Human Reproduction, Vol. 16, No. 8, 1653-1656,
August 2001
© 2001 European Society of Human Reproduction and Embryology
Mutation screening and CAG repeat length analysis of the androgen receptor gene in Klinefelter's syndrome patients with and without spermatogenesis
1 Department of Urology, Yamagata University School of Medicine, Yamagata and 2 Department of Pediatrics, Tokyo Electric Power Company Hospital, Tokyo, Japan
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Abstract |
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BACKGROUND: Mutations of the androgen receptor (AR) gene give rise to a wide array of phenotypic abnormalities. A systematic analysis of the AR gene in patients with 47,XXY has not previously been performed. METHODS: Mutations of the AR gene and expansion of the CAG repeats in exon 1 of the AR gene were studied in 13 patients with Klinefelter's syndrome either with (n = 1) or without (n = 12) spermatogenesis. RESULTS: No abnormalities in the AR gene were detected by single strand conformational polymorphism analysis. The CAG lengths ranged from 17 to 27 (mean ± SD 22.8 ± 3.3, median 23) for Klinefelter patients or from 17 to 28 (mean ± SD 23.2 ± 2.6, median 23) for control subjects. X-inactivation analysis for the methylation status of the AR gene was performed in seven patients who were heterozygous for CAG repeats of different length, showing that the longer CAG repeat alleles underwent random but more frequent inactivation in five patients and skewed inactivation in two. CONCLUSIONS: An AR gene abnormality does not constitute an important factor for impaired spermatogenesis in patients with Klinefelter's syndrome.
Key words: androgen receptor gene/CAG repeat/Klinefelter's syndrome/spermatogenesis/X-inactivation
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Introduction |
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Androgens play an important role in the prenatal virilization of the external genitalia in 46,XY fetuses. Hence mutations of the androgen receptor (AR) gene cause a wide spectrum of phenotypic abnormalities in 46,XY genetic males that vary in severity from phenotypic female external genitalia to phenotypic males with infertility (Griffin and Wilson, 1989
). These variable forms of phenotypic abnormalities due to AR gene mutations are all X-linked disorders and are known collectively as androgen insensitivity syndrome (AIS).
The AR gene has been successfully cloned from chromosome Xq12 (Lubahn et al., 1988, for example). Its protein coding regions are comprised of 8 exons: exon 1 encodes the transactivation domain; exons 2 and 3 encode the DNA binding domain; the 5' portion of exon 4 encodes the hinge domain, and the 3' portion of exon 4 together with exons 5 to 8 encode the ligand binding domain (Quigley et al., 1995).
Exon 1 contains highly polymorphic CAG repeats which encode for the polyglutamine tract of the AR. Results of functional studies of the AR gene with different CAG repeat numbers suggest an inverse relationship between the CAG repeat length and transactivation function or expression of the AR gene (Chamberlain et al., 1994; Tut et al., 1997).
The 47,XXY karyotype causes Klinefelter's syndrome, which is characterized by gynaecomastia, variable degrees of eunuchoidism and atrophic testes with absence of spermatogenesis (Jecht et al., 1984). Since a defect resulting from a mutant allele of the AR on one inactive X chromosome can be masked by the effect of the normal allele on the other active X chromosome (Lyon, 1961; Disteche, 1995), the combination of AIS and 47,XXY karyotype is exceedingly rare, one case having been reported (Muller et al., 1990). However, a systematic analysis of the AR gene in patients with 47,XXY karyotype has not been performed or reported to date. In the present study, we investigated the size or expansion of the CAG repeats—so-called dynamic mutations of the AR gene in 47,XXY patients with or without spermatogenesis.
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Case reports, materials and methods |
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Case reports
Thirteen patients with a full-blown 47,XXY karyotype in their peripheral blood lymphocytes were studied. All were aged between 23 and 49 years. Repeated semen analyses showed azoospermia in 12 cases and oligozoospermia (2.5x106/ml and 2.0x106/ml) in one case (Table I
). Fifty healthy fertile males with a normal 46,XY karyotype, aged from 23 to 42 years, comprised the control group.
