Cancer Prevention

Estrogen Receptor B Polymorphism Is Associated with

Prostate Cancer Risk
Camilla Thellenberg-Karlsson,1 Sara Lindström,1 Beatrice Malmer,1 Fredrik Wiklund,1
Katarina Augustsson-Bälter,3 Hans-Olov Adami,3 Par Stattin,2 Maria Nilsson,4
Karin Dahlman-Wright,4 Jan-Åke Gustafsson,4 and Henrik Grönberg1

Abstract Purpose: After cloning of the second estrogen receptor, estrogen receptor h (ERh) in 1996,
increasing evidence of its importance in prostate cancer development has been obtained. ERh is
thought to exert an antiproliferative and proapoptotic effect. We examined whether sequence
variants in the ERh gene are associated with prostate cancer risk.

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Experimental Design: We conducted a large population-based case-control study (CAncer
Prostate in Sweden, CAPS) consisting of 1,415 incident cases of prostate cancer and 801
controls.We evaluated 28 single nucleotide polymorphisms (SNP) spanning the entire ERh gene
from the promoter to the 3V-untranslated region in 94 subjects of the control group. From this, we
constructed gene-specific haplotypes and selected four haplotype-tagging SNPs (htSNP:
rs2987983, rs1887994, rs1256040, and rs1256062). These four htSNPs were then genotyped
in the total study population of 2,216 subjects.
Results: There was a statistically significant difference in allele frequency between cases and
controls for one of the typed htSNPs (rs2987983), 27% in cases and 24% in controls (P =
0.03). Unconditional logistics regression showed an odds ratio of 1.22 (95% confidence interval,
1.02-1.46) for men carrying the variant allele TC or CC versus the wild-type TT, and an odds ratio
of 1.33 (95% confidence interval, 1.08-1.64) for localized cancer. No association of prostate
cancer risk with any of the other SNPs or with any haplotypes were seen.
Conclusion:We found an association with a SNP located in the promoter region of the ERh gene
and risk of developing prostate cancer. The biological significance of this finding is unclear, but it
supports the hypothesis that sequence variation in the promoter region of ERh is of importance for
risk of prostate cancer.

Prostate cancer is a large global health problem with as many
as half a million new cases each year (1). Genetic susceptibility
is of major importance in the etiology of prostate cancer and
may account for as much as 40% of all cases (2). A recent
segregation analysis (3) suggests that multiple low-penetrant
genes account for a major part of the genetic susceptibility of
prostate cancer and that only a small fraction can be explained
by dominant inheritance of highly penetrant genes.
Authors’ Affiliations: Departments of 1Radiation Sciences/Oncology, and
2
Surgical and Perioperative Sciences, University of Umeå, Umeå, 3Department of
Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, and
4
Department of Medical Nutrition and Biosciences, Karolinska Institutet, Novum,
Huddinge, Sweden
Received 2/4/05; revised 11/8/05; accepted 12/15/05.
Grant support: Lion’s Cancer Research Foundation (Umeå, Sweden), Swedish
Cancer Society, and Spear grant fromthe Umeå University Hospital (Umeå, Sweden).
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with18 U.S.C. Section1734 solely to indicate this fact.
Note: Supplementary data for this article are available at Clinical Cancer Research
Online (http://clincancerres.aacrjournals.org/).
Requests for reprints: Henrik Grönberg, Department of Radiation Sciences/
Oncology, Umeå University, S-901 87 Umeå, Sweden. Phone: 46-90-785-1982;
Fax: 46-90-127-464; E-mail: Henrik.Gronberg@ oc.umu.se.
F 2006 American Association for Cancer Research.
doi:10.1158/1078-0432.CCR-05-0269
The estrogen receptor h (ERh) gene is highly expressed in the
prostate epithelium, suggesting a direct effect of estrogen on the
prostate (4, 5). Deletion of ERh in mice lead to hyperplasia in
the ventral prostate, indicating that ERh has an antiproliferative
role in this tissue (6), a notion that was later confirmed (7). A
number of studies on the expression pattern of ERh in both
normal and malignant prostate tissue have been done. In most
studies of cancer, expression of ERh diminishes with increasing
Gleason score but may reappear in metastatic lesions (8 – 11).
Epigenetic regulation, for example, methylation of the
promoter region seems to cause down-regulation of downstream
genes. It also seems to be a reversible event in tumor progression
because methylation is more common in high-grade compared
with low-grade prostate cancer and normal prostate tissue, and
then decreases again in metastatic lesions compared with
localized high-grade tumors (9, 12, 13). Intense staining with
Ki-67 in the prostate of ERh knockout mice, together with high
expression of the androgen receptor, suggests the regulatory role
of ERh in the androgen receptor. A proposed ligand to ERh,
5a-androstane 3h, 17h-diol (3hAdiol) inhibits the growth of
prostate epithelium in wild-type mice but not in ERh knockout
mice, via down-regulation of androgen receptor (6, 14). Other
studies of prostate cancer cell lines have shown the antiprolifer-
ative and anti-invasion properties of ERh as reintroduction of the
gene inhibits growth and invasion and triggers apoptosis (15).

