Initial Association of NR2E1 With Bipolar Disorder and Identification of Candidate Mutations in Bipolar Disorder, Schizophrenia, and Aggression Through Resequencing

Abstract

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 5' and 3' 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 = 0.77, P = 0.013), bipolar disorder I (OR = 0.77, P = 0.015), bipolar disorder in females (OR = 0.72, P = 0.009), and with age at onset < or = 25 years (OR = 0.67, P = 0.006)], all of 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.

2008 Wiley-Liss, Inc.

Full text

Page 1

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,1,2Kevin A. McGhee,3Stephen Leach,4Russell Bonaguro,1Alan Maclean,3
Rosalia Aguirre-Hernandez,5Brett S. Abrahams,1Emil F. Coccaro,6Sheilagh Hodgins,7
Gustavo Turecki,8Anne Condon,5Walter J. Muir,3Angela R. Brooks-Wilson,2,4Douglas H. Blackwood,3
and Elizabeth M. Simpson1,2*
1Centre for Molecular Medicine and Therapeutics and Child & Family Research Institute, Vancouver,
British Columbia, Canada
2Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada
3Division of Psychiatry, University of Edinburgh, Edinburgh, Scotland
4Canada’s Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, British Columbia, Canada
5Department of Computer Science, University of British Columbia, Vancouver, British Columbia, Canada
6Department of Psychiatry, University of Chicago, Chicago, Illinois
7King’s College London, Institute of Psychiatry, London, United Kingdom
8McGill Group for Suicide Studies, Douglas Hospital Research Centre, Montreal, Quebec, Canada
Nuclearreceptor2E1gene(NR2E1)resideswithin
a 6q21-22 locus for bipolar disorder and schizo-
phrenia. Mice deleted for Nr2e1 show altered
neurogenesis, cortical and limbic abnormalities,
aggression, hyperexcitability,
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 schizo-
phrenia, and 479 controls using six common
markers and haplotypes. We also performed a
comprehensive mutation screen of NR2E1, rese-
quencingitsentirecodingregion,complete50and30
untranslated regions, consensus splice-sites, and
evolutionarily conserved regions in 126 humans
with bipolar disorder, schizophrenia, or aggres-
sivedisorders.NR2E1wasassociatedwithbipolar
disorder I and II [odds ratio (OR¼0.77, P¼0.013),
bipolar disorder I (OR¼0.77, P¼0.015), bipolar
disorderinfemales(OR¼0.72,P¼0.009),andwith
age at onset ?25 years (OR¼0.67, P¼0.006)], all of
andcognitive
which
for multiple comparisons. We identified eight
novel candidate mutations that were absent in
325 controls; four of these were predicted to alter
knownneuraltranscription
sites.Analyses ofNR2E1
brain revealed forebrain-specific transcription.
Thedatapresentedsupport
that genetic variation at NR2E1 may be associa-
ted with susceptibility to brain-behavior disor-
ders.
? 2008 Wiley-Liss, Inc.
remainedsignificant aftercorrecting
factorbinding
humanmRNAin
thehypothesis
KEY WORDS:nuclear receptor; mental illness;
‘‘fierce’’ mice; brain; polymorph-
isms
Pleasecitethisarticleasfollows:KumarRA,McGheeKA,
LeachS,BonaguroR,MacleanA,Aguirre-HernandezR,
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 preva-
lence ofapproximately 1% [Jablensky etal., 1992; Sklar, 2002]
and show variable and sometimes overlapping clinical pre-
sentation [Maier et al., 2006]. In some patients, neurodevelop-
mental phenotypes such as altered neurogenesis, reduced
dendritic branching, impairment in GABAergic interneurons,
ventricular enlargement, or reduced volume of the hippo-
campus, cerebral cortex, corpus callosum, amygdala, and
olfactory bulb have been described [Rapoport et al., 2005;
Strakowski et al., 2005; Ross et al., 2006].
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 Profes-
sor, 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
? 2008 Wiley-Liss, Inc.
Am J Med Genet B Neuropsychiatr Genet (2008), 147B(6), 880-889. doi: 10.1002/ajmg.b.30696

