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Molecular Psychiatry (2004) 9, 28–34
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IMMEDIATE COMMUNICATION
Association of estrogen receptor b gene polymorphisms
with bulimic disease in women
M Nilsson1, S Naessén2, I Dahlman3, A Lindén Hirschberg2, J-Å Gustafsson1 and K Dahlman-Wright1
1
Department of Biosciences, Karolinska Institute, Stockholm, Sweden; 2Department of Obstetrics and Gynaecology,
Karolinska Hospital, Stockholm, Sweden; 3Department of Medicine, Huddinge University Hospital, Karolinska Institute,
Stockholm, Sweden
In this study, we explored the potential association between estrogen receptor b (ERb) and
disease in a group of bulimic women. Eating disorders are much more common in females
than in males, suggesting a possible role for female sex hormone signalling in the
pathogenesis of these diseases. Furthermore, estrogen has been implicated in appetite
regulation. The occurrence of menstrual disturbances is also increased in bulimic women. We
studied 76 bulimic women and 60 controls, and found an association between two common
polymorphisms in the ERb gene with disease in this group of bulimic women. More detailed
characterisation of the ERb gene identified a novel variant changing the primary structure of
ERb protein in one bulimic patient. An initial functional characterization of this variant did not
reveal any differences compared to the wild-type protein. Our findings point towards a
possible role of ERb and/or neighboring genes in the etiology of disease in bulimic patients.
Molecular Psychiatry (2004) 9, 28–34. doi:10.1038/sj.mp.4001402
Keywords: association; eating disorders; ERb; mutation; polymorphisms
Eating disorders are much more common in females
than in males1, suggesting a possible role for female
sex hormone signalling in the etiology of disease.
Estrogen has also been implicated in feeding behavior
and is known to inhibit feeding in animal experiments.2,3 There is evidence that estradiol acts to
increase the activity of the cholecystokinin (CCK)
satiation signalling pathway.2,3 The satiating potency
of endogenous CCK is increased during estrus in
normal rats and by estradiol treatment in ovariectomized rats.3–5 In bulimic women, the occurrence of
menstrual irregularities is increased, despite the
maintenance of normal weight.6 Little is, however,
known about the role of estradiol in disordered eating
in women. Estrogen receptor b (ERb) is expressed in
many areas of the central nervous system in rats.7 In
the hypothalamus, a brain area known to be involved
in the regulation of appetite and satiety, both ERa and
ERb are expressed.7 Recently, ERb was found to be the
receptor regulating the anorectic action of estrogen in
mice.8
The involvement of a genetic component in the
etiology of bulimic disease is suggested from
studies of families with bulimic patients and
twins.9–15 However, the actual genes involved in
Correspondence: Dr M Nilsson, Department of Biosciences,
Novum, SE-141 57 Huddinge, Sweden.
E-mail: [email protected]
Received 07 March 2003; revised 03 June 2003; accepted 14 June
2003
the pre-disposition for disease remain to be elucidated.
So far, only a few variants of the ERb gene have
been reported and characterized with regard to allele
frequency.16–19 Rosenkranz et al16 performed a systematic mutation screening of ERb in probands of
different weight extremes and identified five different
genetic variants. Only two of these appeared at an
allele frequency over 1% for the less common allele.
These SNPs, 1082 G-A and 1730 A-G, did not
display association with the phenotypes under study.
These SNPs have later been analyzed for association
between ERb and ovulatory dysfunctions,19 including
polycystic ovary syndrome (PCOS) and could be
associated with ovulatory defects in patients with
unknown causes of disease. Other studies have
investigated the possible association between a
dinucleotide repeat polymorphism located in the
flanking region of the human ERb gene and systemic
blood pressure or bone mineral density, respectively.17,18 A possible association between this
polymorphism and hypertension in Japanese
women was reported.18 The dinucleotide repeat was
also suggested to be associated with bone mineral
density.17
Based on the potential role of female sex hormone
signalling for the development of binge-eating
together with the fact that bulimic women frequently
present menstrual irregularities, we hypothesized
that a reduction in ERb function might contribute to
disease.
