s t e r o i d s 7 1 ( 2 0 0 6 ) 960–965
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Estrogen receptor alpha polymorphism and
susceptibility to uterine leiomyoma
Fabiola E. Villanova a , Priscila M. Andrade c , Audrey Y. Otsuka a , Mariano T.V. Gomes a ,
Elcio S. Leal b , Rodrigo A. Castro a , Manoel J.B.C. Girão a , Eddy Nishimura d ,
Edmund C. Baracat a , Ismael D.C.G. Silva a,∗
a
Molecular Gynecology Laboratory, Department of Gynecology, Federal University of São Paulo-UNIFESP,
Rua Pedro de Toledo 781, 4th Floor, São Paulo 04039-032, SP, Brazil
b Department of Medicine, Federal University of São Paulo-UNIFESP, São Paulo, Brazil
c Department of Urology, University of São Paulo-USP, São Paulo, Brazil
d Perola Byington Hospital, São Paulo, Brazil
a r t i c l e
i n f o
a b s t r a c t
Article history:
Uterine leiomyoma is the most frequent pelvic tumor found in female genital tract. Some
Received 18 May 2006
studies have suggested an association between single nucleotide polymorphisms (SNPs) in
Received in revised form
estrogen receptors genes with susceptibility in developing uterine leiomyoma. In this work,
11 July 2006
we estimated the frequency of two SNPs: one located in the intron 1 (rs9322331) and other
Accepted 13 July 2006
in the exon 1 (rs17847075) of the estrogen receptor ␣ (ESR1) gene in 125 women with uterine
Published on line 28 August 2006
leiomyoma and 125 healthy women. To do this we used a PCR–RFLP method with MspI and
HaeIII restriction enzymes to respectively detect C/T SNPs in the intron 1 and in the exon
Keywords:
1 of ESR1. To our knowledge this is the first study aimed to investigate the association of
Uterine leiomyoma
ESR1 SNPs with the risk of developing uterine leiomyoma in Brazilian women. Our results
Estrogen receptor ␣
showed that the allele frequencies of the exon 1 and the intron 1 of the ESR1 gene did
Brazilian women
not differ between cases and controls (P = 0.325 and 0.175, respectively). Furthermore, our
Polymorphism
findings provided little support for the association of these SNPs on ESR1 with leiomyoma.
SNP
However, we found that the SNP in the intron 1 of the ESR1 gene was underrepresented in
Genotypes
the Brazilian female population.
© 2006 Elsevier Inc. All rights reserved.
1.
Introduction
Uterine leiomyomas, also known as fibroids or myomas are
one of the most frequent solid pelvic tumors in women. It is
estimated that one in four women during reproductive period
will develop this kind of benign neoplasia [1,2]. Leiomyomas
are the primary indication for hysterectomy, accounting for
over 200,000 surgical interventions each year in the United
States [3,4]. Therefore, leiomyoma is considered one of the
main public health problems [5].
∗
Corresponding author. Tel.: +55 11 55791534; fax: +55 11 55791534.
E-mail address: [email protected] (I.D.C.G. Silva).
0039-128X/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2006.07.005
Estrogen has been reported to be one important risk factor for leiomyomas’ development. In this regard, leiomyomas’ dependency on estrogen is clearly recognized by
the fact that they do not occur before menarche and can
increase in size during pregnancy. Conversely, they reduce
after ovariectomy, in the menopause, or during gonadotropinreleasing hormone agonist therapy [4–6]. On the other hand,
leiomyomas’ growth is variable in women with regular menstrual cycles and even among nodules in the same uterus
[7].
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s t e r o i d s 7 1 ( 2 0 0 6 ) 960–965
The effect of estrogen on leiomyomas’ growth and development is mediated by two ligand-inducible transcription factors
that regulate the target gene expression: (i) estrogen receptor
␣ (ESR1) and (ii) estrogen receptor ␤ (ESR2) [8,9]. Differences
in receptor ␣ and ␤ messenger RNA (mRNA) levels have also
been reported, thus, higher concentration were found in uterine leiomyomas compared with autologous myometrium [10].