Page 2

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 etal., 2005]. Todate, nostudies have found evidence
ofassociationtocandidategenesinthisregion[Mamdanietal.,
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 hyper-
activity, cognitive deficits, increased aggression, abnormal
startle reactivity, reduced anxiety, altered neurogenesis,
impairment in GABAergic interneurons, reduced dentritic
branching, retinopathy, ventricular enlargement, and hypo-
plasia of the hippocampus, cerebral cortex, corpus callosum,
amygdala, and olfactory bulb (Wong and Simpson, unpub-
lished 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 develop-
ment 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 inphenotypes 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 schizo-
phrenia, and 479 ethnically-matched controls using six
common markers and haplotypes comprised of each marker
combination.Second,weperformedacomprehensivemutation
screen of NR2E1 in 126 humans with bipolar disorder,
schizophrenia, or aggressive behaviors. We genotyped candi-
date mutations in 325 healthy controls. We applied bioinfor-
matic 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
BritishColumbia(UBC),ChildandFamilyResearchInstitute,
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
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
andSchizophrenia-Lifetimeversion),hospitalrecordsandwhen
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
withadisorderwithevidenceofgeneticlinkageto6q21-22and/
or presented with features resembling Nr2e1?/?mice, includ-
ing 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/).Thesecondgroup
(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
residentswithinalargeforensicpsychiatrichospital.Thethird
group (aggressive disorders without a diagnosis of bipolar
disorder or schizophrenia) included subjects selected for a
historyofextremeviolentbehaviorincluding: (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 Aggres-
sion, 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-directedaggressivebehaviorswhocommittedsuicidein
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
BDHIadaptedtoproxy-basedinterviews[Dumaisetal.,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)autismandahistoryofviolencewhowereobtained
from the Autism Genetics Resource Exchange (http://www.
agre.org/). Candidate mutations were genotyped in the follow-
ing 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
Association of NR2E1 With Bipolar Disorder881

Page 3

functional importance: g.?1429G>A resides in an evolutio-
narily conserved region in the proximal promoter [Kumar
et al., 2007], rs2233488 resides in the 50UTR, and the (CA)n
microsatellite (D6S1594) that resides within the 30UTR. 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 50with 6-FAMTM(ABI, Foster City, CA). DNA
fragmentswereamplifiedasfollows:2minat948C,35cyclesof
20 sec at 948C, 30 sec at 558C, and 1 min at 728C; and a final
extensionof20minat728C.Amplified products wereruninan
ABI 3730 DNA Analyzer (Warrington, UK) with GS500 LIZ
size standards. Allele sizes were determined using Genemap-
per3.0(ABI,CA).ForSNPgenotyping,thetargetsequencefor
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 50and 30untranslated regions (1,973 bp), exon-
flanking 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 w2test 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
TABLE I. Clinical and Demographic Data on Subjects
Diagnosis
Sample size Mean age (range) Ethnicity
TotalMale FemaleMale FemaleAA CAU
Association analyses
Bipolar disorder
I
II
Schizophrenia
Control
Candidate mutation screen
Bipolar I disorder
Schizophrenia
Aggressive behavior
Psychopathy
Intermittentexplosive disorder
Paraphilic disorder
Suicide and aggression
MR and behavioral problems
Autism and violence
358
33
394
479
162 196
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
8
278
251
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
240
1
0
1
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
24
3
8
3
2
8
4
9n.d.
n.d.
n.d.
n.d. n.d.n.d.
MR, mental retardation; n.a., not applicable; n.d., not determined.
Fig. 1.
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.
Genomic structure of NR2E1 and location of six markers selected for association analyses in bipolar disorder and schizophrenia. A: Schematic of
882Kumar et al.

