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The Prostate 41:253–257 (1999)
Androgen Receptor CAG Repeat Length
Polymorphism in Benign Prostatic Hyperplasia
(BPH): Correlation With Adenoma Growth
Kenji Mitsumori, Akito Terai,* Hiroya Oka, Takehiko Segawa, Keiji Ogura,
Osamu Yoshida, and Osamu Ogawa
Department of Urology, Faculty of Medicine, Kyoto University, Kyoto, Japan
BACKGROUND. The androgen receptor (AR) gene has a polymorphic CAG microsatellite
encoding variable-length glutamine repeats in the AR protein. The purpose of this study was
to evaluate the association between the growth of benign prostatic hyperplasia (BPH) and the
AR gene CAG repeat length.
METHODS. We determined CAG repeat lengths in 176 BPH patients who underwent simple
prostatectomy and in 41 control subjects without benign prostatic enlargement (non-BPE
group).
RESULTS. A statistically significant (P < 0.02) trend for large adenoma size with short CAG
repeat length was found among the adenoma quartiles. CAG repeat length in the fourth
quartile (large adenoma, 21.5 ± 2.7) was significantly shorter than in the first quartile (small
adenoma, 23.3 ± 2.1, P < 0.02). It tended to be shorter than in the non-BPE group (23.1 ± 2.4),
but CAG repeat lengths in the entire BPH (22.4 ± 2.5) and non-BPE groups did not significantly differ. The relative risk of large BPH (the fourth quartile) was 2.75 (95% confidence
interval, 1.05–7.24; P < 0.05) on comparing CAG repeats of ⱕ22–ⱖ23.
CONCLUSIONS. Shorter CAG alleles may be a genetic factor that promotes the growth of
BPH. Prostate 41:253–257, 1999. © 1999 Wiley-Liss, Inc.
KEY WORDS:
CAG microsatellite; androgen receptor; benign prostatic hyperplasia;
adenoma size; relative risk
INTRODUCTION
Benign prostatic hyperplasia (BPH) is a very common pathology in 50% of 50-year-old men and in 90%
of 90-year-old men [1]. Although the pathogenesis and
natural course of BPH are still unclear, androgen is
widely accepted as necessary for differentiation and
growth of the prostatic epithelium and as having a
critical role in pathological conditions of the prostate,
including prostate cancer and BPH [2]. Responses to
androgens are mediated through the androgen receptor (AR), a member of the steroid receptor superfamily
of ligand-dependent transcription factors.
The androgen receptor (AR) gene has a polymorphic CAG microsatellite in exon one that codes for
variable-length glutamine repeats in the N-terminal
domain of the AR protein [3]. Previously reported in
vitro studies showed that the length of the polygluta© 1999 Wiley-Liss, Inc.
mine chain is correlated inversely with the transcriptional activity of the AR [4–6]. Consistent with these
observations, expansion of AR gene CAG repeat
length has been found in patients with partial androgen insensitivity, such as in Kennedy’s disease [7].
CAG repeat lengths have been intensively studied
in patients with prostate cancer [8–14]. The length of
CAG repeats is known to be associated with susceptibility to or age of onset of prostate cancer. Despite
the central role of androgens in the development of
The authors certify that all authors of this manuscript have no financial arrangement with a company whose product figures prominently in the submitted manuscript or with a company making a
competing product.
*Correspondence to: A. Terai, M.D., Department of Urology, Faculty
of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan.
E-mail: akiterai@kuhp.kyoto-u.ac.jp
Received 25 January 1999; Accepted 11 June 1999
254
Mitsumori et al.
BPH, only a few studies have analyzed CAG repeat
lengths in BPH patients. Recently, Giovannucci et al.
[15] found an inverse correlation between the AR gene
CAG repeat length and the prevalence of BPH in men
enrolled from the Health Professionals Follow-Up
Study. Men were defined as having BPH if they had
responded in the affirmative to a questionnaire as
“ever [having been] diagnosed with an enlarged prostate by digital rectal examination (DRE)” and/or “ever
[having] had surgery for BPH.” Additional parameters, such as prostate volume and histopathology,
had not been analyzed in their study.
We hypothesized that variation in the transcriptional activity of the AR gene that is mediated by different CAG repeat lengths may be a genetic factor that
affects the growth of adenomas. We have examined
AR gene CAG repeat lengths in BPH patients who
underwent open prostatectomy and for whom adenoma weight had been measured.