Clin Cancer Res 2006;12(6) March 15, 2006 1936 www.aacrjournals.org

 ERb Polymorphisms Associated with Prostate Cancer Risk

Our hypothesis is that genetic variation in the ERh gene
might alter the expression of the gene and thus affect the risk of
prostate cancer. We tested this hypothesis in a large population-
based epidemiologic study.
Patients and Methods
Briefly, the cases and controls studied came from a large-scale,
population-based case-control study (CAncer Prostate in Sweden,
CAPS). A detailed description of the CAPS study is presented elsewhere
(16). The study population consisted of 1,415 patients with prostate
cancer and 801 control subjects. The case participants were recruited
from four of the six regional cancer registries that cover the entire
population of Sweden. Each of these registries serves one health care
region (Northern, Central, Stockholm, and Southeastern) and altogether
encompasses f6 million inhabitants (67% of Sweden’s population).
The cases were linked to the National Prostate Cancer Registry and
subjects and therefore excluded from further analysis, as were 10 SNPs
with assay failure. From the remaining 22 SNPs, haplotypes were
estimated using a Markov Chain Monte Carlo approach as imple-
mented in the PHASE software package (http://www.stats.ox.ac.uk/
mathgen/software.html).
Four SNPs (rs2987983, rs1887994, rs1256040, and rs1256062),
which captured 99.6% of the haplotype variation among the 94
controls, were selected as htSNPs using the htSNP2 package (http://
www.gene.cimr.cam.ac.uk/clayton/software/stata) for the STATA soft-
ware. These four htSNPs were genotyped under the same conditions
and with the same equipment as described above for all 1,415 cases and
801 control subjects. For all of the htSNPs, three htSNPs in each group
of homozygous and heterozygous subjects were sequenced to produce
internal controls. The primer sequences used to amplify the target DNA
are available on request. PCR conditions were as follows: deoxynucleo-
tide triphosphate 200 Amol/L, 10
PCR buffer, 3 mmol/L MgCl2,
0.5 Amol/L primer, 1 unit Taq Gold enzyme, 50 ng of DNA, cycling at
94jC for 20 seconds, 50jC for 30 seconds, 72jC for 30 seconds,

