Polymorphic CAG repeats of the androgen receptor gene in Japanese male patients with ankylosing spondylitis
K. Mori,
T. Ushiyama,
K. Inoue and
S. Hukuda
Department of Orthopaedic Surgery, Shiga University of Medical Science, Seta, Otsu, 520-2192, Japan
 |
Abstract
|
---|
Objective. In view of a possible role of androgens in the pathogenesis of ankylosing spondylitis (AS), we investigated the association between Japanese male patients with AS and CAG microsatellites of the androgen receptor (AR) gene which related to the AR transactivation function.
Methods. Peripheral blood was collected from 39 men with AS and 305 male control subjects. The number of CAG repeats in exon 1 of the AR gene was determined.
Results. CAG repeat lengths in AS patients were significantly shorter than those in the controls (median value 22 vs 23; P = 0.03). However, there was no significant difference in CAG repeats between HLA-B27-positive and -negative patients (median value 22 vs 22; P = 0.78).
Conclusions. Shorter CAG repeats of the AR gene, presenting high levels of transactivation activity, may play a role in male AS.
KEY WORDS: Ankylosing spondylitis, Androgen, Androgen receptor gene, CAG microsatellites.
 |
Introduction
|
---|
There is a male preponderance in the prevalence of ankylosing spondylitis (AS), and the role of sex hormones such as androgens has been discussed [1, 2]. Previous studies, however, have failed to show altered serum androgen levels in AS [37]. As shown in family and twin studies, there is a strong genetic association with the disease [8, 9]. Although HLA-B27 is known as the most important genetic factor, there is considerable epidemiological evidence implicating non-HLA factors in the genetic susceptibility to AS [810].
The effects of androgens depend on the plasma concentration available for tissue uptake and the interaction of androgens with the androgen receptor (AR). The androgenAR complex activates the expression of other genes (transactivation). This transactivation activity resides in the NH2-terminal domain of the protein, encoded in exon 1, which contains a polymorphic CAG repeat sequence. In vitro, the length of the CAG repeat is inversely related to levels of transactivation by the AR [11]. Men who possess exceptionally long CAG repeat lengths in spinal and bulbar muscular atrophy manifest an androgen insensitivity, probably due to reduced transactivation activity of the AR [12].
In this study, we investigated the possible relationship between Japanese male AS and polymorphic CAG repeats of the AR gene.
 |
Subjects and methods
|
---|
Subjects
Several institutions co-operated for this study, providing blood samples from AS patients who fulfilled the modified New York criteria [13]. Because of the low number, female AS patients were excluded, and a total of 39 Japanese male patients with AS were studied, with a mean age of 43.1 yr (range 1864). Thirty-two patients were HLA-B27 positive and seven were negative.
As a control, 305 Japanese men (mean age 48.9 yr, range 1791), who underwent an annual health check held by several communities, participated in the study. Peripheral blood was collected for analysis of AR gene microsatellites from both the AS patients and control subjects.
Polymorphic CAG repeats of the AR gene
The determination of CAG repeats of the AR gene was based on a previous report with a slight modification [11]. One hundred nanograms of genomic DNA, extracted from peripheral blood, was amplified by two rounds of a polymerase chain reaction using nested primers surrounding the CAG repeat in exon 1 of the AR gene. The final products were analysed by electrophoresis on 6% denaturing polyacrylamide gels, and then the gels were stained using a silver staining method. To determine the number of CAG repeats, all of the different sizes of the final products were sequenced using an ABI 310 sequencer (Perkin-Elmer, CA, USA) according to the manufacturer's protocol.
Statistical analysis
MannWhitney's U-test was applied when appropriate. P values less than 0.05 were regarded as significant. The software application used for the analysis was SPSS (SPSS Inc., Chicago, IL, USA).
 |
Results
|
---|
The number of CAG repeats of the AR gene was from 16 to 27 in patients with AS, and 14 to 32 in the control subjects. The median value of CAG repeats in male AS was significantly smaller than in the male controls (22 vs 23; P = 0.03 by MannWhitney's U-test) (Table 1
). The median value of CAG repeats was also compared between HLA-B27-positive and -negative patients, but no significant difference could be found (22 vs 22; P = 0.78).
 |
Discussion
|
---|
The present study demonstrated the association of polymorphic CAG repeats of the AR gene with male Japanese patients with AS. The number of CAG repeats was significantly smaller in AS than in the controls. The AR gene, which is located in chromosome X, does not map to any of the previously reported hot spots in the whole genome screen of British patients with AS [10].