MATERIALS AND METHODS
Study Population
Between 1980–1996, 530 Japanese patients with
BPH underwent surgery at Kyoto University (204
open prostatectomies, 326 transurethral resections of
the prostate). Weights of the adenomas had been measured for 176 of the 204 patients who underwent open
prostatectomy, and the histopathological diagnosis of
benign prostatic hyperplasia was confirmed using
step sections of whole specimens. The median weight
of the resected adenomas was 40.0 g (25th and 75th
percentiles; 25.0 and 59.3 g, respectively). All 176 specimens were subjected to CAG repeat length analysis.
Men without benign prostatic enlargement (BPE)
were matched against the groups of prostatectomized
patients as the control group. This non-BPE group
consisted of 41 men who met all the following criteria:
age 65 years or older, normal serum PSA level, benign
DRE finding, and normal appearance with <20 ml
prostate volume on transrectal ultrasonography
(TRUS).
Eighteen women selected at random comprised an
additional control group.
DNA Extraction
Because somatic mutations in the lengths of CAG
repeats in the AR gene are rare, we used paraffinembedded tissues of resected adenomas to extract the
target DNA with the standard protocols [14]. Briefly,
15 × 15 mm tissue sections sliced to 10-␮m thickness
were treated twice with xylene to remove paraffin,
and then washed twice with 99.5% ethanol. After being dried in a vacuum, the samples were incubated at
50°C for 48 hr in 100 ␮l of tissue lysis buffer (50 mM
Tris, pH 8.5, 1 mM EDTA, 0.5% Tween-20) containing
200 mg/ml of Proteinase K. To inactivate proteinase,
the samples were boiled for 10 min and then centrifuged. Five microliters of each supernatant were used
as the template in the polymerase chain reaction
(PCR).
Genomic DNAs of the non-BPE subjects and
women were extracted from peripheral blood lymphocytes.
Analysis of CAG Repeat Length in the AR
The CAG region of the AR gene was amplified by
PCR. The primer pairs used, AR-F (5⬘-TGC GCG AAG
TGA TCC AGA AC-3⬘) and AR-R (5⬘-CTT GGG GAG
AAC CAT CCT CA-3⬘), produced fragments of 195 bp
for 17 repeats and 234 bp for 29 repeats.
Each 30 ␮l of reaction mixture contained 50 pmol of
each primer, 200 nM dNTPs, 2.0 mM MgCl2, 1 × PCR
Buffer II (Perkin Elmer, Norwalk, CT), 0.75 U of
AmpliTaq Gold (Perkin Elmer), and 5 ␮l of the target
DNA. Amplification conditions were the initial denaturation and activation of the enzyme at 95°C for 8
min followed by 38 cycles of 94°C for 1 min, 60°C for
1 min, 72°C for 1 min, and final extension at 72°C for
10 min.
After amplification, the PCR products were separated by electrophoresis at 900 v for 5 hr on 8% polyacrylamide gels on nonfluorescence glass plates. The
gels were stained with SYBR Green I (Molecular
Probes, Eugene, OR), and PCR bands were made visible with FluoroImager SI (Amersham Pharmacia Biotech, Buckinghamshire, UK). Repeat sizes were determined by a comparison with a series of previously
determined (by direct sequencing of PCR products)
CAG size standards. All samples were examined at
least twice. At the second electrophoresis, we applied
the samples in ranked order based on size and confirmed the original determinations.
Statistical Methods
Simple regression analysis was used to evaluate the
relationship between CAG repeat length and adenoma
weight. Differences in CAG repeat length distribution
were examined by the Mann-Whitney U test (between
two groups) or the Kruskal-Wallis and Scheffé F tests
(among quartiles and five groups). The relative risk
and Miettinen 95% confidence interval were analyzed
using 2 × 2 tables and the ␹2 test. All statistical analyses were done with StatView-J 4.5 software (Abacus
Concepts, Berkeley, CA).
Androgen Receptor CAG Microsatellite in BPH
255
Fig. 1. A: Association between number of AR gene CAG repeats and adenoma weight. B: Association between patient age and adenoma
weight.
RESULTS
Excluding 18 specimens from which no amplified
products were obtained after repeated PCR, CAG repeat length could be determined for 158 alleles of the
BPH patients, 41 alleles of the non-BPE group, and 36
alleles of the female control group. The associations of
adenoma weight with patient age and CAG repeat
length in the BPH patients were assessed by regression analysis (Fig. 1). Adenoma weight was inversely
related to CAG repeat length, but the coefficient of
determination was low (R2 = 0.075). In contrast, adenoma weight did not have a statistically significant
association with patient age.