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clinical information such as Gleason sum, PSA level at the time of repeated 35 cycles. For optimizing the PCR for SNP rs1256040, the
diagnosis, tumor-node-metastasis stage, means of diagnosis and following changes were made: increased MgCl2 to 4.5 mmol/L and
primary treatment were obtained for 95.3% of the cases. The cases addition of 5% DMSO, annealing temperature 50jC ! 43jC for 14
were thereafter classified as either localized (n = 772); (T1-2 and N0/NX cycles and 40jC for 20 cycles. Sequencing was carried out using the ABI
and M0/MX and grades 1-2/Gleason sum 2-7, and PSA <100) or
advanced (n = 568; prone to progressive disease); (T3/4 or N+ or M+ or
grade 3 or Gleason sum 8-10 or PSA >100). For cases and controls
who reported at least one family member with prostate cancer, a more
detailed family history of prostate cancer was obtained through
additional questionnaires and record linkage to the Swedish Cancer
Registry or through medical records. After retrieving these supplemen-
tary data, a total of 177 cases were classified as familial prostate cancer.
Control subjects were randomly selected from the continuously
updated Swedish Population Registry, frequency-matched according
to the expected age distribution (within 5 years) and geographic origin
of the cases. Mean age (age at diagnosis for case patients and age at
inclusion for control subjects) for the cases and controls were 66.6 and
67.9 years, respectively.
The study was approved by the Ethical Committees at the two
participating academic institutions, Umeå University and Karolinska
Institutet. Written informed consent was obtained from each subject.
Selection of ER-b single nucleotide polymorphisms. To make a
thorough evaluation of sequence variation in the ERh gene, we used
a haplotype-tagging single nucleotide polymorphisms (htSNP) method.
The ER-h gene is located on chromosome 14 q23.2, and is f61.2 kb
including eight exons, together with two untranslated first exons, ON
and OK (Fig. 1). Five isoforms are presently known (17) and even more
mRNA splice variants of unknown importance seem to exist (18). We
conducted a search for known SNPs in the data bases http://
snpper.chip.org/ and http://www.ncbi.nih.gov/SNP/ and selected a
subset of SNPs from the promoter region (15 kb), introns, exons, and
3V-untranslated region (UTR) covering a total length of 68.5 kb. There
are three SNPs in the coding regions, all synonymous. At the time of
selection, not many SNPs in ERh were validated and even fewer had
frequency data, so the main criteria for selection were that the SNPs
were evenly spread throughout the gene.
In total, 37 SNPs were chosen with a mean distance between SNPs
of 1,800 bp (Table 1). The SNP genotyping assays were designed by
using the Assay-by-Design and Assay-on-Demand service (Applied
Biosystems, Foster City, CA). The 37 SNPs were then genotyped in 94
randomly selected control subjects using a 5V-nuclease TaqMan assay
together with fluorescently labeled Minor Groove Binders probes. All
reactions were done in a 25 AL volume consisting of 10 ng of genomic
DNA, 900 nmol/L of each primer, 200 nmol/L of each probe and
12.5 AL of TaqMan universal master mix. PCR cycling conditions
were: 50jC for 2 minutes, 95jC for 10 minutes followed by 40 cycles
of 92jC for 15 seconds, and 60jC for 1 minute. The samples were
analyzed on an ABI 7700 sequence detection system. Five of the Fig. 1. Structure of ERh gene. Position of htSNPs. Shaded boxes, exons; thin line,
genotyped SNPs were monomorphic in the 94 randomly selected introns, promotor, 3V-UTR.

www.aacrjournals.org 1937 Clin Cancer Res 2006;12(6) March 15, 2006

Cancer Prevention

Table 1. Initially selected 37 SNPs for genotyping and construction of haplotypes

dbSNP no. Position Exchange Region Minor allele frequency P

in 94 subjects (%)
rs2987983 13950 T/C Promotor 25.5 0.03
rs1271572 12214 G/T Promotor 49.5
rs1887994 10908 G/T Intron 1 11.7 0.94
rs1952586 9716 A/G Intron 1 Assay failure
rs1256028 7446 A/G Intron 1 Assay failure
rs1256029 5454 A/G Intron 1 Assay failure
rs1256030 2533 C/T Intron 2 50.0
rs1256031 3524 C/T Intron 3 50.0
rs960070 4524 C/G Intron 3 0
rs2776606 5558 C/T Intron 3 45.7
rs1256037 6402 C/T Intron 3 49.5

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rs1256038 8964 A/G Intron 3 Assay failure
rs1273196 10198 C/T Intron 3 0
rs1256040 11309 C/T Intron 3 50.0 0.93
rs2026086 12348 C/T Intron 3 0
rs1256042 15136 C/T Intron 4 0
rs1256044 15676 G/T Intron 4 49.5
rs1256045 19943 G/T Intron 4 48.4
rs1256048 21423 G/T Intron 4 Assay failure
rs1256049 25652 A/G Exon 6 3.7
rs1256050 28264 C/T Intron 6 Assay failure
rs1977487 31573 A/G Intron 6 Assay failure
rs1256053 32748 G/T Intron 6 3.7
rs1256054 33390 C/G Exon 7 0
rs1256055 35530 A/G Intron 7 3.7
rs1952585 37348 A/G Intron 7 3.2
rs953592 38954 A/G Intron 7 Assay failure
rs2776608 40532 A/T Intron 7 Assay failure
rs1256060 41653 A/G Intron 7 3.8
rs944461 44563 C/T Intron 7 3.7
rs1256062 46385 A/G Intron 7 6.9 0.81
rs1256063 47486 C/T Intron 7 Assay failure
rs944045 48257 A/G Intron 8 3.2
rs944050 9 -3 A/G Intron/exon boundary 3.2
rs1256065 50771 A/C 3V-UTR 48.9
rs1152577 52218 G/T 3V-UTR 48.9
rs1152579 54616 A/G 3V-UTR 48.9