Page 4

were therefore combined into a single allele (combined rare
group);comparisonswerethereforemadewithnineallelesand
the combined rare group. Individual SNPs were tested for
association using a w2test. Haplotype association analyses
were performed using UNPHASED [Dudbridge, 2003]. This
program performs genetic association analysis in both nuclear
families and unrelated subjects. It implements maximum-
likelihood inference on haplotype and genotype effects while
dealingwithmissingdatasuchasuncertainphaseandmissing
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 combina-
tions. This resulted in 4,774 haplotype combinations. There-
foreinordertocorrectformultipletesting,thegoldstandardof
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 micro-
satellite. 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
Hybridizationanalysiswasperformedonadulthumanbrain
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 manufac-
turer.
primer labeling using Ready-To-GoTMDNA 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].
32P-radiolabeled probes were generated by random
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 func-
tional, 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 associa-
tionbetweenrs217520andbipolardisorder(IandIIcombined)
[odds ratio (OR) for the A allele¼0.77, confidence interval
(CI)¼0.62–0.95, w2¼6.11, uncorrected P¼0.013, permuted
P¼0.036] but not with any of the other four SNPs (Table II).
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
(OR¼0.77, CI¼0.62–0.96, w2¼5.89, uncorrected P¼0.015,
permuted P¼0.041) but not for bipolar II disorder (w2¼1.540,
P¼0.215). 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 (OR¼0.72,
CI¼0.56–0.93, w2¼6.92, uncorrected P¼0.009, permuted
P¼0.025) and with AAO ?25 years in bipolar disorder
(OR¼0.67, CI¼0.50–0.90, w2¼7.59, uncorrected P¼0.006,
permuted P¼0.017) (Table III). AAO ?25 years in bipolar
disorder was also associated with a second marker, rs588409
before correction (OR¼1.28, CI¼1.00–1.66, w2¼3.90, uncor-
rected P¼0.048, permuted P¼0.13).
Microsatellite Analysis Does Not Detect Association
in Bipolar Disorder or Schizophrenia
We identified 17 alleles from our analyses of the micro-
satellite D6S1594 (data not shown). We did not detect any
significant differences between repeat lengths of the micro-
satellite in bipolar disorder versus controls (w2¼10.3, df¼16,
P¼0.85) nor in schizophrenia versus controls (w2¼22.1,
df¼16, P¼0.14), nor in bipolar disorder type I (w2¼9.31,
P¼0.90) and type II (w2¼15.3, P¼0.50) analyzed separately.
We did not find any evidence of association with schizophrenia
orbipolardisorderineithersex,norwithAAOwithbipolarIor
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 hap-
lotype analyses with all six markers, generating 63 marker
combinations resulting in 4,773 haplotypes (data not shown).
Five haplotypes were associated with bipolar disorder, how-
ever, these associations did not remain significant after
correcting for multiple testing. rs217520, the marker associ-
ated with bipolar disorder, was the only marker common to all
fivehaplotypes.TheuncorrectedPvaluesarepresentedforthe
reader’s information (g.?1429/rs217520, w2¼7.929, df¼2,
uncorrected P¼0.019, rs127503/rs217520, w2¼11.85, df¼3,
uncorrected P¼0.008, rs2233488/rs217520, w2¼6.247, df¼2,
uncorrectedP¼0.044,
12.29,df¼5,
rs588409, w2¼12.29, df¼5, uncorrected P¼0.031). However,
we recognize that we may have insufficient power to detect
significant haplotype associations after multiple testing
correction.
rs127503/g.?1429/rs217520,
uncorrectedP¼0.019,
w2¼
g.?1429/rs217520/
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
thatcodingregiondeletionsarepresentinsomesubjects,given
that exon-based sequencing cannot detect deletions of whole
exons.
Association of NR2E1 With Bipolar Disorder883

Page 5

We identified 12 subjects harboring 11 novel non-coding
variants (Table IV) that have not been previously reported in
apparentlyhealthyhumans[Kumaretal.,2007]norindbSNP
(http://www.ncbi.nlm.nih.gov/projects/snp/; Build 124). One of
these variants (g.?1726C>A) has been previously identified
inapatientwithabnormalcorticaldevelopment[Kumaretal.,
2007]. Two of the novel variants were deletions, one was an
insertion, and eight were single-base substitutions. Each of
these11variants(hereinreferredtoas‘‘patientvariants’’)was
present in the heterozygote state. Of the eight single-base
substitutions,sixweretransitionsandtwoweretransversions.
When possible we obtained DNA samples from relatives of
thecandidatemutationcarriersforfurthergeneticstudies.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.14314-
14319delACTCTandg.20826A>G)wereidentifiedinpatients
TABLE II. NR2E1 SNP Association Analyses in Bipolar Disorder and Schizophrenia
Markera
Case-controlna
Alleleb
w2
OR (95% CI)PCorrected PGA
rs127503Bipolar 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
—
Markera
Case-controlna
Alleleb
w2
OR (95% CI)PGA
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
—
Markera
Case-controlna
Alleleb
w2
OR (95% CI)PGC
rs2233488 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
—
Markera
Case-controlna
Alleleb
w2
OR (95% CI)PAC
rs217520Bipolar 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*
Markera
Case-controlna
Alleleb
w2
OR (95% CI)PAG
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
—
aNumber of subjects examined.
bNumber of alleles; values in parenthesis refer to frequencies.
cg, 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.
884Kumar et al.

