American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 147B:880 –889 (2008)
Initial Association of NR2E1 With Bipolar Disorder
and Identification of Candidate Mutations in
Bipolar Disorder, Schizophrenia, and
Aggression Through Resequencing
Ravinesh A. Kumar,1,2 Kevin A. McGhee,3 Stephen Leach,4 Russell Bonaguro,1 Alan Maclean,3
Rosalia Aguirre-Hernandez,5 Brett S. Abrahams,1 Emil F. Coccaro,6 Sheilagh Hodgins,7
Gustavo Turecki,8 Anne Condon,5 Walter J. Muir,3 Angela R. Brooks-Wilson,2,4 Douglas H. Blackwood,3
and Elizabeth M. Simpson1,2*
1
Centre for Molecular Medicine and Therapeutics and Child & Family Research Institute, Vancouver,
British Columbia, Canada
2
Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada
3
Division of Psychiatry, University of Edinburgh, Edinburgh, Scotland
4
Canada’s Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, British Columbia, Canada
5
Department of Computer Science, University of British Columbia, Vancouver, British Columbia, Canada
6
Department of Psychiatry, University of Chicago, Chicago, Illinois
7
King’s College London, Institute of Psychiatry, London, United Kingdom
8
McGill Group for Suicide Studies, Douglas Hospital Research Centre, Montreal, Quebec, Canada
Nuclear receptor 2E1 gene (NR2E1) resides within
a 6q21-22 locus for bipolar disorder and schizophrenia. Mice deleted for Nr2e1 show altered
neurogenesis, cortical and limbic abnormalities,
aggression, hyperexcitability, and cognitive
impairment. NR2E1 is therefore a positional and
functional candidate for involvement in mental
illness. We performed association analyses in 394
patients with bipolar disorder, 396 with schizophrenia, and 479 controls using six common
markers and haplotypes. We also performed a
comprehensive mutation screen of NR2E1, resequencing its entire coding region, complete 50 and 30
untranslated regions, consensus splice-sites, and
evolutionarily conserved regions in 126 humans
with bipolar disorder, schizophrenia, or aggressive disorders. NR2E1 was associated with bipolar
disorder I and II [odds ratio (OR ¼ 0.77, P ¼ 0.013),
bipolar disorder I (OR ¼ 0.77, P ¼ 0.015), bipolar
disorder in females (OR ¼ 0.72, P ¼ 0.009), and with
age at onset 25 years (OR ¼ 0.67, P ¼ 0.006)], all of
Grant sponsor: Jack and Doris Brown Foundation and British
Columbia Institute for Children’s and Women’s Health; Grant
sponsor: Harry Frank Guggenheim Foundation; Grant sponsor:
UK Medical Research Council; Grant sponsor: Chief Scientist
Office of the Scottish Executive; Grant sponsor: Wellcome Trust;
Grant sponsor: State Hospital Board for Scotland; Grant sponsor:
Canadian Institutes for Health Research (CIHR); Grant sponsor:
CIHR Research and Development; Grant sponsor: Canada
Research Chair in Genetics and Behaviour.
*Correspondence to: Elizabeth M. Simpson, Associate Professor, Department of Medical Genetics, University of British
Columbia, Vancouver, British Columbia, Canada V5Z 4H4; Senior
Scientist, Centre for Molecular Medicine and Therapeutics and
Child & Family Research Institute, 3020-950 West 28th Avenue,
Vancouver, British Columbia, Canada V5Z 4H4.
E-mail: simpson@cmmt.ubc.ca
Received 13 August 2007; Accepted 14 November 2007
DOI 10.1002/ajmg.b.30696
ß 2008 Wiley-Liss, Inc.
which remained significant after correcting
for multiple comparisons. We identified eight
novel candidate mutations that were absent in
325 controls; four of these were predicted to alter
known neural transcription factor binding
sites. Analyses of NR2E1 mRNA in human
brain revealed forebrain-specific transcription.
The data presented support the hypothesis
that genetic variation at NR2E1 may be associated with susceptibility to brain-behavior disorders.
ß 2008 Wiley-Liss, Inc.
KEY WORDS:
nuclear receptor; mental illness;
‘‘fierce’’ mice; brain; polymorphisms
Please cite this article as follows: Kumar RA, McGhee KA,
Leach S, Bonaguro R, Maclean A, Aguirre-Hernandez R,
Abrahams BS, Coccaro EF, Hodgins S, Turecki G,
Condon A, Muir WJ, Brooks-Wilson AR, Blackwood
DH, Simpson EM. 2008. Initial Association of NR2E1
With Bipolar Disorder and Identification of Candidate
Mutations in Bipolar Disorder, Schizophrenia, and
Aggression Through Resequencing. Am J Med Genet
Part B 147B:880–889.
INTRODUCTION
Bipolar disorder and schizophrenia are common and severe
psychiatric disorders and major causes of disability and
morbidity worldwide. Both disorders have a lifetime prevalence of approximately 1% [Jablensky et al., 1992; Sklar, 2002]
and show variable and sometimes overlapping clinical presentation [Maier et al., 2006]. In some patients, neurodevelopmental phenotypes such as altered neurogenesis, reduced
dendritic branching, impairment in GABAergic interneurons,
ventricular enlargement, or reduced volume of the hippocampus, cerebral cortex, corpus callosum, amygdala, and
olfactory bulb have been described [Rapoport et al., 2005;
Strakowski et al., 2005; Ross et al., 2006].
Association of NR2E1 With Bipolar Disorder
The genetic basis of bipolar disorder and schizophrenia is
well documented through family, twin and adoption studies
[Tsuang and Faraone, 1990]. Genome-wide linkage studies
have reproducibly identified several promising susceptibility
loci, including 6q21-22 at 108.5 Mb [Kohn and Lerer, 2005;
McQueen et al., 2005]. To date, no studies have found evidence
of association to candidate genes in this region [Mamdani et al.,
2007].
