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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Key words: androgen receptor gene/CAG repeat/Klinefelter's syndrome/spermatogenesis/X-inactivation
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Introduction |
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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 repeatsso-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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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 MannWhitney 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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No abnormal SSCP band patterns in exons 18 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 1727) for the 13 Klinefelter's patients, 19 and 26 for case 1 with spermatogenesis, 22.8 + 3.2 (median 23, 1727) for the 12 patients without spermatogenesis, and 23.2 ± 2.6 (median 23, range 1728) 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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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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References |
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Submitted on January 3, 2001; accepted on April 26, 2001.