ERb gene variants in patients with eating disorders
M Nilsson et al
Materials and methods
Subjects
Blood samples from 76 Swedish patients diagnosed
with bulimia nervosa (BN) or eating disorders not
otherwise specified (EDNOS), with bulimia as the
predominating symptom (referred to as bulimic
patients in this paper), and 60 healthy female controls
(body mass index (BMI) and age were matched to the
bulimic patients) were collected at the Department of
Obstetrics and Gynaecology at the Karolinska Hospital. Patients were diagnosed by interview and met the
criteria for BN and EDNOS with bulimic symptoms
according to DSM-IV-R.20 A total of 48 patients were
diagnosed with BN and 28 patients were diagnosed
with EDNOS. Menstrual status was classified as
regular monthly periods, amenorrhea; no bleeding
for the last 3 months or oligomenorrhea; periods at an
interval exceeding 6 weeks. Among the bulimic
patients, 27 (36%) had menstrual disturbances (amenorrhea or oligomenorrhea), whereas only two (3%) of
the controls had oligomenorrhea. The average ages of
the bulimic patients with or without menstrual
irregularities were 26 and 28 years, respectively, and
their BMI values were 22.4 and 22.2 kg/m2, respectively. The average ages of the controls with or
without menstrual irregularities were 31 and 29 years,
respectively, and their BMI values were 22.3 and
22.9 kg/m2, respectively. All subjects gave their
informed consent, and the study was approved by
the ethical committee of the Karolinska Hospital.
Available sample size provided 80% power to detect a
disease susceptibility allele with a frequency of 40%
among controls, odds ratio¼2 and multiplicative
impact on phenotype, applying a threshold of
Po0.05 and a two-tailed test (H. Cordell, Cambridge
Institute for Medical Research, University of Cambridge, personal communication).
Mutation analysis
DNA amplification Genomic DNA was prepared
using a QIAamps DNA MiniKit (QIAGEN). All
polymerase chain reaction (PCR) amplifications
were performed using AmpliTaq Gold (Roche)
according to standard protocols. All primers were
Table 1
Exon
1
2
3
4
5
6
7
8
CX
designed to cover intron–exon borders of the ERb
gene; see Table 1.
29
SNP scoring using RFLP analysis Three ERb
polymorphisms were studied: a G-A change at
position 1082 in exon 5 (reported as rs1256049 in
the SNPper database (http://snpper.chip.org/))
creating a recognition site for RsaI, a G-A change
at position 1730 in the 30 UTR of exon 8 creating an
AluI site, and a G-A change at ERb cx þ 56
(rs928554) generating a restriction site for Tsp509I.
ERb cx þ 56 is located 56 bases 30 of the coding part of
ERb cx exon 9. Restriction enzyme assays were
performed according to standard procedures.
Genotypes were scored twice.
Statistical analysis Allele frequencies of the ERb
polymorphisms were compared between the patient
and control groups using the w2 test. The statistical
analyses were performed using Epi Info 2000
software. Haplotypes in population samples were
inferred using the Phase software.21 Linkage
disequilibrium between adjacent markers was
estimated as r2¼(pAB-pApB)2/(pA*pa*pB*pb), where a
and b are two loci with allele frequencies pA, pa, pB, pb
and haplotype frequencies pAB, pab, pAb, paB.22
Denaturing high-performance liquid chromatography
Mutation analysis was undertaken using temperaturemodulated heteroduplex analysis (TMHA) on an
automated high-performance liquid chromatography
(HPLC) instrument equipped with a DNASep column
(Transgenomic). Heteroduplex and homoduplex generations were performed by heating the PCR products
to 951C for 5 min and slowly cooling by 1.51C/min for
47 cycles. A volume of 5 ml of each PCR product was
analyzed using the WAVEs system (Transgenomic).