By this reason, leiomyomatous tissue may be characterized by
increased levels of estrogen receptor mRNA higher than that
seen in normal myometrial tissue [11–13]. This feature could
represent a major predisposing factor for development and
growth of leiomyomas.
It has been hypothesized that single nucleotide polymorphisms (SNP) on estrogen receptor genes might correlate with
leiomyomas’ development. In this regard, the best-studied
polymorphisms on ESR1 are two SNPs in the intron 1, commonly detected by RFLP assays with the PvuII (rs2234693)
and XbaI (rs9340799) restriction enzymes [14–17] plus a dinucleotide repeat (TAn) upstream of the ESR1 [18] gene. The
later study found an association between TA12 and TA13 dinucleotide repeats with increased risk of leiomyoma in Asiatic
women [18]. However, the same polymorphism was not correlated to a higher risk of developing leiomyoma in Italian
Caucasian women [16,17]. Because of these discrepancies we
therefore speculated whether this polymorphism was indeed
a risk factor to developing leiomyoma or the association was
biased due to the genetic background of the population. Thus,
we explored these possibilities through the study of single
nucleotide polymorphisms (SNP) on exon 1 and intron 1 of the
ESR1 gene in a highly heterogeneous population from southeast of Brazil [19]. To do this, we evaluated ESR1 allele variants and their potential association with uterine leiomyoma.
We used the MspI (C to T substitution, Ser → Ser, refSNP ID:
rs1784705) and HaeIII (C to T substitution, refSNP ID: rs9322331)
RFLPs that are located, respectively, in the exon 1 and the
intron 1 of the estrogen receptor ␣ gene. We used uterine biopsies from women with leiomyoma and compared the allele
and genotype frequencies with healthy women.
2.
Patients and methods
2.1.
Patients
A case-control study was conducted in 250 eligible women
where the frequencies of polymorphisms of the ESR1 gene
were compared. Patients were categorized as follow: (i) 125
women with surgical and histological confirmation of leiomyoma and (ii) 125 normal control women. The 125 cases with
leiomyoma (mean age 43.9 ± 7.3 years) were admitted and
treated at Perola Byington Hospital in São Paulo, Brazil, from
2003 to 2004.
To provide the control-group we also included 125 women
(mean age 56.9 ± 7.4 years) without any evidence of leiomyoma, according to ultrasound exam. All the participants were
ethnically classified as white or non-white according to selfreported ethnicity. The main characteristics of the women
included in this study are shown in Table 1.
Informed consent was obtained from all participants prior
to their inclusion in the study, and the Ethics Committees of
the Perola Byington Hospital and the Federal University of São
Paulo approved all procedures.
2.2.
DNA extraction
Sample pieces of the adjacent normal uterine tissue (1 cm3 )
were collected from women with leiomyoma after hysterectomy or myomectomy. These samples were frozen in liquid
N2 and stored at −80 ◦ C until use. For DNA extraction, pieces
of 50 mg were incubated overnight at 50 ◦ C in 500 ␮l of digestion buffer containing proteinase K (Tris–HCl, 10 mM, pH 8;
EDTA, 0.25 mM, pH 8; SDS, 0.25%; NaCl, 100 mM; proteinase
K, 200 ␮g). After incubation the enzyme was heat inactivated
at 70 ◦ C, for 15 min. Then the lysate was deposited into a column (GFXTM Genomic Blood Purification Kit, GE, Healthcare,
Nova Jersey, EUA). All reactions were performed according to
manufacturer’s specifications.
For the control group, whole blood samples were collected
in tubes containing EDTA and kept at 4 ◦ C before extraction.
Genomic DNA was isolated by using the GFXTM Genomic Blood
Purification Kit (GE, Healthcare, Nova Jersey, EUA), according
to manufacturer’s instructions.
