefevre, Y. Morel, and P. Bougneres Impaired Sexual Activity in Male Adults with Partial Androgen Insensitivity J. Clin. Endocrinol. Metab., September 1, 2006; 91(9): 3310 - 3315. [Abstract] [Full Text] [PDF]

				J. D. Veldhuis, A. Bae, R. S. Swerdloff, A. Iranmanesh, and C. Wang Experimentally Induced Androgen Depletion Accentuates Ethnicity-Related Contrasts in Luteinizing Hormone Secretion in Asian and Caucasian Men J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1632 - 1638. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chu, J. Articles by Luo, Z. Search for Related Content PubMed PubMed Citation Articles by Chu, J. Articles by Luo, Z.