The orphan nuclear receptor 2E1 gene (NR2E1; previously
Tlx [MIM 603849]) on 6q21-22 is a positional and functional
candidate gene for bipolar disorder and schizophrenia. NR2E1
at 108.6 Mb is located directly under the genome-wide linkage
peak obtained for bipolar I disorder. Genes known or proposed
to interact with Nr2e1 [Stenman et al., 2003; Shi et al., 2004;
Zhang et al., 2006] have themselves been implicated in
schizophrenia, including PAX6 [Stober et al., 1999] and
NR4A2 (NURR1) [Buervenich et al., 2000; Chen et al., 2001].
Strikingly, mice deleted for Nr2e1 (Nr2e1/) display hyperactivity, cognitive deficits, increased aggression, abnormal
startle reactivity, reduced anxiety, altered neurogenesis,
impairment in GABAergic interneurons, reduced dentritic
branching, retinopathy, ventricular enlargement, and hypoplasia of the hippocampus, cerebral cortex, corpus callosum,
amygdala, and olfactory bulb (Wong and Simpson, unpublished data) [Monaghan et al., 1997; Roy et al., 2002; Young
et al., 2002; Land and Monaghan, 2003; Roy et al., 2004].
Hypothesized to underlie these behavioral and anatomical
phenotypes are defects in neural stem cells during development and in adult Nr2e1/ mice [Roy et al., 2004; Shi et al.,
2004; Christie et al., 2006]. Notably, we have corrected the
brain and behavioral abnormalities of Nr2e1/ mice using a
genomic clone of human NR2E1 [Abrahams et al., 2005],
thereby demonstrating functional equivalence of the human
and mouse genes in mice. This suggests that mutations in
human NR2E1 may result in phenotypes resembling Nr2e1/
mice, which is supported by the demonstration that mutations
in human and mouse NR2E3, the closest genomic relative to
NR2E1, result in a similar eye abnormality in both species
[Akhmedov et al., 2000; Haider et al., 2000].
Based on the support for NR2E1 in bipolar disorder and
schizophrenia, we hypothesize that some humans with these
disorders will harbor at-risk genetic variants of NR2E1, which
we systematically tested using two convergent strategies.
First, we performed case-control association analyses in
394 patients with bipolar disorder, 396 patients with schizophrenia, and 479 ethnically-matched controls using six
common markers and haplotypes comprised of each marker
combination. Second, we performed a comprehensive mutation
screen of NR2E1 in 126 humans with bipolar disorder,
schizophrenia, or aggressive behaviors. We genotyped candidate mutations in 325 healthy controls. We applied bioinformatic approaches and expression studies to support our
hypothesis that NR2E1 may be associated with susceptibility
to brain-behavior disorders.
MATERIALS AND METHODS
Human Subjects
Approval for this study was obtained from The University of
British Columbia (UBC), Child and Family Research Institute,
UBC Department of Medical Genetics, and the Central Office
for Research Ethics Committees (UK). The research followed
Canada’s Tri-Council Statement on ‘‘Ethical Conduct for
Research Involving Humans.’’ Clinical and demographic data
for subjects are reported in Table I.
For the association analyses, schizophrenia and bipolar
disorder subjects were contacted through their consultant
psychiatrist and written informed consent to the study was
881
obtained before a sample of blood was donated for DNA
extraction. Diagnoses were reached by consensus between two
trained psychiatrists and were based on DSM-IV criteria
[American Psychiatric Association, 2000]. Clinical data were
obtained by direct interview of the patient using the SADS-L
semi structured interview (Schedule for Affective Disorders
and Schizophrenia-Lifetime version), hospital records and when
possible from relatives or caregivers. Ethnically matched
controls were recruited through the South of Scotland
Blood Transfusion Service (BTS). Controls were not directly
screened to exclude those with a personal or family history of
psychiatric illness; however, BTS does not accept blood
donations from patients on regular medication or with a
history of major illness.
The screen for candidate mutations was conducted in three
main groups. Subjects were included if they were diagnosed
with a disorder with evidence of genetic linkage to 6q21-22 and/
or presented with features resembling Nr2e1/ mice, including aggressive behavior. The first group (bipolar I disorder)
included subjects who were (1) part of the association analyses
described above; and (2) obtained from the Coriell Cell
Repository (CCR) (http://coriell.umdnj.edu/). The second group
(schizophrenia) included subjects who were (1) part of the
association analyses described above; (2) obtained from the
CCR; (3) members of a pedigree collection of the National
Institute of Mental Health Schizophrenia Genetics Initiative
showing linkage to 6q21-22 [Cao et al., 1997; Martinez et al.,
1999; Levinson et al., 2000]; or (4) violently aggressive and
residents within a large forensic psychiatric hospital. The third
group (aggressive disorders without a diagnosis of bipolar
disorder or schizophrenia) included subjects selected for a
history of extreme violent behavior including: (1) psychopathic
behavior and severe personality disorders who were residents
within forensic psychiatric hospitals and detained in a secure
environment specializing in the treatment and rehabilitation
of violent offenders; (2) DSM-IV intermittent explosive
disorder who were assessed with the Life History of Aggression, the Buss-Durkee Hostility Inventory (BDHI), the Motor
Aggression and Research Criteria for Intermittent Explosive
Disorder, the Eysenck Personality Questionnaire II, and the
Barratt Impulsiveness Scale as previously described [Goveas
et al., 2004]; (3) paraphilic disorders who attended a sexual
disorder clinic in Baltimore, Maryland [Berlin et al., 1991];
(4) self-directed aggressive behaviors who committed suicide in
the Montreal metropolitan area; each had a very high score in
measures of aggressive behavior, as assessed by the Brown
Goodwin lifetime history of aggression questionnaire and the
BDHI adapted to proxy-based interviews [Dumais et al., 2005];
(5) mild to severe mental retardation and behavioral problems
and/or psychosis who were recruited from the Greenwood
Genetics Centre, Greenwood, South Carolina [Schuback et al.,
1999]; or (6) autism and a history of violence who were obtained
from the Autism Genetics Resource Exchange (http://www.