NOTE: htSNPs in boldface. Position is relative to the ATG start codon according to the National Center for Biotechnology Information genomic contig NT___026437.

Prism Big dye terminator kit v1.1/3.1 from Applied Biosystems and
following the manufacturer’s instructions. When genotyping the whole
study group, we placed two positive controls for each genotype and two
negative controls on each plate. In addition, 29 blind duplicates were
spread among the plates.
Statistical analysis. Hardy-Weinberg Equilibrium tests for each
sequence variant and pair-wise linkage disequilibrium tests for all
sequence variants were done using a replication method as imple-
mented in the GENETICS package (http://lib.stat.cmu.edu/R/CRAN/
index.html) for the R programming language. For each test, 10,000
permutations were done. Associations between genotypes and prostate
cancer were assessed by the score test in unconditional logistic
regression assuming a dominant genetic model. Genotype-specific risks
were estimated as odds ratios with associated 95% confidence intervals
by unconditional logistic regression. When testing for association and
estimating odds ratios, the unconditional logistic regression was
stratified by each combination of age (5-year age groups) and
geographic region to adjust for the matching conducted in collecting
control subjects.
Tests for association between haplotypes and prostate cancer risk
were done using a score test developed by Schaid et al. (19), using the
HAPLO.STAT program (http://www.mayo.edu/hsr/Sfunc.html) for the
R programming language. This method, based on the generalized
linear model framework, allows adjustment for possible confounding
variables and provides both global tests and haplotype-specific tests.
In these analyses, age and geographic region were adjusted
for through indicator variables representing each combination of
age category (5-year age groups) and geographic region (northern and
central part of Sweden versus southeastern part of Sweden and the
area of Stockholm). Haplotypes with estimated frequencies <0.005
were pooled into a single group. Empirical P values were based
on randomly permuting the trait and covariates and computing

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 ERb Polymorphisms Associated with Prostate Cancer Risk

haplotype statistics adjusted for the covariates as described in ref.

(20). Precision criteria for the P values were set to a sample SE of one
fourth of the estimated P values but at least 1,000 permutations were
run for each simulation. All P values are two-sided.
Results
Four selected htSNPs (rs2987983, rs1887994, rs1256040, and
rs1256060) were genotyped in all study subjects, 1,415 cases
and 801 controls. All SNPs were in Hardy-Weinberg equilibrium
among both cases and controls (P > 0.05). The pair-wise linkage
disequilibrium between these SNPs was 0.05 between
rs2987983 and rs1256062 and 0.61 to 0.99 for the other SNP
combinations. No genotyping error was detected among the
duplicates, corresponding to an estimated error rate of 0.0%.
The promoter SNP C/T 13950 (rs2987983) differed signif-
icantly in genotype frequency among cases and controls (P =
Discussion
To test our hypothesis that sequence variation in the ERh gene
is associated with prostate cancer risk, we did a systematic
and comprehensive analysis of the promoter, introns, exon, and
3V-UTR region of the gene in a large population-based study. To
our knowledge, this is the first study evaluating sequence
variations in ERh and risk of prostate cancer. In a previous study
on ERh and risk of breast cancer, we found no significant
association (21), whereas a larger study found an association
with sequence variants in ERh (22). Our finding that one SNP in
the promoter region (13950 C/T) is associated with prostate
cancer risk, suggests that genetic variation in the promoter of
ERh plays a part in the etiology of prostate cancer.
The increased risk of 23% overall and 35% for localized
cancer may seem modest but is not unexpected, in a complex
disease such as prostate cancer. Multiple genes in different