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Materials and methods
Genomic DNA was obtained from peripheral lymphocytes using the Qiagen Blood and Cell Culture DNA Midi Kit (Qiagen, Hilden, Germany). For mutation screening of the AR gene, exons 1–8 and their flanking intron sequences, except the CAG and GGC repeat regions at exon 1, were amplified by polymerase chain reaction (PCR) (Lubahn et al., 1989
). The lengths of the PCR products analysed were 314 base pairs (bp), 599 bp and 320 bp in exon 1 (which was divided into three regions due to excessive length), 379 bp in exon 2, 413 bp in exon 3, 394 bp in exon 4, 285 bp in exon 5, 294 bp in exon 6, 416 bp in exon 7 and 347 bp in exon 8. A 5 µl PCR product was mixed with 5 µl of 95% formamide stop buffer for single strand conformational polymorphism (SSCP). Samples were denatured at 95°C for 5 min and thereafter placed directly on ice to prevent reannealing of the single-stranded product. They were loaded onto a 0.5 mm thick 122x110 mm 15% polyacrylamide gel and electrophoresced for 80 min at 600 V (25 mA) on a GenePhor (Pharmacia Biotech, Wikstroms, Sweden) at 23°C. Following electrophoresis, the gels were silver-stained using a DNA Silver Staining Kit (Pharmacia Biotech) with an automated gel stainer (Hoefer; Pharmacia Biotech).
The CAG repeat length in the AR gene was determined from leukocytic genomic DNA of each subject. The CAG repeat region was amplified by PCR with primers flanking the polymorphic CAG repeat region. Amplification was performed in a reaction volume of 20 µg containing 0.1 µg genomic DNA, 8pmol fluorescently labelled forward primer (5'-TCCAGAATCTGTTCCAGAGCGTGC3'), 8 pmol unlabelled reverse primer (5'-GCTGTGAAGG- TTGCTGTTCCTCAT-3'), 0.1 mmol/l dNPTs, and 1 U Taq polymerase (Allen et al., 1992). PCR was performed in 30 cycles for 45 s at 94°C, 45 s at 55°C, and 45 s at 72°C. The PCR products were mixed with internal control size markers and were electrophoresced on an autosequencer (ABI Prism 310; Applied Biosystems, Perkin Elmer, Norwalk, CT, USA). The size of the PCR products was determined by GeneScan software. Furthermore, to confirm the correct CAG repeat regions of 10 subjects with different CAG, repeat numbers were subjected to direct sequencing on the autosequencer.
X-inactivation analysis for methylation status of the AR gene was performed for patients heterozygous for the CAG repeat lengths. In brief, leukocytic genomic DNA was amplified by PCR, as described in the CAG repeat length analysis, before and after HpaII digestion, and the PCR products were examined for fragment size and area under curve on the autosequencer. Since the region subject to PCR amplification contains two methylation sensitive HpaII sites in addition to the CAG repeats, PCR products are obtained from both active and inactive X chromosomes before and from inactive X chromosomes alone after HpaII digestion (Allen et al., 1992).
In all patients with a 47,XXY karyotype, plasma concentrations of LH, FSH and testosterone were determined by solid-tube radioimmunoassay (LH, FSH: Daiichi Radioisotope Laboratory, Tokyo, Japan; testosterone: Diagnostic Products Corporation, Los Angeles, CA, USA).
All results were expressed as the mean ± SD. Statistical analysis was carried out using the Statview 4.0 program (Abacus Concept, Berkeley, CA, USA). The Mann–Whitney U-test was used for comparison of hormonal profiles of 47,XXY patients and the control group. P < 0.05 was considered statistically significant.
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Results |
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Table I
shows the clinical, hormonal and seminal parameters in the Klinefelter's syndrome patients with and without spermatogenesis. Plasma LH and FSH concentrations were abnormally high and plasma testosterone concentrations were relatively low in all patients. These hormone concentrations were significantly different between Klinefelter's patients and controls (P < 0.001).