agre.org/). Candidate mutations were genotyped in the following groups of control subjects who had no known history
of behavioral or psychiatric disorders: (1) 110 individuals of
African descent obtained from the CCR; (2) 27 individuals
of African descent obtained from Dr. M.R. Hayden (UBC,
Vancouver, Canada); (3) 94 Caucasians from the CCR; and
(4) 94 Caucasian patients enrolled in a genetic study of Gilbert
Syndrome who did not present with any known behavioral or
psychiatric disorder. Six additional family members (one from
a patient with schizophrenia and five from three unrelated
patients with bipolar disorder) were also examined.
Selection of Polymorphic Markers
Six markers were examined in the association analyses
(Fig. 1). Three markers were chosen based on their putative
882
Kumar et al.
TABLE I. Clinical and Demographic Data on Subjects
Sample size
Diagnosis
Association analyses
Bipolar disorder
I
II
Schizophrenia
Control
Candidate mutation screen
Bipolar I disorder
Schizophrenia
Aggressive behavior
Psychopathy
Intermittent explosive disorder
Paraphilic disorder
Suicide and aggression
MR and behavioral problems
Autism and violence
Total
Male Female
Mean age (range)
Ethnicity
Male
Female
AA
CAU
358
33
394
479
162
8
278
251
196
25
116
228
24 (13–55)
18 (14–21)
53 (22–88)
50 (25–89)
24 (10–63)
19 (11–70)
49 (21–89)
48 (19–86)
0
0
0
0
358
33
394
479
39
33
15
23
24
10
38 (26–52)
39 (15–82)
40 (24–67)
40 (24–58)
0
5
39
28
24
4
8
4
9
5
24
3
8
3
n.d.
n.d.
0
1
0
1
n.d.
n.d.
34 (14–57)
32 (24–38)
30 (21–56)
45 (42–49)
n.d.
n.d.
n.a.
54
n.a.
21
n.d.
n.d.
0
2
0
0
0
n.d.
24
2
8
4
9
n.d.
MR, mental retardation; n.a., not applicable; n.d., not determined.
functional importance: g.1429G > A resides in an evolutionarily conserved region in the proximal promoter [Kumar
et al., 2007], rs2233488 resides in the 50 UTR, and the (CA)n
microsatellite (D6S1594) that resides within the 30 UTR. The
final three markers were chosen as tag SNPs based on the
HapMap data using the ‘‘Solid spine of LD’’ method: the entire
NR2E1 gene fell within a single haplotype block from which
rs217520 was chosen; rs127503 and rs588409 were chosen
from adjacent blocks. Two additional tag SNPs, rs217538 and
rs385029, were also selected however, we were technically
unable to genotype with these markers.
DNA Amplification, Sequencing, Genotyping
For case-control association analyses, we genotyped the
D6S1594 microsatellite by performing polymerase chain
reaction (PCR) in 12-ml reactions containing 2.5 pmol of each
primer, 1 Sigma PCR Buffer, 0.2 mM dNTPs, 0.25 units of
Sigma Taq and 20 ng of genomic DNA. Primers were
synthesized by Invitrogen (Paisley, UK) with one of the two
primers labeled 50 with 6-FAMTM (ABI, Foster City, CA). DNA
fragments were amplified as follows: 2 min at 948C, 35 cycles of
20 sec at 948C, 30 sec at 558C, and 1 min at 728C; and a final
extension of 20 min at 728C. Amplified products were run in an
ABI 3730 DNA Analyzer (Warrington, UK) with GS500 LIZ
size standards. Allele sizes were determined using Genemapper 3.0 (ABI, CA). For SNP genotyping, the target sequence for
each SNP was submitted to the Assay By Design Service for
Custom SNP Genotyping Assays (ABI, Warrington, UK), or
when possible the SNP assay was ordered as an Assay On
Demand Assay. Genotyping was performed in 384-well plates
using the TaqMan PCR-based method (ABI, UK).
For mutation screening, we sequenced genomic NR2E1
using 20 PCR amplicons that covered the coding (1,146 bp),
complete 50 and 30 untranslated regions (1,973 bp), exonflanking regions including consensus splice-sites (1,719 bp),
and evolutionarily conserved regions including core and
proximal promoter (1,528 bp) as previously described [Kumar
et al., 2007]. Ethnically matched controls were sequenced for
amplicons containing variants in patients.
Haplotype, Bioinformatic, and Statistical Analyses
For association analyses the microsatellite marker and SNP
distributions in patients and control subjects were compared
by the w2 test using the CLUMP program [Sham and Curtis,
1995]. For the microsatellite, alleles 105, 107, 109, 113, 119,
123, 137, 139 were too infrequent for separate analyses and
Fig. 1. Genomic structure of NR2E1 and location of six markers selected for association analyses in bipolar disorder and schizophrenia. A: Schematic of
NR2E1 and closest neighboring gene SNX3. B: LD map generated from the HAPMap data CEU population set. LD blocks (1, 2, and 3) were generated using
the ‘‘Solid spine of LD’’ method. Markers that are tag SNPs are indicated in red.