Page 6

withschizophreniabelongingtofamilieswithevidenceof6q21-
linkage; 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.
Weamplifiedandsequenced theamplicons correspondingto
each of the 11 patient variants in ethnically-matched controls
of African (274 chromosomes) and European (376 chromo-
somes)descent.Iftheethnicityofthesubjectwasunknown,the
patient variants were genotyped in chromosomes of both
African and European descent (650 chromosomes).
Theresultsaresurprising,
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
252,505bp ofsequence.No candidate
found in the controls whereas the null hypothesis would
expect13variants inthe
the remaining eight novel variants identified in other pheno-
types, 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
geneticheterogeneitywithinsibshipsispoorlyunderstoodand
particularlyfor bipolar
variantswere
controls(TableIV). Of
remains to be determined. Two patient variants were found in
controls, and are thus are not considered candidates. Impor-
tantly, 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 30UTR (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
transcriptionfactorbinding,weperformedinsilicoanalyseson
previously experimentally-validated consensus sequences for
TFBS [Quandt et al., 1995]. We restricted our analyses to
TABLE III. NR2E1 Association Analyses in Bipolar Disorder Stratified by Sex and Age of Onset
MarkerCase-controlna
Alleleb
w2
OR (95% CI)PCorrected PAC
rs217520Bipolar disorder
Females
Males
AAO?25
AAO>25
214
159
130
55
232 (0.54)
183 (0.58)
136 (0.52)
64 (0.58)
196 (0.46)
135 (0.42)
124 (0.48)
46 (0.42)
6.92
1.90
7.59
0.61
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*
aNumber of subjects examined.
bNumber of alleles; values in parenthesis refer to frequencies.
*Significant P values.
TABLE IV. Characterization of NR2E1 Patient Variants in Families and Control Subjects
Patient ID PhenotypeLocationa
Nucleotide variantb
GenotypeFrequency of
candidate
mutation in
control
subjectsc
Patient
Unaffected
father
Unaffected
mother
Affected
sibling
3000
929
3542
gEMS465
gEMS680
3
Bipolar disorder I
Bipolar disorder I
Bipolar disorder I
Schizophrenia
Schizophrenia
Schizophrenia—6qd
PPR
Intron 5
30UTR
CE12A
CE13A
Intron 7
g.?3079A>G
g.11594T>C
g.20920G>A
g.?1726C>A
g.?1220–1221insT
g.14314-14319del-
ACTCT
g.20826A>G
g.?3079A>G
g.21850C>T
g.2078G>C
g.21332-21335delCTT CTT/—
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
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
9
gEMS453
gEMS455
SD4
CMS4989
CMS4989
Schizophrenia—6q
Psychopathy
Psychopathy
Paraphilic offender
MR and psychosis
MR and psychosis
30UTR
PPR
30UTR
Intron 1
30UTR
30UTR
A/G
A/G
C/T
G/C
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
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
0/524G/A
n.d., not determined (i.e., parents unavailable for genotyping and/or patient does not have an affected sibling.); MR, mental retardation.
aPPR, 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.
bg, 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.
cNumbers represent total number of successfully sequenced chromosomes.
d6q, subject belongs to a family that showed evidence of linkage to 6q21-22 [Cao et al., 1997; Martinez et al., 1999].
eThe 188 chromosomes examined for subject gEMS680 represented ethnically-diverse chromosomes [Kumar et al., 2007].
Association of NR2E1 With Bipolar Disorder885

Page 7

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,intwoinstances(g.?1726C>Aandg.2078G>C),the
majorhuman 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.
Todetermine whether the NR2E1 candidate mutations may
reside within cis-acting UTR motifs, we searched for the
presence of experimentally-validated 50and 30UTR 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 30UTR
binding for microRNAs (miRNA), we aligned the 30UTR of
NR2E1 against known miRNA motifs [Xie et al., 2005], but we
did not detect a motif that included a candidate mutation.
No Predicted Alterations in NR2E1 Secondary
Structure by Candidate Mutations
To determine whether the 30UTR candidate mutations
(g.20920G>A, g.20826A>G, g.21850C>T, and g.21839G>
A) may affect the 30UTR secondary structure, we predicted
theminimumfreeenergy(MFE)secondarystructuresforeach.
The MFE structure of all four variants did not differ from the
MFEstructuregeneratedfromaconsensuswild-typesequence
(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 approx-
imately 4.0 kb was detected in cerebral cortex, occipital pole,
frontal lobe, temporal lobe, and putamen, but not in cerebel-
lum, medulla, and spinal cord (Fig. 2).
DISCUSSION
ThepresentstudyisthefirstgeneticinvestigationofNR2E1
in bipolar disorder, schizophrenia, and aggressive disorders.
A strength of our study is that it was designed to elucidate the
role of both common and rare susceptibility variants. Impor-
tantly, 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
shownthatagenome-widesignificantlinkagepeak(LOD4.19)
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
TABLE V. NR2E1 Candidate Mutations Predicted to Alter Neural Transcription Factor Consensus Binding Sites
Nucleotide
varianta
Locationb
Transcription
factor
binding site
Transcription
factorRole in brain
Orthologous nucleotide in other species
Human Apes Macaque Mouse Fugu
g.?1726C>A CE12A
g.2078G>C
Abolished
Abolished
SP1
GTF3A (AP2) Regulator of neural gene expression
and development
Pou3f2 (Bm2) Regulator of neural cell differentiation
NfatRegulator of neuronal survivial
Foxa2Expressed in brain
n.a.n.a.
n.a. n.a.
n.a. n.a.
Regulator of neuronal survivalC
G
C
G
C
G
C
G
C
GIntron 1
g.11595T>C
g.20826A>G 30UTR
g.21857C>T
g.?3079A>G PPR
g.20920G>A 30UTR
g.21846G>A 30UTR
Intron 5Abolished
Abolished
Created
n.a.
n.a.
n.a.
T
A
C
A
G
G
T
A
C
A
G
G
C
A
C
A
G
G
Tn.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
30UTR
n.d., not determined (i.e., ortholgous region does not align with human sequence); n.a., not applicable.
ag, 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.
bPPR, 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.
Fig. 2.
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.]
NR2E1 is expressed in human adult forebrain. A: Northern blot
886 Kumar et al.