DNA was eluted with a mixture containing 0.1 mol/l
triethylamine acetate and 0.1 mol/l triethylamine
acetate þ 25% acetonitrile at a flow rate of 0.9 ml/
min. The WAVEMAKERTM Software package (Transgenomic) suggested mobile-phase gradients and a
melting temperature based on the exon sequence.
Exons with an abnormal TMHA profile were sequenced and compared with the published genomic
sequence of the ERb gene.
Primers used for amplifying each ERb exon and surrounding intron sequences in both TMHA and RFLP analysis
Forward primers, 50 -30
Reverse primers, 50 -30
TTATACTTGCCCACGAATCTTT
GCTTTGCTGTATCAGATTTCCGGG
TGGCTTTGTACCTGTACTGGTCAT
CCCCACAGGCTCCAGAAAATA
GTTGCGCAGCTTAACTTCAAAGTTTTCTTC
GTGTGGTCTCATTAACACCCTGTTGTAG
TGCATTAGGCCAGGCTTCTCTTCT
GGTTTAGGGGTGGGGTAGACTG
CTCTACTTAAGGGCAGAAAAGGCCTCTC
CCTGTGTTTTGGGTGCTGT
ATTTCTGCCAAGTCATCTCTGC
GCCAAAATCTGCCTCCCATAATC
ATGCCAAGACAGAAGGAAGGAACA
GAAGGAGCTGATGATGCTCTGATC
CCAGACATAAGTATAAATGCATGCCAAC
GTGCCCATCTTTGCTTACAGGTG
CCAAGCCTGCCATCACCAAATGAG
GTTGGATTGATAATAGAAAGGAAGGTG
Molecular Psychiatry
ERb gene variants in patients with eating disorders
M Nilsson et al
30
DNA sequencing Amplified products were purified
using a QIAquicks PCR purification kit (QIAGEN).
Sequencing reactions were performed using a
BigDyeTM Terminator Cycle Sequencing kit (Applied
Biosystems). Samples were resolved in an ABI377
automatic sequencer (Applied Biosystems).
212 bp, respectively, for the heterozygous G/A samples, and two bands of 243 and 212 bp, respectively,
in samples homozygous for the less common allele
A/A; see Figure 2a. Analysis of the 1730 G-A
polymorphism by AluI digestion produced one band
of 420 bp for the homozygous G/G samples, three
Plasmid constructs hERb 530 cDNA cloned into the
EcoRI sites of the pSG5 expression vector (Stratagene,
La Jolla, CA, USA) was used as template for
mutagenesis. Mutant construct (pSG5-hERb 530
R221G) was produced by the introduction of a 1 bp
substitution (A-G) using the QuickChange XL sitedirected
mutagenesis
kit
(Stratagene).
The
substitution was confirmed by DNA sequencing.
Cell culture and transfection HEK 293 cells were
cultured in a 1 : 1 ratio of Ham’s Nutrient mixture F12
(Invitrogen) and DMEM (Invitrogen) supplemented
with 5% FBS and 100 U penicillin/ml and 100 mg
streptamycin/ml. Cells were plated in 24-well plates
24 h prior to transfection. Transfections using the
Superfect reagent (QIAGEN) were performed
according to the manufacturer’s protocol. 0.8 mg of
2 ERE-TK-LUC was cotransfected with 0.032 mg of
pSG5-hERb 530 or pSG5-hERb 530 R221G. A pRL-TK
control plasmid, containing the Renilla luciferase
gene, was used as an internal control. The pSG5
vector was used to equalize plasmid concentrations.
Upon transfection, the medium was changed to a
phenol-red-free 1 : 1 mixture of F12 and DMEM
containing 5% dextran-coated charcoal-treated FBS
and 100 U penicillin/ml and 100 mg streptamycin/ml.
Hormone or vehicle in 0.1% EtOH was added
simultaneously. Cells were harvested 24 h after
transfection
and
luciferase
activities
were
determined using the Dual Luciferase Reporter
Assay System (Promega) according to the
manufacturer’s protocol.