Association of NR2E1 With Bipolar Disorder
were therefore combined into a single allele (combined rare
group); comparisons were therefore made with nine alleles and
the combined rare group. Individual SNPs were tested for
association using a w2 test. Haplotype association analyses
were performed using UNPHASED [Dudbridge, 2003]. This
program performs genetic association analysis in both nuclear
families and unrelated subjects. It implements maximumlikelihood inference on haplotype and genotype effects while
dealing with missing data such as uncertain phase and missing
genotypes. Many of the commonly performed analyses include
global and individual tests of haplotype association and
permutation tests. In this study, six markers were used (five
SNPs and one microsatellite). Haplotype analysis for all
combinations of marker (i.e., all 2, 3, 4, 5, and 6 marker
haplotypes) were conducted resulting in 63 marker combinations. This resulted in 4,774 haplotype combinations. Therefore in order to correct for multiple testing, the gold standard of
permutation testing was carried out (1,000 iterations) using
UNPHASED. In unrelated subjects, the trait values are
randomly shuffled between subjects. The randomization is
held constant over all analyses determined by the user’s
parameters. In each permutation, the minimum P-value is
compared to the minimum P-value over all the analyses in the
original data. This allows for multiple testing corrections over
all tests performed in a run.
Linkage disequilibrium analyses for all six markers were
carried out using GOLD because one marker was a microsatellite. However, in the case of identifying tag SNPs, the
program Tagger [de Bakker et al., 2005] implemented in
Haploview [Barrett et al., 2005] was used.
Transcription factor binding site (TFBS) analyses were
performed using MatInspector [Quandt et al., 1995]. We
analyzed the minor and major alleles at each variant site
together with 50 bp of surrounding sequence using the
Optimized Matrix Similarity thresholds. RNA folding was
performed using Vienna RNA Package (http://www.tbi.univie.
ac.at/rna/).
Expression Analyses
Hybridization analysis was performed on adult human brain
multi-tissue northern blot (Clontech, Palo Alto, CA) as per the
manufacturer’s instructions using an NR2E1 partial cDNA
(pEMS741) [Young et al., 2002]. Prehybridization (528C,
90 min) and hybridization (518C, 15 hr) were performed using
ExpressHyb Solution (Clontech) as described by the manufacturer. 32P-radiolabeled probes were generated by random
primer labeling using Ready-To-GoTM DNA Labeling Beads
(Amersham, Piscataway, NJ). Washes included: twice with
Wash Solution 1 (Clontech) at room temperature for 30 min;
and twice with Wash Solution 2 (Clontech) at 508C for 40 min.
The membrane was exposed to autoradiographic film with an
intensifying screen at 808C for 2 days. The blot was stripped
and re-probed with GAPDH (pHcGAP) [Tso et al., 1985].
RESULTS
SNP Analyses Detect Significant
Associations in Bipolar Disorder
To determine whether NR2E1 SNPs associate with bipolar
disorder or schizophrenia, we performed association analyses
using five polymorphisms comprised of two putative functional, and three tag SNPs located within and around NR2E1
(Fig. 1). All SNPs were in Hardy–Weinberg equilibrium in
controls and cases (P > 0.05). We found a significant association between rs217520 and bipolar disorder (I and II combined)
[odds ratio (OR) for the A allele ¼ 0.77, confidence interval
(CI) ¼ 0.62–0.95, w2 ¼ 6.11, uncorrected P ¼ 0.013, permuted
P ¼ 0.036] but not with any of the other four SNPs (Table II).
883
We examined bipolar I disorder and bipolar II disorder
separately and found evidence of a significant association
between the same marker, rs217520, for bipolar I disorder
(OR ¼ 0.77, CI ¼ 0.62–0.96, w2 ¼ 5.89, uncorrected P ¼ 0.015,
permuted P ¼ 0.041) but not for bipolar II disorder (w2 ¼ 1.540,
P ¼ 0.215). However, the lack of association with bipolar II
disorder could be due to the small sample size in this group.
To determine whether sex-specific or age at onset (AAO)
associations exist between NR2E1 and bipolar disorder or
schizophrenia, we repeated the association analyses with all
five markers by stratifying for sex and AAO (25 years vs.
>25 years). The same marker, rs217520, was significantly
associated with bipolar disorder in females (OR ¼ 0.72,
CI ¼ 0.56–0.93, w2 ¼ 6.92, uncorrected P ¼ 0.009, permuted
P ¼ 0.025) and with AAO 25 years in bipolar disorder
(OR ¼ 0.67, CI ¼ 0.50–0.90, w2 ¼ 7.59, uncorrected P ¼ 0.006,
permuted P ¼ 0.017) (Table III). AAO 25 years in bipolar
disorder was also associated with a second marker, rs588409
before correction (OR ¼ 1.28, CI ¼ 1.00–1.66, w2 ¼ 3.90, uncorrected P ¼ 0.048, permuted P ¼ 0.13).
Microsatellite Analysis Does Not Detect Association
in Bipolar Disorder or Schizophrenia
We identified 17 alleles from our analyses of the microsatellite D6S1594 (data not shown). We did not detect any
significant differences between repeat lengths of the microsatellite in bipolar disorder versus controls (w2 ¼ 10.3, df ¼ 16,
P ¼ 0.85) nor in schizophrenia versus controls (w2 ¼ 22.1,
df ¼ 16, P ¼ 0.14), nor in bipolar disorder type I (w2 ¼ 9.31,
P ¼ 0.90) and type II (w2 ¼ 15.3, P ¼ 0.50) analyzed separately.
We did not find any evidence of association with schizophrenia
or bipolar disorder in either sex, nor with AAO with bipolar I or
II, alone or combined.
NR2E1 Two- and Three-Marker
Haplotype Association
To determine whether NR2E1 haplotypes are associated
with bipolar disorder or schizophrenia, we performed haplotype analyses with all six markers, generating 63 marker
combinations resulting in 4,773 haplotypes (data not shown).