All BPH specimens were divided into quartiles by
adenoma weight (first quartile, ⱕ29.0 g; second, >29.0
g and ⱕ40.0 g; third, >40.0 g and ⱕ59.3 g; and fourth,
>59.3 g) to further analyze the association between the
weight of adenomas and CAG repeat length. The
trend for larger adenoma size with shorter CAG repeat length was statistically significant (P < 0.02), and
CAG repeat length in the fourth quartile (21.5 ± 2.7)
was significantly shorter than that in the first quartile
(23.3 ± 2.1, P < 0.02). When the non-BPE group was
considered, CAG repeat length did not differ significantly between the BPH (22.4 ± 2.5) and non-BPE
groups (Table I). CAG repeat length in the fourth
quartile, however, was still significantly shorter than
that in the first quartile (P < 0.05) and tended to be
shorter than in the non-BPE group (23.1 ± 2.4, P =
0.0584). Furthermore, CAG repeat length in the 199
men analyzed (22.6 ± 2.5) agreed with that in the randomly selected female control group (23.3 ± 2.6).
The relative risk of BPH was calculated, using the
median repeat length in the non-BPE group as the
arbitrary cutoff point (ⱕ22 and ⱖ23 CAG repeats,
Table II). There was a 1.97-fold nonsignificant excess
risk for BPH associated with ⱕ22 CAG repeats. These
repeats, however, showed the highest relative risk for
large BPH (the fourth quartile), which reached statistical significance (relative risk, 2.75; 95% confidence
interval, 1.05–7.24; P < 0.05).
DISCUSSION
BPH seems to be a pathobiological process that, at
least in part, depends on androgen. Although androgen does not cause BPH, its development requires the
presence of testicular androgens during prostate development, puberty, and aging. AR levels in the prostate remain high throughout aging, unlike in other
androgen-dependent organs [2]. The prostate, therefore, retains the capacity for further growth, despite
the age-related decreasing levels of androgen in peripheral circulation. Because AR gene CAG repeat
length is inversely correlated with the transcriptional
activity of the receptor, it also may affect the development of BPH. Recently, Giovannucci et al. [15] found
an inverse correlation between AR gene CAG repeat
length and the prevalence of BPH in men enrolled
256
Mitsumori et al.
TABLE I. Distribution of CAG Repeat Length in Adenoma Quartiles of BPH and in Control Groups*
BPH
No. of cases
analyzed
Median adenoma
weight (g)
(minimum–
maximum)
Mean age
No. of CAG repeats
15–16
17–18
19–20
21–22
23–24
25–26
27–28
29–30
Noninformative
Minimum–maximum
Mean
All BPH
First quartile
Second quartile
Third quartile
Fourth quartile
Non-BPE
(control)
Womena
176
44
44
44
44
41
18
40.0
(4.2–155.0)
17.0
(4.2–25.0)
33.5
(26.0–40.0)
49.5
(40.3–59.0)
75.0
(60.0–155.0)
N.A.
N.A.
71.3 ± 6.8
(55–87)
1
69.9 ± 6.5
(57–85)
0
71.2 ± 7.2
(55–87)
0
71.5 ± 6.2
(59–83)
0
72.1 ± 7.1
(58–85)
1
77.1 ± 7.8
(65–93)
0
N.A.
6
28
49
43
20
9
2
18
15–29
22.4 ± 2.5
0
4
12
14
9
2
1
2
20–29
23.3 ± 2.1
0
6
16
8
3
4
1
6
19–29
22.6 ± 2.6
2
4
13
9
5
1
0
10
17–27
22.4 ± 2.3
4
14
8
12
3
2
0
0
15–28
21.5 ± 2.7
0
5
10
16
7
2
1
0
19–29
23.1 ± 2.4
2
2
10
11
6
5
0
0
17–28
23.3 ± 2.6
0
*N.A., not applied. Results of statistical analysis: 1) Among the 4 quartiles of BPH: P < 0.02 by the Kruskal-Wallis test. First vs. fourth
quartiles: P < 0.02 by the Scheffé F test. 2) All BPH vs. non-BPE: not significant by Mann-Whitney U test. 3) Among the 5 groups
(non-BPE and the 4 quartiles of BPH): P < 0.02 by Kruskal-Wallis test. First vs. fourth quartiles: P < 0.05 by Scheffé F test. Fourth
quartile vs. non-BPE: P = 0.0584 by the Scheffé F test.
a
Thirty-six alleles were analyzed from 18 randomly selected women.