2.3.
MspI genotyping
The amplifications for the exon 1 of the ESR1 gene were
performed using primers previously published [20] (Table 2).
Positive controls with known genotypes and negative controls (reaction mixtures without DNA templates) were also
included in each reaction. PCR reactions had a final volume
of 30 ␮l, containing 50–200 ng of genomic DNA, 10 pmol of
each primer, 15 ␮l of PCR Mix (final concentration: 1× reaction buffer pH 8.5, 1.5 mM MgCl2 , 200 ␮M of each dNTP and
0.75 U of Taq DNA polymerase; Promega, Madison, USA). The
Table 1 – Descriptive characteristics of leiomyoma and control groups
Variable
Category
Groups
Cases (n = 125)
Age (years) (mean ± S.D.)
UV (cm3 ) (mean ± S.D.)
Parity (mean ± S.D.)
Ethnicity
UV: uterine volume.
White
Non-white
Controls (n = 125)
43.9 (±7.3)
418.8 (±293.3)
3.3 (±3.6)
56.9 (±7.4)
37.7 (±21.6)
2.1 (±1.8)
63 (50.4%)
62 (49.6%)
97 (77.6%)
28 (22.4%)
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Table 2 – Description of primers pairs used for PCR of ESR1
Primer sequence 5 –3
Sizes of undisgested
PCR (bp)
Sizes of digested
fragments (bp)
5 -ATGCGCTGCGTCGCCTCTAA-3 ,
5 -CTGCAGGAAAGGCGACAGCT-3
5 -CATCTACTCCTATGTCTGGT-3 ,
5 -CGTGTAGACTGAAGGGCAT-3
479
C allele: 198, 121, 90, 53, 18;
T allele: 288, 121, 53, 18
C allele: 205, 22; T allele: 227
Locationa
SNP
MspI (rs17847075)
Exon 1 (2369 bp)
HaeIII (rs9322331)
Intron 1 (35609 bp)
227
rs: refSNP ID at the NCBI.
a
According to GenBank accession no. AY425004.
reactions were incubated at 95 ◦ C for 10 min, followed by 40
cycles at 94 ◦ C for 1 min, 56 ◦ C for 1 min, 72 ◦ C for 1 min, and a
final extension at 72 ◦ C for 10 min. Ten microliters of the 479 bp
PCR products were digested overnight with 5 U of MspI (Invitrogen, California, USA) according to manufacturer’s instructions. The digestion products were analyzed by electrophoresis in a 3% low melting point agarose gel stained with ethidium bromide (2 ␮g/ml) in 1× TBE (44.5 mM Tris, 44.5 mM boric
acid and 1 mM Na2 EDTA) for 30 min at 125 V. The alleles of
the MspI polymorphism were defined as C and T, denoting
respectively the presence and the absence of the restriction
site [20].
2.4.
3.
Results
3.1.
MspI digestion profile
HaeIII genotyping
The amplifications for the intron 1 of the ESR1 gene were performed using primers previously published [20] (Table 2). The
PCR reaction was carried out in a final volume of 25 ␮l containing 50–200 ng of genomic DNA, 10 pmol of each primer
and 12.5 ␮l of PCR Mix (final concentration: 1× reaction buffer
pH 8.5, 1.5 MgCl2 , 200 ␮M of each dNTP and 0.625 U of Taq
DNA polymerase; Promega, Madison, EUA). The PCR conditions comprised an initial denaturing step at 94 ◦ C for 2 min,
followed by 40 cycles at 94 ◦ C for 30 s, 54 ◦ C for 45 s, 72 ◦ C for
45 s and a final extension at 72 ◦ C for 10 min. The 227 bp PCR
products were digested overnight with 5 U of HaeIII restriction
enzyme (Invitrogen, California, USA), according to manufacturer’s instructions.