Five haplotypes were associated with bipolar disorder, however, these associations did not remain significant after
correcting for multiple testing. rs217520, the marker associated with bipolar disorder, was the only marker common to all
five haplotypes. The uncorrected P values are presented for the
reader’s information (g.1429/rs217520, w2 ¼ 7.929, df ¼ 2,
uncorrected P ¼ 0.019, rs127503/rs217520, w2 ¼ 11.85, df ¼ 3,
uncorrected P ¼ 0.008, rs2233488/rs217520, w2 ¼ 6.247, df ¼ 2,
uncorrected P ¼ 0.044, rs127503/g.1429/rs217520, w2 ¼
12.29, df ¼ 5, uncorrected P ¼ 0.019, g.1429/rs217520/
rs588409, w2 ¼ 12.29, df ¼ 5, uncorrected P ¼ 0.031). However,
we recognize that we may have insufficient power to detect
significant haplotype associations after multiple testing
correction.
Candidate NR2E1 Mutations Identified
in Behavioral and Psychiatric Disorders
Resequencing of NR2E1 in 126 unrelated subjects with
schizophrenia, bipolar disorder, and impulsive-aggressive
disorders generated approximately 844,200 bp of sequence
data. We did not detect any coding region variants in the 126
subjects tested. This does not, however, exclude the possibility
that coding region deletions are present in some subjects, given
that exon-based sequencing cannot detect deletions of whole
exons.
884
Kumar et al.
TABLE II. NR2E1 SNP Association Analyses in Bipolar Disorder and Schizophrenia
Alleleb
Markera
Case-control
na
G
A
w2
OR (95% CI)
P
rs127503
Bipolar disorder (I and II)
Bipolar I disorder
Bipolar II disorder
Schizophrenia
Control
337
309
27
335
386
350 (0.52)
319 (0.52)
30 (0.56)
358 (0.53)
427 (0.55)
324 (0.48)
299 (0.48)
24 (0.44)
312 (0.47)
345 (0.45)
1.66
1.88
0.00
0.51
—
0.87 (0.71–1.08)
0.86 (0.69–1.07)
1.01 (0.56–1.82)
0.93 (0.93–1.15)
—
0.198
0.170
0.972
0.475
—
Corrected P
Alleleb
Markera
Case-control
na
G
A
w2
OR (95% CI)
P
g.1429c
Bipolar disorder (I and II)
Bipolar I disorder
Bipolar II disorder
Schizophrenia
Control
359
327
31
380
400
686 (0.96)
626 (0.96)
58 (0.94)
735 (0.97)
772 (0.97)
32 (0.04)
28 (0.04)
4 (0.06)
25 (0.03)
28 (0.03)
0.91
0.59
1.40
0.05
—
0.78 (0.45–1.34)
0.81 (0.46–1.43)
0.53 (0.17–1.83)
1.07 (0.60–1.91)
—
0.339
0.441
0.236
0.819
—
Alleleb
Markera
rs2233488
Case-control
na
G
C
w2
OR (95% CI)
P
Bipolar disorder (I and II)
Bipolar I disorder
Bipolar II disorder
Schizophrenia
Control
355
321
33
378
405
677 (0.96)
614 (0.96)
61 (0.92)
730 (0.97)
775 (0.96)
33 (0.04)
28 (0.04)
5 (0.08)
26 (0.03)
35 (0.04)
0.09
0.00
1.48
0.81
—
0.93 (0.56–1.55)
0.99 (0.58–1.70)
0.55 (0.17–1.54)
1.27 (0.74–2.19)
—
0.758
0.970
0.223
0.367
—
Alleleb
Markera
Case-control
na
A
C
w2
OR (95% CI)
P
rs217520
Bipolar disorder (I and II)
Bipolar I disorder
Bipolar II disorder
Schizophrenia
Control
374
341
34
376
369
417 (0.56)
380 (0.56)
37 (0.54)
438 (0.58)
458 (0.62)
331 (0.44)
302 (0.44)
31 (0.46)
314 (0.42)
280 (0.38)
6.11
5.89
1.54
2.26
—
0.77 (0.62–0.95)
0.77 (0.62–0.96)
0.73 (0.43–1.24)
0.85 (0.69–1.06)
—
0.013*
0.015*
0.215
0.133
—
0.036*
0.041*
Alleleb
Markera
Case-control
na
A
G
w2
OR (95% CI)
P
rs588409
Bipolar disorder (I and II)
Bipolar I disorder
Bipolar II disorder
Schizophrenia
Control
368
334
33
382
390
496 (0.67)
452 (0.68)
44 (0.67)
508 (0.67)
502 (0.64)
240 (0.33)
216 (0.32)
22 (0.33)
256 (0.34)
278 (0.36)
1.55
1.75
0.14
0.78
—
1.14 (0.92–1.43)
1.16 (0.93–1.45)
1.11 (0.67–1.94)
1.10 (0.88–1.43)
—
0.213
0.186
0.707
0.378
—
a
Number of subjects examined.
Number of alleles; values in parenthesis refer to frequencies.
g, numbering based on Antonarakis and the Nomenclature Working Group [1998], where A of the initiator Met codon in exon 1 is denoted nucleotide þ1.
Human genomic NR2E1 sequence: NCBI AL078596.
*Significant P values.
b
c
We identified 12 subjects harboring 11 novel non-coding
variants (Table IV) that have not been previously reported in
apparently healthy humans [Kumar et al., 2007] nor in dbSNP
(http://www.ncbi.nlm.nih.gov/projects/snp/; Build 124). One of
these variants (g.1726C > A) has been previously identified
in a patient with abnormal cortical development [Kumar et al.,
2007]. Two of the novel variants were deletions, one was an
insertion, and eight were single-base substitutions. Each of
these 11 variants (herein referred to as ‘‘patient variants’’) was
present in the heterozygote state. Of the eight single-base
substitutions, six were transitions and two were transversions.