TABLE II. Relative Risk of BPH Based on CAG Repeat Length†
Case
Control
No. of CAG repeats
No. of cases
No. of controls
RR
95% CI
All BPH
Non-BPE
Non-BPE
Second quartile
Non-BPE
Third quartile
Non-BPE
Fourth quartile
Non-BPE
84
74
16
26
22
16
19
15
27
17
15
26
15
26
15
26
15
26
15
26
1.97
1.00
1.07
1.00
2.38
1.00
2.20
1.00
2.75*
1.00
0.91–4.27
First quartile
ⱕ22
ⱖ23
ⱕ22
ⱖ23
ⱕ22
ⱖ23
ⱕ22
ⱖ23
ⱕ22
ⱖ23
0.24–4.79
0.85–6.64
0.75–6.46
1.05–7.24
†
RR, relative risk; CI, confidence interval.
*P < 0.05.
from the Health Professionals Follow-Up Study. Men
with ⱕ19 CAG repeat lengths had odds ratio for BPH
of 1.92 relative to those with ⱖ25 repeats. Because the
definition of BPH was based on questionnaire responses of a history of diagnosis of an enlarged prostate by DRE and/or a history of surgery for BPH, the
additional parameters of prostate volume and histopathology were not analyzed in their study.
In the study reported here, we found an inverse
correlation between adenoma weight and AR gene
CAG repeat length (Fig. 1), although the coefficient of
determination in the regression line was low (R2 =
0.075). We also found a statistically significant trend
among the adenoma quartiles for larger adenoma
sizes with shorter CAG repeat lengths (Table I). These
findings support our hypothesis that short CAG repeats may promote the growth of prostatic adenoma,
possibly through modification of the transcriptional
activity of the AR. Furthermore, this is the first study
to demonstrate unambiguously that there is an inverse
correlation between prostatic adenoma size and AR
gene CAG repeat length.
Androgen Receptor CAG Microsatellite in BPH
With regard to BPH, defining the control group
may be difficult because it is unusual to find a histologically normal prostate in men over 70 years of age.
Berry et al. [1] reported that the average weight of an
adult human prostate with normal histology is 20 ± 6
g and remains constant with age. In a healthcare examination, Kitagawa et al. [16] found that prostatic
size as a function of age is separable into two groups;
increasing and no-change (20.0 ± 5.0 g). On the basis of
these reports, we defined the control subjects in our
study as men ⱖ65 years old, of normal echogenicity
with <20 ml prostate volume on TRUS, benign DRE
findings, and normal serum PSA levels. Although we
did not examine the pathology of the prostate in the
control group, these criteria would exclude nearly all
prostatic diseases of clinical significance in aged men.
In this study, CAG repeat length did not differ significantly between the BPH and non-BPE groups, but the
fourth quartile (large adenoma) group tended to have
shorter CAG repeats than the non-BPE group (Table I).
On the basis of these findings, we conclude that
short CAG repeats may promote the growth of prostatic adenoma through modification of the transcriptional activity of the AR. Their impact, however, may
become overt only in patients with large BPH, probably because of great interindividual variability in
other risk factors such as androgens. A familial form
of BPH was reported in several recent studies, indicative of the presence of genes contributing to the pathogenesis of the disease [17,18]. Segregation analysis
showed that a Mendelian dominant model of transmission is consistent with the observed familial clustering of BPH [18]. The possibility of maternal transmission also agrees with the finding [18] that brothers
carried a greater age-specific relative risk of BPH requiring surgery (6.1) than fathers (3.5). Our findings
support the hypothesis that the human AR gene,
which has been mapped to chromosome Xq11–q12,
may be one of the candidate genes responsible for this
disorder. Further study is needed to establish molecular markers by which to identify men at high risk of
BPH because, in combination with traditional epidemiologic studies, the stratification of genetic predisposition may make it possible to clarify related environmental risk factors.
CONCLUSIONS
We investigated the association between prostatic
adenoma weight and AR gene CAG repeat length in
patients who underwent open prostatectomy. A statistically significant trend for large adenomas with
short CAG repeats was found among the adenoma
quartiles. Our findings suggest that short CAG alleles
may constitute a genetic factor which has a promoting
influence on the growth of benign prostatic hyperplasia.
257
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