Results
Association of polymorphisms in the ERb gene with
disease in bulimic women
Frequencies for three polymorphisms were scored.
The locations of these polymorphisms are displayed
in Figure 1. Restriction enzyme digestion with RsaI
was used to score the 1082 G-A variant. This yielded
one band of 455 bp for samples homozygous for the
most common allele, G/G, three bands of 455, 243 and
Figure 1 Locations of the three polymorphisms in the ERb
gene analyzed in this study. (1) 1082G-A, (2) 1730 G-A,
(3) ERb cx þ 56 G-A. CX is an alternatively spliced ninth
exon.
Molecular Psychiatry
Figure 2 RFLP analysis of RsaI, AluI and Tsp509I polymorphisms of the human ERb gene. (a) RsaI polymorphism;
lane 1: 1 kb DNA ladder; lane 2: uncleaved PCR product;
lane 3: homozygous G/G samples; lane 4: heterozygous G/A;
lane 5: homozygous A/A samples. (b) AluI polymorphism;
lane 1 : 1 kb DNA ladder; lane 2: homozygous G/G samples;
lane 3: heterozygous G/A samples; lane 4: homozygous A/A
samples. (c) Tsp509I polymorphism; lane 1: 50 bp DNA
ladder; lane 2: homozygous G/G samples; lane 3: heterozygous G/A samples; lane 4: homozygous A/A samples.
ERb gene variants in patients with eating disorders
M Nilsson et al
Table 2
31
Allele and genotype frequencies in 76 bulimic patients and 60 controls
Polymorphism
Study group
1082 G-A
Bulimia
Controls
Bulimia
Controls
Bulimia
Controls
1730 G-A
ERb cx þ 56 G-A
Number of
samples
Variant allele
frequencya
76
60
76
60
76
60
0.046 (7)
0.017 (2)
0.43 (66)
0.26 (31)
0.61 (93)
0.43 (52)
Genotype distribution
wt/wt
wt/var
var/var
0.92 (70)
0.97 (58)
0.28 (21)
0.52 (31)
0.12 (9)
0.33 (20)
0.065 (5)
0.03 (2)
0.58 (44)
0.45 (27)
0.54 (41)
0.47 (28)
0.013 (1)
—
0.14 (11)
0.033 (2)
0.34 (26)
0.2 (12)
w2b
P-valueb
ORc
1.81
NS
2.85
9.04
0.0026
2.20
9.14
0.0033
2.06
a
Frequency (number of variant alleles).
w and P-value for the differences in allelic distribution in bulimic patients and controls.
c
Odds ratio¼(D1/D0)/(H1/H0), where D1¼number variant allele among cases, D0¼number wild-type (wt) alleles among cases,
H1¼number variant alleles among controls, and H0 number wt alleles among controls.
NS¼not significant.
b 2
Table 3 Estimation of haplotypes (1730 G-A and ERb cx
þ 56 G-A) in cases and controls
Loci
LIV-CX
Haplotypea Controls (n)
GGb
GA
AA
AG
%
Cases (n)
%
RRc
67
22
30
1
56
18
25
1
56
30
63
3
37
20
41
2
—
1.6
2.5
3.5
120
100
152
100
a
Shown are haplotypes inferred using the Phase software.21
b
Wild-type haplotype.
c
Relative risk¼(D1/D0)/(H1/H0), where D1¼number of variant
haplotypes among cases, D0¼number of wt haplotypes
among cases, H1¼number of variant haplotypes among
controls, and H0¼number of wt haplotypes among
controls.
bands of 420, 336 and 84 bp, respectively, for the
heterozygous G/A samples, and two bands of 336 and
84 bp, respectively, for the homozygous A/A samples;
see Figure 2b. Analysis of the ERb cx þ 56 polymorphism by Tsp509I digestion produced two bands
of 187 and 78 bp, respectively, for the homozygous
G/G samples. Four bands of 187, 149, 78 and 38 bp,
respectively, are produced in the heterozygous G/A
samples. Three bands of 149, 78 and 38 bp, respectively, are seen in the homozygous A/A samples; see
Figure 2c.