When possible we obtained DNA samples from relatives of
the candidate mutation carriers for further genetic studies. We
amplified and sequenced the regions corresponding to four of
the 11 patient variants in five additional family members
(Table IV). For g.3079A > G, the unaffected mother harbored
the variant whereas the unaffected father did not, indicating
that g.3079A > G was not de novo. For g.11594T > C and
g.20290G > A, only one unaffected parent was available for
each and neither harbored the variant. For one of these,
g.11594T > C, the variant was also detected in an affected
family member (bipolar cousin of the father). For the patient
with g.1220–1221insT, an affected sibling did not harbor the
variant, indicating that the variant does not segregate with
disease. Interestingly, two of the 11 patient variants (g.1431414319delACTCT and g.20826A > G) were identified in patients
Association of NR2E1 With Bipolar Disorder
885
TABLE III. NR2E1 Association Analyses in Bipolar Disorder Stratified by Sex and Age of Onset
Alleleb
Marker
rs217520
Case-control
Bipolar disorder
Females
Males
AAO 25
AAO > 25
na
214
159
130
55
A
232 (0.54)
183 (0.58)
136 (0.52)
64 (0.58)
w2
C
196 (0.46)
135 (0.42)
124 (0.48)
46 (0.42)
6.92
1.90
7.59
0.61
OR (95% CI)
P
Corrected P
0.72 (0.56–0.93)
0.83 (0.63–1.09)
0.67 (0.50–0.90)
0.85 (0.56–1.30)
0.009*
0.168
0.006*
0.435
0.025c
0.017*
a
Number of subjects examined.
Number of alleles; values in parenthesis refer to frequencies.
*Significant P values.
b
with schizophrenia belonging to families with evidence of 6q21linkage; DNA from the parents of these two patients was
unavailable for typing. The g.14314-14319delACTCT variant
represents the largest deletion at NR2E1 reported to date.
We amplified and sequenced the amplicons corresponding to
each of the 11 patient variants in ethnically-matched controls
of African (274 chromosomes) and European (376 chromosomes) descent. If the ethnicity of the subject was unknown, the
patient variants were genotyped in chromosomes of both
African and European descent (650 chromosomes).
The results are surprising, particularly for bipolar
disorder. For example, the three candidate mutations found
in 39 European patients with Bipolar Disorder occurred in
three amplicons (g.3079A > G, g.11594T > C, g.20920G > A),
equivalent to 56,043 bp of sequence. Those three amplicons
were sequenced in 165–185 European controls generating
252,505 bp of sequence. No candidate variants were
found in the controls whereas the null hypothesis would
expect 13 variants in the controls (Table IV). Of
the remaining eight novel variants identified in other phenotypes, six (g.1726C > A, g.1220–1221insT, g.20826A > G,
g.21850C > T, g.2078G > C, and g.21839G > A) were not
detected in any control subjects (Table IV). Of these, we
excluded g.1220–1221insT as a candidate mutation given
that it was absent in an affected sibling, although the role of
genetic heterogeneity within sibships is poorly understood and
remains to be determined. Two patient variants were found in
controls, and are thus are not considered candidates. Importantly, in sequencing 1.18 Mb of control data, no variants were
found that were specific to controls.
Therefore, in total we identified eight novel variants specific
to families with behavioral or psychiatric disorders that
represent candidate mutations. One of these candidate
mutations (g.3079A > G) was identified in two subjects, one
with bipolar I disorder and the other with psychopathy. This
candidate mutation resides within the proximal promoter
region (PPR) of NR2E1 and therefore constitutes a reasonable
candidate for a regulatory mutation. The seven remaining
candidate mutations were identified separately in unrelated
subjects. One of these (g.1726C > A) resides in the PPR in a
100-bp element that is conserved between mouse and human,
four reside in the 30 UTR (g.20920G > A, g.20826A > G,
g.21850C > T, and g.21839G > A), and two (g.11594T > C and
g.2078G > C) reside in intronic regions.
Alterations of Consensus Transcription Factor
Binding Sites by NR2E1 Candidate Mutations
To predict the impact of the eight candidate mutations on
transcription factor binding, we performed in silico analyses on
previously experimentally-validated consensus sequences for
TFBS [Quandt et al., 1995]. We restricted our analyses to
TABLE IV. Characterization of NR2E1 Patient Variants in Families and Control Subjects
Genotype
Patient ID
Phenotype
Locationa
3000
929
3542
gEMS465
gEMS680
3
Bipolar disorder I
Bipolar disorder I
Bipolar disorder I
Schizophrenia
Schizophrenia
Schizophrenia—6qd
PPR
Intron 5
30 UTR
CE12A
CE13A
Intron 7
9
gEMS453
gEMS455
SD4
CMS4989
CMS4989
Schizophrenia—6q
Psychopathy
Psychopathy
Paraphilic offender
MR and psychosis
MR and psychosis
30 UTR
PPR
30 UTR
Intron 1
30 UTR
30 UTR
Affected
sibling
Frequency of
candidate
mutation in
control
subjectsc
A/G
T/T
G/G
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
G/G
n.d.
0/330
0/550
0/350
0/532
0/188e
1/460
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0/536
0/330
0/524
0/370
1/182
0/524
Unaffected Unaffected
father
mother
Nucleotide variantb
Patient
g.3079A > G
g.11594T > C
g.20920G > A
g.1726C > A
g.1220–1221insT
g.14314-14319delACTCT
g.20826A > G
g.3079A > G
g.21850C > T
g.2078G > C
g.21332-21335delCTT
g.21839G > A
A/G
T/C
G/A
C/A
G/insT
ACTCT/—
A/A
n.d.
n.d.
n.d.
n.d.
n.d.
A/G
A/G
C/T
G/C
CTT/—
G/A
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d., not determined (i.e., parents unavailable for genotyping and/or patient does not have an affected sibling.); MR, mental retardation.
a
PPR, proximal promoter region (defined as a 2.0-kb region upstream of the initiator Met codon); CE, evolutionary conserved element within PPR [as
described in Abrahams et al., 2002]; UTR, untranslated region.
b
g, genomic; numbering based on Antonarakis and the Nomenclature Working Group [1998], where A of the initiator Met codon in exon 1 is denoted
nucleotide þ1. Human genomic NR2E1 sequence: NCBI AL078596. Note that g.30179A > G was identified in two subjects.
c
Numbers represent total number of successfully sequenced chromosomes.
d
6q, subject belongs to a family that showed evidence of linkage to 6q21-22 [Cao et al., 1997; Martinez et al., 1999].
e
The 188 chromosomes examined for subject gEMS680 represented ethnically-diverse chromosomes [Kumar et al., 2007].