Fragments were separated by electrophoresis in a 2.5%
low melting point agarose gel stained with ethidium bromide
(2 ␮g/ml) in 1× TBE (44.5 mM Tris, 44.5 mM boric acid and 1 mM
Na2 EDTA) for 40 min at 100 V. The alleles of the HaeIII polymorphism were defined as C and T, denoting respectively the
presence and the absence of the restriction site [20].
All PCR amplifications were performed in a programmable
thermal cycler GeneAmp PCR system 9700 (Applied Biosystems Inc., USA). Digestion profiles were documented with a
digital camera (DC120, Eastman Kodak Co., Rochester, NY)
under UV illumination.
2.5.
Allele and genotype frequencies were associated with the
risk of leiomyoma by Fisher exact odds ratio (OR). Stratification
analysis was used to estimate the risk for subgroups by race
and parity.
We also estimate the corresponding 95% confidence intervals (CIs) using the unconditional logistic regression model.
The 2 -test was also used to compare variables with P-value
<0.05 considered statistically significant. All P-values were two
sided. These statistical analyses were carried out using the
SPSS Version 11.0 for Windows (SPSS Inc., Chicago, IL, USA).
Statistical analysis
The expected genotype and allele proportions according to
the Hardy–Weinberg equilibrium were compared against the
observed genotypes and alleles using the genetics package of
the R software (http://cran.r-project.org). Because of the low
prevalence of homozygote variants, we combined heterozygotes and homozygotes to increase statistical efficiency.
Results of the MspI digestion profiles of the ESR1 gene were
summarized in Fig. 1. After digestion, homozygous women
with C/C alleles presented fragments of 198, 121, 90 and 53 bp
on low melting point agarose gel. Homozygous individuals
with T/T alleles presented fragments of 288, 121 and 53 bp.
On the other hand, heterozygous individuals with C/T alle-
Fig. 1 – PCR–RFLP profile of exon 1 of the ESR1 gene
digested with MspI. (A) Both lanes show the digestion of
two distinct PCR products of homozygous women for the C
allele (C/C). In this reaction the MspI enzyme generates
fragments of 198, 121, 90 and 53 base pairs (bp). (B)
Digestion of PCR products of homozygous for the T allele
(T/T) generates fragments of 288, 121 and 53 bp. (C)
Digestion of PCR products of heterozygous (C/T) individuals
generates fragments of 288, 198, 121, 90 and 53 bp. (D)
Molecular ladder of 100 bp. (E) Undigested PCR products of
479 bp.
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s t e r o i d s 7 1 ( 2 0 0 6 ) 960–965
les showed the combination of fragments of all previous sizes
(Fig. 1).
Allele and genotype frequencies of the ESR1 exon 1 C/T
polymorphism of women with uterine leiomyoma and controls are shown in Table 3. The calculated frequencies for MspI
alleles were 52% and 47.6% for the C allele and 48% and 52.4%
for the T allele, respectively, for women with uterine leiomyoma and women of the control group. This difference was not
statistically significant (2 = 0.97 and P = 0.325).
The proportions of homozygous women for the C allele
(C/C), heterozygous (C/T) and homozygous for the T allele (T/T)
were respectively 24.8%, 54.4% and 20.8% in the leiomyoma
group and 20.8%, 53.6% and 25.6% in the control group. Statistical analysis of the exon 1 MspI polymorphism revealed no
significant difference between genotyped cases and the control group with regard to genotype frequencies (2 = 1.07 and
P = 0.586) (Table 3).
The observed frequencies of the C/C, C/T and T/T genotypes
of the MspI polymorphism did not differ from the expected
frequencies according to the Hardy–Weinberg equilibrium for
both the leiomyoma group (2 = 1.15 and P = 0.561) and the control group (2 = 0.51 and P = 0.773).
We did not find statistical support for the association of any
genotype frequency and specific strata defined by parity and
race (data not shown).
3.2.