Page 8

haplotypewerethwarted,inpart,bytheunusuallylowgenetic
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
thatincludeshormonereceptorsforestrogen,whichhavebeen
implicated in female-specific genetic associations with mental
illness [Westberg et al., 2003]. One possibility for the female-
specific 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
NR2E1andearlyAAOinbipolardisorder,whichmaycomprise
a genetically distinct subgroup [Bellivier et al., 2001; Visscher
etal.,2001;Linetal.,2006].Grigoroiu-Serbanescuetal.[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,suggestiveevidencethat6qmayharbor
asusceptibility gene(s) specific forearly-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
stemcellproliferation[Royetal.,2004;Shietal.,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 beenshown toenhance neurogenesis inthe 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 candi-
dates’’ for bipolar disorder [Ogden et al., 2004].
Ourcandidatemutationscreenindicatesthatprotein-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
regionsexaminedinthepresentstudy[Kumaretal.,2007].We
interpret these observations to suggest that the rare variants
identifiedhereascandidatemutationsdonotrepresentprivate
(i.e., population-specific) polymorphisms.
The eight heterozygote candidate mutations identified here
may operate via a hypomorphic mechanism, which is sup-
portedbystudiesinmiceheterozygousforNr2e1deletionsthat
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. Impor-
tantly, 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-
standingthemolecularmechanismsunderlyingbraindevelop-
ment 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. Impor-
tantly, functional neuroimaging studies provide evidence
to suggest that genetically-mediated metabolic disturbances
of the frontal lobe may predispose to violence [Filley et al.,
2001].
Inconclusion,thepresentstudyprovidesevidencetosupport
the hypothesis that genetic variation at NR2E1 may be
associated with susceptibility to human behavioral and
psychiatric disorders. In particular, our study indicates that
NR2E1isapromising candidate geneforbipolar 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 underrepre-
sented 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 Foun-
dation (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).
REFERENCES
Abrahams BS, Mak GM, Berry ML, Palmquist DL, Saionz JR, Tay A, Tan
YH, Brenner S, *Simpson EM, *Venkatesh B. 2002. Novel vertebrate
genes and putative regulatory elements identified at kidney disease
and NR2E1/fierce loci. Genomics 80(1):45–53 (*authors contributed
equally).
Abrahams BS, Kwok MC, Trinh E, Budaghzadeh S, Hossain SM, Simpson
EM. 2005. Pathological aggression in ‘‘fierce’’ mice corrected by human
nuclear receptor 2E1. J Neurosci 25(27):6263–6270.
Akhmedov NB, Piriev NI, Chang B, Rapoport AL, Hawes NL, Nishina PM,
Nusinowitz S, Heckenlively JR, Roderick TH, Kozak CA, et al. 2000. A
deletion in a photoreceptor-specific nuclear receptor mRNA causes
retinal degeneration in the rd7 mouse. Proc Natl Acad Sci USA 97(10):
5551–5556.
American Psychiatric Association. 2000. Diagnostic and statistical manual
of mental disorders, 4th edition. Washington: American Psychiatric
Association.
ArinamiT,GaoM,HamaguchiH,ToruM.1997.Afunctionalpolymorphism
in the promoter region of the dopamine D2 receptor gene is associated
with schizophrenia. Hum Mol Genet 6(4):577–582.
Barrett JC, Fry B, Maller J, Daly MJ. 2005. Haploview: Analysis and
visualization of LD and haplotype maps. Bioinformatics 21(2):263–265.
Association of NR2E1 With Bipolar Disorder 887