886
Kumar et al.
TABLE V. NR2E1 Candidate Mutations Predicted to Alter Neural Transcription Factor Consensus Binding Sites
Nucleotide
varianta
Transcription
factor
Transcription
Locationb binding site
factor
g.1726C > A CE12A
g.2078G > C Intron 1
Abolished
Abolished
g.11595T > C
g.20826A > G
g.21857C > T
g.3079A > G
g.20920G > A
g.21846G > A
Abolished
Abolished
Created
n.a.
n.a.
n.a.
Intron 5
30 UTR
30 UTR
PPR
30 UTR
30 UTR
Orthologous nucleotide in other species
Role in brain
SP1
Regulator of neuronal survival
GTF3A (AP2) Regulator of neural gene expression
and development
Pou3f2 (Bm2) Regulator of neural cell differentiation
Nfat
Regulator of neuronal survivial
Foxa2
Expressed in brain
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
Human Apes Macaque Mouse Fugu
C
G
C
G
C
G
C
G
C
G
T
A
C
A
G
G
T
A
C
A
G
G
C
A
C
A
G
G
T
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d., not determined (i.e., ortholgous region does not align with human sequence); n.a., not applicable.
a
g, genomic; numbering based on Antonarakis and the Nomenclature Working Group [1998], where A of the initiator Met codon in exon 1 is denoted
nucleotide þ1.
b
PPR, proximal promoter region (defined as a 2.0-kb region upstream of the initiator Met codon); CE, evolutionary conserved element within PPR [as
described in Abrahams et al., 2002]; UTR, untranslated region.
transcription factors expressed in the brain. Of the eight
candidate mutations, five were predicted to create or abolish
binding of transcription factors known to have roles in brain
development (Table V).
To determine whether functional constraint may exist at
sites corresponding to the eight candidate mutations, we
determined the orthologous nucleotide at each of the sites in
chimpanzee, gorilla, orangutan, macaque, mouse, and Fugu.
Notably, in two instances (g.1726C > A and g.2078G > C), the
major human nucleotide was conserved to Fugu (Table V). The
absence of nucleotide variability at these non-coding sites
between human and Fugu, which are separated by 900
million years [Kumar and Hedges, 1998], suggests strong
functional constraint.
To determine whether the NR2E1 candidate mutations may
reside within cis-acting UTR motifs, we searched for the
presence of experimentally-validated 50 and 30 UTR motifs
using UTRscan [Mignone et al., 2005]. We did not identify any
motifs that included a candidate mutation. To determine
whether any of the candidate mutations may alter 30 UTR
binding for microRNAs (miRNA), we aligned the 30 UTR of
NR2E1 against known miRNA motifs [Xie et al., 2005], but we
did not detect a motif that included a candidate mutation.
A strength of our study is that it was designed to elucidate the
role of both common and rare susceptibility variants. Importantly, several of the markers chosen for association analyses
and the regions we chose to examine for the presence of
candidate mutations were selected based on their putative
functional and regulatory roles.
We report significant associations between bipolar disorder
and NR2E1 with marker rs217520, which resides in the
haplotype block that harbors all nine NR2E1 exons [TIHMC,
2005]. The significant association of NR2E1 with bipolar I
disorder is consistent with McQueen et al. [2005] who have
shown that a genome-wide significant linkage peak (LOD 4.19)
for bipolar I disorder (but not bipolar II disorder) exists on
Chromosome 6q in a linkage region that peaks above NR2E1.
The bipolar I association is also consistent with the mania-like
behaviors in Nr2e1/ mice (Wong and Simpson, unpublished
data). Although the case and control samples used for the
association analyses were of Caucasian origin, we cannot
formally exclude the possibility that population structure may
confound the interpretation of our findings. Attempts to
extend this association to neighboring SNPs and to develop a
No Predicted Alterations in NR2E1 Secondary
Structure by Candidate Mutations
To determine whether the 30 UTR candidate mutations
(g.20920G > A, g.20826A > G, g.21850C > T, and g.21839G >
A) may affect the 30 UTR secondary structure, we predicted
the minimum free energy (MFE) secondary structures for each.
The MFE structure of all four variants did not differ from the
MFE structure generated from a consensus wild-type sequence
(ENST230083; www.ensembl.org) (data not shown).
NR2E1 Expression in Normal Human Adult
Brain Is Forebrain-Specific
To examine expression of NR2E1 in regions of the normal
adult human brain not specifically examined previously, we
performed northern analyses. A single transcript of approximately 4.0 kb was detected in cerebral cortex, occipital pole,
frontal lobe, temporal lobe, and putamen, but not in cerebellum, medulla, and spinal cord (Fig. 2).
DISCUSSION
The present study is the first genetic investigation of NR2E1
in bipolar disorder, schizophrenia, and aggressive disorders.
Fig. 2. NR2E1 is expressed in human adult forebrain. A: Northern blot
analysis of an unaffected human brain with an NR2E1-specific cDNA probe
demonstrates transcription in cerebral cortex (whole), occipital lobe, frontal
lobe, temporal lobe, and putamen. NR2E1 transcription was not detected in
the cerebellum, medulla, and spinal cord. B: Probing with GAPDH
demonstrates approximately equal loading of RNA. [Color figure can be
viewed in the online issue, which is available at www.interscience.
wiley.com.]
Association of NR2E1 With Bipolar Disorder
haplotype were thwarted, in part, by the unusually low genetic
diversity at this locus [Kumar et al., 2007].