No abnormal SSCP band patterns in exons 1–8 of any patient could be detected. The CAG repeat lengths and the X-inactivation patterns are summarized in Table II. The mean ± SD CAG repeat length was 22.8 ± 3.3 (median 23, range 17–27) for the 13 Klinefelter's patients, 19 and 26 for case 1 with spermatogenesis, 22.8 + 3.2 (median 23, 17–27) for the 12 patients without spermatogenesis, and 23.2 ± 2.6 (median 23, range 17–28) in the control males. Thus, the CAG repeat lengths did not differ between 47,XXY males and the controls. X-inactivation analysis in seven patients heterozygous for the CAG repeat lengths showed that X chromosomes with longer CAG repeat alleles underwent random but more frequent inactivation in five patients and skewed inactivation in two patients (Figure 1).
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Discussion |
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TopAbstractIntroductionCase reports, materials and...ResultsDiscussionReferences |
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Some cases of male infertility are thought to be a result of a minimal androgen insensitivity (Aiman and Griffin, 1982
). To date, some mutations of the AR gene have been reported in infertile males: a deletion in exon 4 of the AR gene in azoospermic males (Akin et al., 1991); point mutations of exons 6 and 8 in patients with minimal androgen insensitivity and spermatogenic defects (Tsukada et al., 1994; Yong et al., 1998); missense mutations of exons 1, 5 and 8 in patients with severe oligozoospermia (Yong et al., 1994; Wang et al., 1998; Knoke et al., 1999); as well as being listed in the AR mutation database (Gottlieb et al., 1999). However, the present study showed no abnormal SSCP band pattern of the AR gene in Klinefelter's syndrome patients either with or without spermatogenesis. In this regard, it might be possible that a mutation has remained undetected because the sensitivity of SSCP analysis has not been completed, especially when applied to fragments larger than 300 bp, under one set of experimental conditions (Ellis et al., 2000), as has been done in this study. It might also be possible that a mutation exsisted in an unexamined region(s) such as the CAG and GGC repeat regions, the promoter region, or the intron sequences. Overall, however, the results suggest that a mutation of the AR gene is rare, if at all, in patients with Klinefelter's syndrome. The CAG repeat length in men with variety of spermatogenic disorders has been studied by several investigators and the association between expanded CAG repeat length and defective spermatogenesis was found by some investigators to be positive but this could not be collaborated by others (Tut et al., 1997; Giwercman et al., 1998; Dowsing et al., 1999; Yoshida et al., 1999; Dadze et al., 2000). Since the causes of unobstructive azoospermia and various degrees of defective spermatogenesis are highly heterogeneous, such a discrepancy is not surprising. Thus the CAG repeat length of expansion may play a relatively important role in some azoospermic patient populations but not in others. In the present study of Japanese patients, the CAG repeat length of control subjects was not significantly different from those of Klinefelter's syndrome patients. It appears that the variation in the CAG repeat length of patients with Klinefelter's syndrome is not related to the quality of spermatogenesis, although it should be stressed that our patient population comprised only one with spermatogenesis. Furthermore, X-inactivation analysis revealed that the longer CAG repeat alleles underwent random but more frequent inactivation or skewed inactivation. Although the X-inactivation pattern may more or less be different between peripheral leukocytes analysed in the present study and genital region where the AR gene is expressed, the results argue against the possibility that the predominant expression of the longer CAG repeat alleles may be relevant to spermatogenic failure in Klinefelter's syndrome. In conclusion, the present study suggests that AR gene abnormality does not constitute an important factor for impaired spermatogenesis in patients with Klinefelter's syndrome. However, more studies of larger patient samples are required to further examine the relevance, if any, of AR gene abnormalities to spermatogenesis in 47,XXY individuals.
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Notes |
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3 To whom correspondence should be addressed at: Department of Urology, Yamagata University School of Medicine,2–2–2 Iidanishi, Yamagata-shi, Yamagata 990-9585, Japan. E-mail: isasaga{at}med.id.yamagata-u.ac.jp
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Submitted on January 3, 2001; accepted on April 26, 2001.
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