Allele and genotype frequencies are shown in
Table 2. Genotype frequencies were in Hardy–Weinberg equilibrium among cases and controls, respectively. Estimated haplotypes and associated relative
risk are shown in Table 3. Haplotype analysis
supports the fact that both studied ERb polymorphisms are associated with increased risk of disease
independently.
Linkage disequilibrium based on inferred haplotypes between 1730 and ERb cx were 0.41 and 0.37
in controls and cases, respectively. Linkage disequilibrium based on inferred haplotypes between
1082 and 1730 were o0.05 for both cases and
controls.
This study shows an association between disease
and the 1730 G-A and ERb cx þ 56 G-A ERb gene
variants for this group of bulimic women.
Identification of novel variants of the ERb gene
The association described above could exist either
because changes at the nucleotide positions in
question affect ERb function or because they are in
linkage disequilibrium with functional changes that
might occur in ERb or a neighboring gene. To
investigate the latter possibility, we screened all
exons of the ERb gene in the bulimic patients and
controls for novel variants. TMHA was chosen to
screen the exons encoding the full-length ERb protein
for potential new variants. Artificial mutations were
generated to validate methods. Direct sequencing was
used to confirm abnormal TMHA profiles and to
determine the identity of the variant. The polymorphisms in exons 5 and 8, studied above result in a
background in this analysis when trying to identify
novel variants. However, this background of heterozygotes for known SNPs can be eliminated using
subsequent restriction enzyme analysis to eliminate
the known polymorphisms. Figure 3 shows the
TMHA profiles for exons 5 and 8, homozygous and
heterozygous patterns. The variants, including frequencies, identified in this study are shown in Table 4.
Interestingly, in one bulimia patient, a 661 A-G
change was identified. The patient is heterozygous for
the variant that leads to an amino-acid substitution,
arginine 221 for glycine (R221G) (Figure 4). This
variant has not been detected in our studies of 4200
subjects who were not part of this study (data not
shown). As an initial test of the functional implications of this amino-acid substitution, this mutant
receptor was analyzed in a transient transfection
system (Figure 5). However, this assay revealed no
differences between the ERb wild type (WT) and ERb
R221G proteins with regard to transcriptional activation. The other variants mentioned above do not affect
the primary structure of the ERb protein.
Molecular Psychiatry
ERb gene variants in patients with eating disorders
M Nilsson et al
32
Number of
Controls
1
2
3
661 A-G
ERb 8 4 A-Ga
ERb 8 68 C-T
1
4
1
—
1
1
0
2
3
4
Retention Time (min)
0.51
1.2
6
5
4.43
4.66
1
a
The variant has been reported in the SNPper database
(http://snpper.chip.org/) as rs944050.
a
Intensity (mV)
1.0
0.8
0.6
0.4
0.2
8
7
6
5
4
3
2
1
0
7.39
0.5
0
0.0
3.28
2.0
1.5
0.0
Intensity (mV)
Number of
bulimic patients
2.5
1.0
b
ERb sequence
variation
3.0
6.51
Intensity (mV)
3.5
Table 4 Novel ERb gene variants identified in this study
1.19
4.0
0.51
a
1
2
3
4
5
Retention Time (min)
6
8
7
-0.2
5
4
3
2
1
1.5
0
4.35
Intensity (mV)
2.0
7
6
7.24
2.5
b
3.16
6
5
3.37
3
4
Retention Time (min)
2.45
2
0.51
c
1
Intensity (mV)
0
1.0
1
2
3
4
5
Retention Time (min)
6
7
8
c
0.5
0
Intensity (mV)
4
3
Retention Time (min)
2
1.5
5
6
4.38
4.62
0.53
d
1
1.0
0.5
0.0
0
1
2
4
3
Retention Time (min)
5
6
Figure 3 TMHA profiles for ERb exons 5 and 8. (a) TMHA
profile for a sample homozygous for exon 5 (1082 G/G).