Previous studies have shown that the mean CAG repeat length was shorter in patients with prostate cancer [1416] and in younger onset male rheumatoid arthritis (RA) [17] than in control subjects. In addition, shorter CAG repeats (
18) of the AR gene have been shown as a significant risk factor for prostate cancer, especially for an aggressive phenotype [14], while they (<18) are also significant for the development of RA in younger men [17]. In view of these studies, we separately analysed the association between shorter CAG repeats (
18) and male AS, and found that shorter repeats were also related to developing AS [odds ratio (OR) = 4.34; 95% confidence interval (CI) = 1.4013.4]. Therefore, the increased transactivation levels of those having shorter CAG repeats might be related to disease occurrence through some biological mechanisms. Synovial cells are androgen responsive, as are prostate cells [18], and ARs play a role in sex steroid regulation of B lymphopoiesis [19]. From these observations, it is possible to speculate that cells expressing AR and with shorter CAG repeats are prone to irreversible pathological changes, such as uncontrolled cell proliferation, leading to prostate cancer on some occasions and to chronic inflammation in others.
As reviewed by James [2], a suspected role of higher serum testosterone levels in AS has been suggested, whereas an aetiological role of lower testosterone levels has been indicated in RA [20]. In our previous report showing the association between shorter CAG repeats and younger onset male RA [17], we postulated that shorter CAG repeats might lead to low levels of testosterone. Thus, at the beginning of the present study, we anticipated that patients with AS might have longer CAG repeats. However, considering our results, the assumption that the repeat length of the AR is related to these disorders through altered levels of testosterone is unjustified.
It would be enlightening to determine whether HLA-B27-positive normal controls have a similar alteration in the number of CAG repeats of the AR gene. However, the prevalence of HLA-B27 is extremely low in Japan [21], and AS is an uncommon disorder with an estimated prevalence of 0.03% [22]. Considering the small number of AS patients enrolled in this study and inter-racial differences in the mean lengths of CAG repeats [16], the association between AS and AR gene polymorphisms should be addressed in a larger study.
 |
Acknowledgments
|
---|
We thank Dr H. Inoue, Assistant Professor of Orthopaedic Surgery at Juntendo University, and Dr K. Shichikawa, President Emeritus of Yukioka Hospital, for their kind assistance along with the members of the Japanese Association of Ankylosing Spondylitis Patients (AS tomonokai) for their generous co-operation in providing blood samples. This study was supported in part by a Grant-in-Aid for Scientific Research (10671355) from the Ministry of Education, Science, and Culture of Japan.
 |
Notes
|
---|
Correspondence to: K. Mori. 
 |
References
|
---|
- Silman AJ, Hochberg MC. Epidemiology of the rheumatic diseases. Oxford: Oxford Medical Publications, 1993.