HaeIII digestion profile
Results of the HaeIII digestion profiles of the ESR1 gene were
summarized in Fig. 2. Homozygous women with C/C alleles
for the HaeIII polymorphism showed a fragment of 205 bp on
agarose gel, while homozygous individuals with T/T alleles
showed only the undigested fragment of 227 bp. On the other
hand, C/T heterozygous individuals showed fragments of 227
and 205 bp after HaeIII digestion (Fig. 2).
The ESR1 intron 1 HaeIII allele and genotype frequencies
of leiomyoma and control groups are shown in Table 4. The
calculated allele frequencies for C and T alleles were 66.4% and
33.6% in the leiomyoma and 72% and 28% in the control group,
respectively. This difference was not statistically significant
(2 = 1.84 and P = 0.175).
The proportions of homozygous individuals for the C allele
(C/C), heterozygous (C/T), and homozygous for the T allele
(T/T) were 47.2%, 49.6% and 3.2%, respectively, in the control group, and 36%, 60.8% and 3.2% in the leiomyoma group
Fig. 2 – PCR–RFLP profile of intron 1 of the ESR1 gene
digested with HaeIII. (A) Digestion of PCR products of
homozygous individuals for the C allele (C/C) generates
fragments of 205 base pairs (bp). (B) Digestion of PCR
products of heterozygous (C/T) generates fragments of 205
and 227 bp. (C) Homozygous individuals for the T allele
(T/T) show undigested PCR fragments of 227 bp. (D)
Molecular ladder of 100 bp.
(Table 4). There were no significant differences in the frequencies between cases and controls (2 = 3.3 and P = 0.191).
Notably, genotype frequencies for the C and T alleles were not
in Hardy–Weinberg equilibrium for both leiomyoma (2 = 16.1
and P = 0.001) and control group (2 = 6.62 and P = 0.01).
No significant differences were found with reference to the
genotype distribution between the uterine leiomyoma group
and the control group. The results remained statistically not
significant after stratifying by parity and race for all genotypes,
in cases and controls (data not shown).
4.
Discussion
Leiomyomas are the most common tumors in women, found
in up to 25% of women in active reproductive life. While the
Table 3 – Genotypes and allele frequencies of the ESR1 exon 1 MspI C/T polymorphism between women with leiomyoma
and controls
Genotypes
Genotype frequency
C/C
C/T
T/T
Allele frequency
C
T
Controls (n = 125)
2
P-Value
OR (95% CI)
31 (24.8%)
68 (54.4%)
26 (20.8%)
26 (20.8%)
67 (53.6%)
32 (25.6%)
1.07
0.586
1.00 (ref.)
0.85 (0.46–1.58)
0.68 (0.33–1.42)
130 (52.0%)
120 (48.0%)
119 (47.6%)
131 (52.4%)
0.97
0.325
1.00 (ref.)
0.84 (0.59–1.19)
Leiomyoma (n = 125)
2 : chi-square; OR: odds ratio; CI: confidence interval.
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Table 4 – Genotypes and allele frequencies of the ESR1 intron 1 HaeIII C/T polymorphism between women with
leiomyoma and controls
Genotypes
Genotype frequency
C/C
C/T
T/T
C/T + T/Ta
Allele frequency
C
T
Controls (n = 125)
2
P-Value
OR (95% CI)
45 (36.0%)
76 (60.8%)
4 (3.2%)
80 (64.0%)
59 (47.2%)
62 (49.6%)
4 (3.2%)
66 (52.8%)
3.3
0.191
1.00 (ref.)
1.60 (0.96–2.68)
1.31 (0.31–5.53)
1.59 (0.96–2.64)
166 (66.4%)
84 (33.6%)
180 (72.0%)
70 (28.0%)
1.84
0.175
1.00 (ref.)
1.30 (0.89–1.90)
Leiomyoma (n = 125)
2 : chi-square; OR: odds ratio; CI: confidence interval.
a
Due to the reduced frequency of T/T homozygous they were combined with C/T heterozygous.
clinical observations that leiomyomas grow during the reproductive years and regress after menopause have supported an
important role for estrogen in the promotion of leiomyomas’
growth, its etiology remains unclear.