Page 9

BellivierF,GolmardJL,HenryC,LeboyerM,SchurhoffF.2001.Admixture
analysis of age at onset in bipolar I affective disorder. Arch Gen
Psychiatry 58(5):510–512.
Berlin F, Hunt W, Malin H, Dyer A, Lahne G,Dean S.1991. A five-year plus
follow-up survey of criminal recidivism within a treated cohort of 406
pedophiles, 111 exhibitionists and 109 sexual aggressive: Issues and
outcomes. Am J Forensic Psychol 12:5–28.
BuervenichS,CarmineA,ArvidssonM,XiangF,ZhangZ,SydowO,Jonsson
EG, Sedvall GC, Leonard S, Ross RG, et al. 2000. NURR1 mutations in
cases of schizophrenia and manic-depressive disorder. Am J Med Genet
96(6):808–813.
Cao Q, Martinez M, Zhang J, Sanders AR, Badner JA, Cravchik A, Markey
CJ, Beshah E, Guroff JJ, Maxwell ME, et al. 1997. Suggestive evidence
for a schizophrenia susceptibility locus on chromosome 6q and a
confirmationinanindependentseriesofpedigrees.Genomics43(1):1–8.
Caspi A, McClay J,Moffitt TE, MillJ,Martin J,Craig IW, Taylor A,Poulton
R. 2002. Role of genotype in the cycle of violence in maltreated children.
Science 297(5582):851–854.
Chen YH, Tsai MT, Shaw CK, Chen CH. 2001. Mutation analysis of the
human NR4A2 gene, an essential gene for midbrain dopaminergic
neurogenesis, in schizophrenic patients. Am J Med Genet 105(8):753–
757.
Christie BR, Li AM, Redila VA, Booth H, Wong BK, Eadie BD, Ernst C,
Simpson EM. 2006. Deletion of the nuclear receptor Nr2e1 impairs
synaptic plasticity and dendritic structure in the mouse dentate gyrus.
Neuroscience 137(3):1031–1037.
de Bakker PI, Yelensky R, Pe’er I, Gabriel SB, Daly MJ, Altshuler D. 2005.
Efficiency and power in genetic association studies. Nat Genet
37(11):1217–1223.
DudbridgeF.2003.Pedigreedisequilibriumtestsformultilocushaplotypes.
Genet Epidemiol 25(2):115–121.
Dumais A, Lesage AD, Lalovic A, Seguin M, Tousignant M, Chawky N,
Turecki G. 2005. Is violent method of suicide a behavioral marker of
lifetime aggression? Am J Psychiatry 162(7):1375–1378.
Faraone SV, Lasky-Su J, Glatt SJ, Van Eerdewegh P, Tsuang MT. 2006.
Early onset bipolar disorder: Possible linkage to chromosome 9q34.
Bipolar Disord 8(2):144–151.
Filley CM, Price BH, Nell V, Antoinette T, Morgan AS, Bresnahan JF,
Pincus JH, Gelbort MM, Weissberg M, Kelly JP. 2001. Toward an
understanding of violence: Neurobehavioral aspects of unwarranted
physical aggression: Aspen Neurobehavioral Conference consensus
statement. Neuropsychiatry Neuropsychol Behav Neurol 14(1):1–14.
Goveas JS, Csernansky JG, Coccaro EF. 2004. Platelet serotonin content
correlates inversely with life history of aggression in personality-
disordered subjects. Psychiatry Res 126(1):23–32.
Grigoroiu-Serbanescu M, Martinez M, Nothen MM, Grinberg M, Sima D,
Propping P, Marinescu E, Hrestic M. 2001. Different familial trans-
mission patterns inbipolar Idisorderwith onsetbefore andafter age25.
Am J Med Genet 105(8):765–773.
Haider NB, Jacobson SG, Cideciyan AV, Swiderski R, Streb LM, Searby C,
Beck G, Hockey R, Hanna DB, Gorman S, et al. 2000. Mutation of a
nuclear receptor gene, NR2E3, causes enhanced S cone syndrome, a
disorder of retinal cell fate. Nat Genet 24(2):127–131.
HaoY,CresonT,ZhangL,LiP,DuF,YuanP,GouldTD,ManjiHK,ChenG.
2004. Mood stabilizer valproate promotes ERK pathway-dependent
cortical neuronal growth and neurogenesis. J Neurosci 24(29):6590–
6599.
JablenskyA,SartoriusN,ErnbergG,AnkerM,KortenA,CooperJE,DayR,
Bertelsen A. 1992. Schizophrenia: Manifestations, incidence and course
in different cultures. A World Health Organization ten-country study.
Psychol Med Monogr Suppl 20:1–97.
Kim JS, Chang MY, Yu IT, Kim JH, Lee SH, Lee YS, Son H. 2004. Lithium
selectivelyincreasesneuronal
neural progenitor cells both in vitro and in vivo. J Neurochem 89(2):
324–336.
differentiationofhippocampal
Kohn Y, Lerer B. 2005. Excitement and confusion on chromosome 6q: The
challenges of neuropsychiatric genetics in microcosm. Mol Psychiatry
10(12):1062–1073.
Kumar S, Hedges SB. 1998. A molecular timescale for vertebrate evolution.
Nature 392(6679):917–920.
KumarRA,LeachS,BonaguroR,ChenJ,YokomDW,AbrahamsBS,Seaver
L, Schwartz CE, Dobyns W, Brooks-Wilson A, et al. 2007. Mutation and
evolutionary analyses identify NR2E1-candidate-regulatory mutations
in humans with severe cortical malformations. Genes Brain Behav