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0.03). The genotype frequency of TC or CC was 47.6% in cases
and 42.2% in controls. The difference was more pronounced
among heterozygous carriers. The frequency for the C allele was
27% in cases and 24% in controls. For the other three htSNPs,
no significant difference was found in genotype frequencies.
Logistic regression analyses revealed a 23% increased risk of
prostate cancer (odds ratios, 1.23; 95% confidence interval,
1.02-1.49) among men with the TC or CC compared with the
genotype TT (adjusted for age and geographic region).
Subgroup analysis based on localized or advanced cancer
revealed an increased risk of 35% (odds ratios, 1.35; 95%
confidence interval, 1.09-1.68) for being diagnosed with
localized cancer (Table 2). Subgroup analysis by age (<65 or
z65 years) or family history did not indicate any heterogeneity
(data not shown). No significant association was found for the
three other analyzed htSNPs and risk of prostate cancer in
subgroup analyses. Seven haplotypes with a frequency of z1%
were found in this population, based on the four htSNPs. We
found no evidence of association with any of the haplotypes.
The overall global test gave P = 0.10 (Table 3).
pathways are probably involved and each contributes to a small
increase in risk. Our view is consistent with metaanalysis of
association studies in other complex diseases (23). For most of
the diseases (seven of eight) where genetic associations were
replicated, the excess risk was in the range of 7% to 76%. The
strengths of our study include its large size and strictly
population-based design. In addition, the population in
Sweden is genetically homogenous, reducing the risk of
confounding due to population stratification. The multiple
testing problems are common in genetic association studies
and is difficult to adjust for when SNPs are in strong linkage
disequilibrium. Using false-positive report probability is one
approach to accommodate this problem (24). In our study, the
prior probability must be considered high for the ERh gene due
to its documented importance in the progression of prostate
cancer and in the regulation of proliferation (12, 15). Assuming
a prior probability of 0.05 to 0.1, the false-positive report
probability is 0.24 to 0.40 if the ERh confers an odds ratio of
1.5 to prostate cancer risk. The range of false-positive report
probability is then below the suggested criterion of 0.5.

Table 2. Genotype frequencies and odds ratios for prostate cancer among ERh SNPs

SNP and position Genotypes Frequencies, n (%) Odds ratio (95% confidence interval)
All cases Controls All cases Localized Advanced
13950 T/C rs2987983 TT 692 (52.4) 445 (57.8) 1.00 1.00 1.00
TC 536 (40.6) 277 (36.0) 1.23 (1.02-1.49) 1.35 (1.09-1.68) 1.14 (0.90-1.45)
CC 92 (7.0) 48 (6.2) 1.16 (0.80-1.69) 1.22 (0.79-1.87) 1.18 (0.75-1.86)
TC/CC 628 (47.6) 325 (42.2) 1.22 (1.02-1.47) 1.33 (1.08-1.64) 1.15 (0.92-1.44)
10908 G/T rs1887994 GG 1,082 (78.3) 619 (78.0) 1.00 1.0 1.0
GT 287 (20.8) 166 (20.9) 1.01 (0.81-1.25) 1.0 (0.77-1.28) 1.08 (0.83-1.41)
TT 13 (0.9) 9 (1.1) 0.75 (0.31-1.79) 0.63 (0.23-1.75) 0.77 (0.25-2.32)
GT/TT 300 (21.7) 175 (22.0) 0.99 (0.80-1.23) 0.97 (0.76-1.25) 1.07 (0.82-1.39)
11309 A/G rs1256040 AA 365 (26.8) 205 (26.2) 1.0 1.0 1.0
AG 699 (51.3) 400 (51.2) 1.0 (0.81-1.24) 0.99 (0.77-1.26) 1.02 (0.78-1.33)
GG 298 (21.9) 177 (22.6) 0.97 (0.75-1.25) 0.85 (0.63-1.15) 1.09 (0.80-1.49)
AG/GG 997 (73.2) 577 (73.8) 0.99 (0.81-1.21) 0.94 (0.75-1.19) 1.04 (0.81-1.34)
46385 C/T rs1256062 CC 1,124 (80.8) 629 (80.6) 1.0 1.0 1.0
CT 255 (18.3) 148 (18.9) 0.97 (0.77-1.22) 0.98 (0.75-1.27) 0.90 (0.68-1.20)
TT 13 (0.9) 7 (0.9) 1.01 (0.39-2.57) 1.45 (0.53-3.94) 0.55 (0.14-2.21)
CT/TT 268 (19.2) 155 (19.8) 0.97 (0.78-1.22) 1.00 (0.77-1.29) 0.89 (0.67-1.17)