Although several single nucleotide polymorphisms have
been described in the estrogen receptor gene [21–24], there
is little information regarding possible roles of these polymorphisms as a risk factor to leiomyoma development. By
this reason, we studied two single nucleotide polymorphisms
(SNP) located on exon 1 and intron 1 of the ESR1 gene.
Although a previous study suggested that TA repeats (TA12
and TA13) on ESR1 might increase the chances of developing leiomyoma in Chinese women [18], this association was
not confirmed in another study in Italian Caucasian women
[17]. Considering that both studies were carried out in homogeneous racial groups, it is conceivable that this association
might be biased due to ethnical background of the population.
Our study, on the other hand, was performed in a population
composed by a well mix of European, African and Amerindian
ancestries [19]. Consequently, the genetic component of the
population may have little effect on the estimated frequencies.
To our knowledge this is the first report investigating the association between exon 1 MspI (dbSNP: rs17847075) and intron 1
HaeIII (dbSNP: rs9322331) polymorphisms of the ESR1 gene and
the risk of uterine leiomyoma development in women from an
ethnically heterogeneous population.
Our results, however, revealed that genotype and allele
frequencies for the MspI and HaeIII polymorphism did not differ between leiomyoma and control groups. Furthermore, we
found that there is no statistical support to the hypothesis
that these SNPs increase the risk of development of leiomyoma. Although limited in number we believe that our analysis, performed in highly heterogeneous population, was not
biased.
Interestingly, although frequencies of ESR1 SNPs did not
differ between controls and women with leiomyoma, we
found that the Hardy–Weinberg test for the intron 1 HaeIII
polymorphism deviates from the equilibrium in both groups.
This implies that homozygous T/T frequency is below that
expected in Brazilian women. Nevertheless, it might represent
a negative selection pressure on the homozygote T/T genotype, the biological significance of this finding needs to be
better addressed.
Acknowledgements
We are grateful to Drs. Julio Katsutani and Walter Oda from the
Perola Byington Hospital, for their support in patient selection.
F.E.V. was supported by a CNPq scholarship grant.
references
[1] Stewart EA. Uterine fibroids. Lancet 2001;357:293–8.
[2] Flake GP, Andersen J, Dixon D. Etiology and pathogenesis of
uterine leiomyomas: a review. Environ Health Perspect
2003;111:1037–54.
[3] Schwartz SM. Epidemiology of uterine leiomyomata. Clin
Obstet Gynecol 2001;44:316–26.
[4] Walker CL, Stewart EA. Uterine fibroids: the elephant in the
room. Science 2005;308:1589–92.
[5] Lethaby A, Vollenhoven B. Fibroids (uterine myomatosis,
leiomyomas). Clin Evid 2005:2264–82.
[6] Maruo T, Ohara N, Wang J, Matsuo H. Sex steroidal
regulation of uterine leiomyoma growth and apoptosis. Hum
Reprod Update 2004;10:207–20.
[7] Kataoka S, Yamada H, Hoshi N, Kudo M, Hareyama H,
Sakuragi N, et al. Cytogenetic analysis of uterine leiomyoma:
the size, histopathology and GnRHa-response in relation to
chromosome karyotype. Eur J Obstet Gynecol Reprod Biol
2003;110:58–62.
[8] Wise LA, Palmer JR, Harlow BL, Spiegelman D, Stewart EA,
Adams-Campbell LL, et al. Reproductive factors, hormonal
contraception, and risk of uterine leiomyomata in
African–American women: a prospective study. Am J
Epidemiol 2004;159:113–23.
[9] Wang H, Mahadevappa M, Yamamoto K, Wen Y, Chen B,
Warrington JA, et al. Distinctive proliferative phase
differences in gene expression in human myometrium and
leiomyomata. Fertil Steril 2003;80:266–76.