Our findings indicate a significant association between
marker rs217520 at NR2E1 and bipolar disorder in females.
Female-specific associations in bipolar disorder have been
reported for other loci, including the orphan G protein-coupled
receptor GPR50 [Thomson et al., 2005b], and markers and
haplotypes within the TRAX/DISC locus [Thomson et al.,
2005a]. NR2E1 belongs to the nuclear receptor superfamily
that includes hormone receptors for estrogen, which have been
implicated in female-specific genetic associations with mental
illness [Westberg et al., 2003]. One possibility for the femalespecific pattern of association observed in the present study
includes hormonal interaction involving NR2E1 and its as yet
unidentified endogenous ligand.
Our results indicate a significant association between
NR2E1 and early AAO in bipolar disorder, which may comprise
a genetically distinct subgroup [Bellivier et al., 2001; Visscher
et al., 2001; Lin et al., 2006]. Grigoroiu-Serbanescu et al. [2001]
reported that early-onset bipolar disorder (<25 years) is best
explained by a non-Mendelian major gene with a polygenic
component, which would be consistent with our findings at
NR2E1. Interestingly, suggestive evidence that 6q may harbor
a susceptibility gene(s) specific for early-onset bipolar disorder
has been reported [Faraone et al., 2006].
The neurogenic role of Nr2e1, including its negative
influence on adult neurogenesis, neurite growth, and neural
stem cell proliferation [Roy et al., 2004; Shi et al., 2004; Christie
et al., 2006], is consistent with its involvement in bipolar
disorder given that drugs used to treat this illness have been
shown to promote neurogenesis, neurite growth, and cell
survival [Ogden et al., 2004]. In particular, both valproate and
lithium have been shown to enhance neurogenesis in the adult
dentate gyrus of the hippocampus as well as in cultured cells
[Hao et al., 2004; Kim et al., 2004]. Thus, genes influencing
neurogenesis, such as NR2E1, are ‘‘high-probability candidates’’ for bipolar disorder [Ogden et al., 2004].
Our candidate mutation screen indicates that protein-coding
NR2E1 mutations are unlikely to contribute to bipolar
disorder, schizophrenia, or impulsive-aggressive disorders in
the subjects examined here. However, we identified eight
candidate non-coding and putative regulatory mutations.
Importantly, no rare variants were found that were specific
to the controls, supporting the high level of conservation and
low genetic diversity previously described for this gene
[Abrahams et al., 2002: Kumar, 2007 #1327]. Furthermore,
none of these rare variants have been previously reported in
ethnically-diverse subjects sequenced for exactly the same
regions examined in the present study [Kumar et al., 2007]. We
interpret these observations to suggest that the rare variants
identified here as candidate mutations do not represent private
(i.e., population-specific) polymorphisms.
The eight heterozygote candidate mutations identified here
may operate via a hypomorphic mechanism, which is supported by studies in mice heterozygous for Nr2e1 deletions that
show premature cortical neurogenesis early in development
[Roy et al., 2004], thereby suggesting dosage sensitivity for
NR2E1. Six of the eight candidate mutations reside within
regions that could conceivably influence transcription of
NR2E1, including the proximal promoter and UTRs. Importantly, genetic studies of promoter and UTR regulatory SNPs
in other genes have been shown to influence psychiatric
disorders and impulsive-aggressive disorders [Arinami et al.,
1997; Okuyama et al., 1999; Caspi et al., 2002]. Future studies
may involve studying these mutations in functional assays to:
(1) demonstrate alteration of transcription factor binding
in vitro; and/or (2) demonstrate altered function in vivo using
genetic mouse models since the characterization of novel
mouse models expressing human alleles can aid in under-
887
standing the molecular mechanisms underlying brain development and function.
The detection of NR2E1 in the normal human adult
forebrain is consistent with the forebrain-specific expression
patterns observed in adult mice [Shi et al., 2004]. Expression
of NR2E1 in the frontal and temporal lobes supports a role for
this gene in mental illness and aggressive disorders. Importantly, functional neuroimaging studies provide evidence
to suggest that genetically-mediated metabolic disturbances
of the frontal lobe may predispose to violence [Filley et al.,
2001].
In conclusion, the present study provides evidence to support
the hypothesis that genetic variation at NR2E1 may be
associated with susceptibility to human behavioral and
psychiatric disorders. In particular, our study indicates that
NR2E1 is a promising candidate gene for bipolar disorder. Our
work contributes to a rapidly growing body of literature that
suggests the involvement of genetic determinants in complex
disorders of human brain and behavior, including phenotypes
such as aggressive behaviors that are largely underrepresented in human genetic studies. Critically, these initial
positive association and candidate mutation studies provide a
new focus for future research efforts towards replication
studies and the development of functional assays for human
variation in NR2E1.
ACKNOWLEDGMENTS
The authors acknowledge the patients and families who
made this study possible by donating their time and blood
samples. We thank Dr. P. Gejman (Northwestern University,
United States) and Drs. X. Breakefield and D. Schuback
(Harvard University, United States) for providing patient
DNA samples. The authors are grateful to Kathleen G. Banks
and Tracey D. Weir (Centre for Molecular Medicine and
Therapeutics, Canada) and Dr. Andrew MacLeod (University
of Edinburgh) for helpful comments on the manuscript. This
work was supported by grants from Jack and Doris Brown
Foundation and British Columbia Institute for Children’s &
Women’s Health (to RAK); Harry Frank Guggenheim Foundation (to BSA); and the UK Medical Research Council, The
Chief Scientist Office of the Scottish Executive, The Wellcome
Trust, and The State Hospital Board for Scotland (to DB, WM,
KM, and AM); and Canadian Institutes for Health Research
(CIHR), CIHR Research and Development, and Canada
Research Chair in Genetics and Behaviour (to EMS).
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