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Cancer Prevention

Table 3. Haplotypes in prostate cancer cases and controls

13950 T/C 10908 G/T 11309A/G 46385 C/T All cases, haplotype Controls, haplotype Haplotype Simulated
frequency (%) frequency (%) score P values
T G G C 45.4 46.7 0.54 0.56
C G A C 23.9 21.4 1.68 0.11
T T A C 11.4 11.6 0.16 0.88
T G A C 9.0 9.9 1.24 0.23
T G A T 4.6 6.0 1.81 0.06
C G A T 3.5 2.7 1.34 0.16
T G G T 2.0 1.6 0.72 0.46

NOTE: Estimated haplotype frequencies in all cases and controls and association with prostate cancer. Global statistical test (P = 0.10).

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The 13950 T/C polymorphism is located upstream in the
promoter region and it might be disputed if it has any functional
implication. Previous studies on methylation of ERh receptor
have investigated CpG islands in the region of the untranslated
exon 0N and the foregoing promoter (9, 12). There is no
detailed study of the more distant region of the promoter 0K.
However, that distant region harbors several potential transcrip-
tion factor binding sites (25). As shown by Zhao et al. (26), the
promoter 0K lies f44 kb upstream from the ATG of exon 1 and
gives another possible implication as the 13950 T/C polymor-
phism is then actually located within an intron and might be
located at and affect the binding site for a transcription factor.
The 0K promoter is GC-rich with f65% GC and encompass
several CpG sites but otherwise it is not yet well characterized.
Alternatively, the polymorphism is a mere marker, which is in
strong linkage disequilibrium with the functional polymor-
phism or even another gene. The gene closest upstream to the
ERh located at a distance of 41.5 kb, is a hypothetical gene which
has not been further characterized (hypothetical gene supported
by BX247957). Recently, a study on lactase non-persistence
found a segregating noncoding polymorphism, 13910 bp 5Vof
the initiation codon of the gene for lactase-phlorizin hydrolase
(27). This promoter variant affected the binding site of a
transcription factor. The lactase-phlorizin hydrolase variant is
now used clinically for diagnosing the condition.
The HapMap project has just published haplotypes and
haplotype blocks together with frequency data of a substantial
number of SNPs in the ERh region (http://www.hapmap.org/
index.html.en). Three of the four htSNPs we identified seem
to belong to one haplotype block. The first one (rs2987983)
located in the promoter, lies in a breaking point, between two
blocks (Fig. 2). This may explain why we do not see any
significant association with our haplotypes in the ERh gene and
prostate cancer. The weak linkage disequilibrium (0.05)
between rs2987983 and the other genotyped htSNPs further
supports this interpretation. The ERh gene is a strong candidate
with respect to the proposed function. With the results that we
now present, genetic variation in the intron and exon regions of
ERh is unlikely to be of importance for the risk of prostate
cancer. In contrast, the promoter region and the region further
upstream need more evaluation.
In summary, we found an association between prostate cancer
and a SNP located 13950 from the initiating codon of ERh with
unknown functional implications. Additional genetic analysis of
this region is necessary, with more densely positioned markers as
well as further characterization of the upstream region with
respect to its function. Preferentially, functional/expression
studies in cell lines with the variant allele versus the common
one should be done. Also, more studies in independent
populations that could replicate our findings are warranted.
Acknowledgments
We thank all study participants in the CAPS study, Ulrika Lund for skillfully coor-
dinating the study center at Karolinska Institute, all urologists including their patients
in the CAPS study, and all urologists providing clinical data to the national registry of
prostate cancer; Karin Andersson, Susan Lindh, Gabriella Thore¤n, and Margareta
Åswa«rd at the Regional Cancer Registries; and Sören Holmgren and the personnel
at the Medical Biobank in Umeå for skillfully handling the blood samples.

Fig. 2. Haplotype blocks over ERh region

spanning 200 kb, from the HapMap project.
Position of ERh outlined.Vertical arrow,
location of rs2987983 in the breaking point
between haplotype blocks. Blocks are
constructed by the confidence interval
method according to ref. (28).

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