6:503–516 [Epub ahead of print Oct 20 2006].
Land PW, Monaghan AP. 2003. Expression of the transcription factor,
tailless, is required for formation of superficial cortical layers. Cereb
Cortex 13(9):921–931.
Levinson DF, Holmans P, Straub RE, Owen MJ, Wildenauer DB, Gejman
PV,PulverAE,LaurentC,KendlerKS,WalshD,etal.2000.Multicenter
linkage study of schizophrenia candidate regions on chromosomes 5q,
6q, 10p, and 13q: Schizophrenia linkage collaborative group III. Am J
Hum Genet 67(3):652–663.
Lin PI, McInnis MG, Potash JB, Willour V, MacKinnon DF, DePaulo JR,
Zandi PP. 2006. Clinical correlates and familial aggregation of age at
onset in bipolar disorder. Am J Psychiatry 163(2):240–246.
Maier W, Zobel A, Wagner M. 2006. Schizophrenia and bipolar disorder:
Differences and overlaps. Curr Opin Psychiatry 19(2):165–170.
Mamdani F, Sequeira A, Alda M, Grof P, Rouleau G, Turecki G. 2007. No
association between the PREP gene and lithium responsive bipolar
disorder. BMC Psychiatry 26:7–9.
MartinezM,GoldinLR,CaoQ,ZhangJ,SandersAR,NancarrowDJ,Taylor
JM, Levinson DF, Kirby A, Crowe RR, et al. 1999. Follow-up study on a
susceptibility locus for schizophrenia on chromosome 6q. Am J Med
Genet 88(4):337–343.
McQueenMB,DevlinB,FaraoneSV,NimgaonkarVL,SklarP,SmollerJW,
Abou Jamra R, Albus M, Bacanu SA, Baron M, et al. 2005. Combined
analysis from eleven linkage studies of bipolar disorder provides strong
evidence of susceptibility Loci on chromosomes 6q and 8q. Am J Hum
Genet 77(4):582–595.
Mignone F, Grillo G, Licciulli F, Iacono M, Liuni S, Kersey PJ, Duarte J,
Saccone C, Pesole G. 2005. UTRdb and UTRsite: A collection
of sequences and regulatory motifs of the untranslated regions
of eukaryotic mRNAs. Nucleic Acids Res 33(Database issue):D141–
D146.
Monaghan AP, Bock D, Gass P, Schwager A, Wolfer DP, Lipp HP, Schutz G.
1997. Defective limbic system in mice lacking the tailless gene. Nature
390(6659):515–517.
OgdenCA,RichME,SchorkNJ,PaulusMP,GeyerMA,LohrJB,Kuczenski
R, Niculescu AB. 2004. Candidate genes, pathways and mechanisms for
bipolar (manic-depressive) and related disorders: An expanded con-
vergent functional genomics approach. Mol Psychiatry 9(11):1007–
1029.
Okuyama Y, Ishiguro H, Toru M, Arinami T. 1999. A genetic polymorphism
in the promoter region of DRD4 associated with expression and
schizophrenia. Biochem Biophys Res Commun 258(2):292–295.
Quandt K, Frech K, Karas H, Wingender E, Werner T. 1995. MatInd and
MatInspector: New fast and versatile tools for detection of consensus
matches in nucleotide sequence data. Nucleic Acids Res 23(23):4878–
4884.
Rapoport JL, Addington AM, Frangou S, Psych MR. 2005. The neuro-
developmental model of schizophrenia: Update 2005. Mol Psychiatry
10(5):434–449.
Ross CA, Margolis RL, Reading SA, Pletnikov M, Coyle JT. 2006. Neuro-
biology of Schizophrenia. Neuron 52(1):139–153.
Roy K, Thiels E, Monaghan AP. 2002. Loss of the tailless gene affects
forebrain development and emotional behavior. Physiol Behav 77(4–
5):595–600.
Roy K, Kuznicki K, Wu Q, Sun Z, Bock D, Schutz G, Vranich N, Monaghan
AP. 2004. The Tlx gene regulates the timing of neurogenesis in the
cortex. J Neurosci 24(38):8333–8345.
Schuback DE, Mulligan EL, Sims KB, Tivol EA, Greenberg BD, Chang SF,
Yang SL, Mau YC, Shen CY, Ho MS, et al. 1999. Screen for
MAOA mutations in target human groups. Am J Med Genet 88(1):
25–28.
Sham PC, Curtis D. 1995. An extended transmission/disequilibrium test
(TDT) for multi-allele marker loci. Ann Hum Genet 59(Pt 3):323–336.
Shi Y, Chichung Lie D, Taupin P, Nakashima K, Ray J, Yu RT, Gage FH,
Evans RM. 2004. Expression and function of orphan nuclear receptor
TLX in adult neural stem cells. Nature 427(6969):78–83.
Sklar P. 2002. Linkage analysis in psychiatric disorders: The emerging
picture. Annu Rev Genomics Hum Genet 3:371–413.
Stenman J, Yu RT, Evans RM, Campbell K. 2003. Tlx and Pax6 co-operate
genetically to establish the pallio-subpallial boundary in the embryonic
mouse telencephalon. Development 130(6):1113–1122.
Stober G, Syagailo YV, Okladnova O, Jungkunz G, Knapp M, Beckmann H,
Lesch KP. 1999. Functional PAX-6 gene-linked polymorphic region:
Potential association with paranoid schizophrenia. Biol Psychiatry
45(12):1585–1591.
888Kumar et al.