(b) TMHA profile for sample heterozygous for exon 5 (1082
G/A). (c) TMHA profile for sample homozygous for exon
8 (1730 G/G). (d) TMHA profile for sample heterozygous
for exon 8 (1730 G/A).
Discussion
In this study, we describe an association between
polymorphisms in the ERb gene and disease in
bulimic patients. Further analysis showed that both
BN and EDNOS were independently associated with
the ERb polymorphisms (data not shown). FurtherMolecular Psychiatry
Figure 4 TMHA analysis identified a bulimic patient
heterozygous for an arginine-to-glycine amino-acid change.
(a) Wild-type TMHA pattern. (b) Abnormal TMHA pattern.
(c) Direct sequencing revealed a double peak at position 661
corresponding to both A and C. The heterozygous position
is indicated with an arrow. The PCR and TMHA analysis
were repeated twice to assure that the variant is not a result
of an error occurring during PCR.
more, menstrual irregularities were not independently associated with the ERb polymorphisms (data
not shown). Our clinical observations show no
distinction between bulimic women with the risk
allele and those without. Interestingly, ERb is located
on chromosome 14q, in a region recently identified to
meet the criterion for genome-wide suggestive linkage
ERb gene variants in patients with eating disorders
M Nilsson et al
Figure 5 Transient transfection analysis of the hERb 530
and hERb 530 R221G variants, respectively. The experiment
was performed three times. Shown is one representative
experiment analyzed in triplicate.
with BN.23 However, a German study did not provide
evidence for an association between BN and ERb
polymorphisms.16 Our finding points towards a
possible role of ERb and/or neighboring genes in the
etiology of disease in bulimic patients. Whether this
role derives from its functions in peripheral and/or
central systems will need to be addressed. It remains a
formal possibility that these polymorphisms per se
contribute to ERb function and the etiology of disease.
As they are located in the 30 UTR, they could affect
mRNA stability. However, more likely, these polymorphisms are in linkage disequilibrium with a
functional change in the ERb gene or other genes. To
test the latter possibility, we screened the ERb gene of
bulimic patients and controls for variants. Our results
are in accordance with previous studies showing a
low degree of variation for the ERb gene, identifying
only very few novel polymorphisms. Two new and
rare intron variants were identified, ERb 8 4 A-G
(rs944050) and ERb 8 –68 C-T. However, since they
do not change the primary structure of the ERb
protein, and are most probably not involved in the
splicing of ERb, they are not likely to be relevant
for ERb protein function. Furthermore, they occur
in both bulimic patients and control subjects
(Table 4).
The 661 A-G variant identified in one bulimic
patient changes the primary structure of the ERb
protein. This patient was diagnosed EDNOS. This
particular amino acid is conserved in the ERb protein
in all sequenced species. Furthermore, this position is
also conserved in ERa with regard to charge (arginine
in ERb and lysine in ERa). The R221G mutation is
situated in the hinge region of ERb. This part of the
receptor is thought to constitute a flexible hinge
between the DNA-binding domain and the ligandbinding domain. Although the R221G mutation is
clearly not a common genetic variant causing EDNOS,
this mutation might be of importance in the etiology
of EDNOS in this particular patient who also had
menstrual disturbance. Since this allele is rare, final
proof for a causative effect of this mutation would be
to show association between this allele and disease in
a family study and to demonstrate a difference
compared to the wild-type receptor in a functional
assay. Evaluation of the functional characteristics of
this variant in a transient transfection assay did not
reveal impaired receptor function. However, this
assay only measures a subset of ERb functions, and
further studies will be needed to determine whether
or not this variant behaves differently from the wildtype ERb protein. The fact that we only identified one
rare variant of ERb does not exclude the possibility
that ERb function is impaired in a larger group of
these patients. Variations at the DNA level, affecting
primary protein structure, are not the only cause of
abnormal ERb receptor function; dysregulation at
the transcriptional and/or translational levels leading
to abnormal ERb protein levels remain other possibilities.
33
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