- James WH. Sex ratio and hormones in HLA related rheumatic diseases. Ann Rheum Dis1991;50:4014.[Abstract]
- Gordon D, Beastall GH, Thomson JA, Sturrock RD. Androgenic status and sexual function in males with rheumatoid arthritis and ankylosing spondylitis. Q J Med1986;60:6719.[ISI][Medline]
- Spector TD, Ollier W, Perry LA, Silman AJ, Thompson PW, Edwards A. Free and serum testosterone levels in 276 males: a comparative study of rheumatoid arthritis, ankylosing spondylitis and healthy controls. Clin Rheumatol1989;8:3741.[ISI][Medline]
- Bronson WD, Walker SE, Hillman LS, Keisler D, Hoyt T, Allen SH. Bone mineral density and biochemical markers of bone metabolism in ankylosing spondylitis. J Rheumatol1998;25:92935.[ISI][Medline]
- Giltay EJ, Popp-Snijders C, van Schaardenburg D, Dekker-Saeys BJ, Gooren LJG, Dijkmans BAC. Serum testosterone levels are not elevated in patients with ankylosing spondylitis. J Rheumatol1998;25:238994.[ISI][Medline]
- Tapia-Serrano R, Jimenez-Balderas FJ, Murrieta S, Bravo-Gatica C, Guerra R, Mintz G. Testicular function in active ankylosing spondylitis. Therapeutic response to human chorionic gonadotrophin. J Rheumatol1991;18:8418.[ISI][Medline]
- Rubin LA, Amos CI, Wade JA et al. Investigating the genetic basis for ankylosing spondylitis: linkage studies with the major histocompatibility complex region. Arthritis Rheum1994;37:121220.[ISI][Medline]
- Brown MA, Kennedy LG, MacGregor AJ et al. Susceptibility to ankylosing spondylitis in twins: the role of genes, HLA, and the environment. Arthritis Rheum1997;40:18238.[ISI][Medline]
- Brown MA, Pile KD, Kenedy G et al. A genome-wide screen for susceptibility loci in ankylosing spondylitis. Arthritis Rheum1998;41:58895.[ISI][Medline]
- Chamberlain NL, Driver ED, Miesfeld RL. The length and location of CAG trinucleotide repeats in the androgen receptor N-terminal domain affect transactivation function. Nucleic Acids Res1994;22:31816.[Abstract]
- Mhatre AN, Trifiro MA, Kaufman M et al. Reduced transcriptional regulatory competence of the androgen receptor in X-linked spinal and bulbar muscular atrophy. Nat Genet1993;5:1848.[ISI][Medline]
- van der Linden S, Valkenburg HA, Cats A. Evaluation of diagnostic criteria for ankylosing spondylitis: a proposal for modification of the New York criteria. Arthritis Rheum1984;27:3618.[ISI][Medline]
- Giovannucci E, Stampfer MJ, Krithivas K et al. The CAG repeat within the androgen receptor gene and its relationship to prostate cancer. Proc Natl Acad Sci USA1997;94:33203.[Abstract/Free Full Text]
- Stanford JL, Just JJ, Gibbs M et al. Polymorphic repeats in the androgen receptor gene: Molecular markers of prostate cancer risk. Cancer Res1997;57:11948.[Abstract]
- Irvine RA, Yu MC, Ross RK, Coetzee GA. The CAG and GGC microsatellites of the androgen receptor gene are in linkage disequilibrium in men with prostate cancer. Cancer Res1995;55:193740.[Abstract]
- Kawasaki T, Ushiyama T, Ueyama H et al. Polymorphic CAG repeats of the androgen receptor gene and rheumatoid arthritis. Ann Rheum Dis1999;58:5002.[Abstract/Free Full Text]
- Cutolo M, Accardo S, Villaggio B et al. Evidence for the presence of androgen receptors in the synovial tissue of rheumatoid arthritis patients and healthy controls. Arthritis Rheum1992;35:100715.[ISI][Medline]
- Smithson G, Couse JF, Lubahn DB, Korach KS, Kincade PW. The role of estrogen receptors and androgen receptors in sex steroid regulation of B lymphopoiesis. J Immunol1998;161:2734.[Abstract/Free Full Text]
- Cutolo M, Masi AT. Do androgens influence the pathophysiology of rheumatoid arthritis? Facts and hypotheses. J Rheumatol1998;25:10417.[ISI][Medline]
- Khan MA, Khan MK. HLA-B27 as an aid to diagnosis of ankylosing spondylitis. In: Khan MA, ed. Spine, state of the art review, Vol. 4. Philadelphia: Hanley and Belfus,1990:61725.
- Shichikawa K, Takenaka Y, Inoue K et al. Epidemiological study of rheumatic diseases in Japan. J Shiga Univ Med Sci1989;4:113 (in Japanese).
Submitted 2 June 1999;
revised version accepted 14 December 1999.