[10] Li S, McLachlan JA. Estrogen-associated genes in uterine
leiomyoma. Ann NY Acad Sci 2001;948:112–20.
[11] Bjornstrom L, Sjoberg M. Mechanisms of estrogen receptor
signaling: convergence of genomic and nongenomic actions
on target genes. Mol Endocrinol 2005;19:833–42.
[12] Brandon DD, Erickson TE, Keenan EJ, Strawn EY, Novy MJ,
Burry KA, et al. Estrogen receptor gene expression in human
uterine leiomyomata. J Clin Endocrinol Metab
1995;80:1876–81.
[13] Benassayag C, Leroy MJ, Rigourd V, Robert B, Honore JC,
Mignot TM, et al. Estrogen receptors (ER␣/ER␤) in normal
s t e r o i d s 7 1 ( 2 0 0 6 ) 960–965
[14]
[15]
[16]
[17]
[18]
and pathological growth of the human myometrium:
pregnancy and leiomyoma. Am J Physiol 1999;276:E1112–8.
Kitawaki J, Obayashi H, Ishihara H, Koshiba H, Kusuki I, Kado
N, et al. Oestrogen receptor-␣ gene polymorphism is
associated with endometriosis, adenomyosis and
leiomyomata. Hum Reprod 2001;16:51–5.
Denschlag D, Bentz EK, Hefler L, Pietrowski D, Zeillinger R,
Tempfer C, et al. Genotype distribution of estrogen
receptor-alpha, catechol-O-methyltransferase, and
cytochrome P450 17 gene polymorphisms in Caucasian
women with uterine leiomyomas. Fertil Steril 2006;85:462–7.
Massart F, Becherini L, Gennari L, Facchini V, Genazzani AR,
Brandi ML. Genotype distribution of estrogen receptor-␣
gene polymorphisms in Italian women with surgical uterine
leiomyomas. Fertil Steril 2001;75:567–70.
Massart F, Becherini L, Marini F, Noci I, Piciocchi L, Del Monte
F, et al. Analysis of estrogen receptor (ER␣ and ER␤) and
progesterone receptor (PR) polymorphisms in uterine
leiomyomas. Med Sci Monit 2003;9:BR25–30.
Hsieh YY, Chang CC, Tsai FJ, Tsai HD, Yeh LS, Lin CC, et al.
Estrogen receptor thymine–adenine dinucleotide repeat
polymorphism is associated with susceptibility to
leiomyoma. Fertil Steril 2003;79:96–9.
965
[19] Parra FC, Amado RC, Lambertucci JR, Rocha J, Antunes CM,
Pena SD. Color and genomic ancestry in Brazilians. Proc Natl
Acad Sci USA 2003;100:177–82.
[20] Herrington DM, Howard TD, Hawkins GA, Reboussin DM, Xu
J, Zheng SL, et al. Estrogen-receptor polymorphisms and
effects of estrogen replacement on high-density lipoprotein
cholesterol in women with coronary disease. N Engl J Med
2002;346:967–74.
[21] Gold B, Kalush F, Bergeron J, Scott K, Mitra N, Wilson K, et al.
Estrogen receptor genotypes and haplotypes associated with
breast cancer risk. Cancer Res 2004;64:8891–900.
[22] Zofkova I, Zajickova K, Hill M. The estrogen receptor alpha
gene determines serum androstenedione levels in
postmenopausal women. Steroids 2002;67:
815–9.
[23] Iwamoto I, Fujino T, Douchi T, Nagata Y. Association of
estrogen receptor ␣ and ␤3-adrenergic receptor
polymorphisms with endometrial cancer. Obstet Gynecol
2003;102:506–11.
[24] Malamitsi-Puchner A, Tziotis J, Evangelopoulos D, Fountas L,
Vlachos G, Creatsas G, et al. Gene analysis of the N-terminal
region of the estrogen receptor alpha in preeclampsia.
Steroids 2001;66:695–700.