Page 10

Strakowski SM, Delbello MP, Adler CM. 2005. The functional neuro-
anatomy of bipolar disorder: A review of neuroimaging findings. Mol
Psychiatry 10(1):105–116.
Thomson PA, Wray NR, Millar JK, Evans KL, Hellard SL, Condie A, Muir
WJ,BlackwoodDH,PorteousDJ.2005a.AssociationbetweentheTRAX/
DISC locus and both bipolar disorder and schizophrenia in the Scottish
population. Mol Psychiatry 10(7):657–668, 616.
Thomson PA, Wray NR, Thomson AM, Dunbar DR, Grassie MA, Condie A,
Walker MT, Smith DJ, Pulford DJ, Muir W, et al. 2005b. Sex-specific
association between bipolar affective disorder in women and GPR50, an
X-linked orphan G protein-coupled receptor. Mol Psychiatry 10(5):470–
478.
The International HapMap Consortium. 2005. A haplotype map of the
human genome. Nature 437(7063):1299–1320.
Tso JY, Sun XH, Kao TH, Reece KS, Wu R. 1985. Isolation and character-
ization of rat and human glyceraldehyde-3-phosphate dehydrogenase
cDNAs: Genomic complexity and molecular evolution of the gene.
Nucelic acid Res 13:2485–2502.
Tsuang MT, Faraone SV. 1990. The genetics of mood disorders. Baltimore:
John Hopkins Press.
Visscher PM, Yazdi MH, Jackson AD, Schalling M, Lindblad K, Yuan QP,
PorteousD,MuirWJ,Blackwood DH.2001. Genetic survivalanalysis of
age-at-onset of bipolar disorder: Evidence for anticipation or cohort
effect in families. Psychiatr Genet 11(3):129–137.
Westberg L, Melke J, Landen M, Nilsson S, Baghaei F, Rosmond R,
Jansson M, Holm G, Bjorntorp P, Eriksson E. 2003. Association
between a dinucleotide repeat polymorphism of the estrogen receptor
alpha gene and personality traits in women. Mol Psychiatry 8(1):118–
122.
XieX,LuJ,KulbokasEJ,GolubTR,MoothaV,Lindblad-TohK,Lander ES,
Kellis M. 2005. Systematic discovery of regulatory motifs in human
promoters and 30UTRs by comparison of several mammals. Nature
434(7031):338–345.
Young KA, Berry ML, MahaffeyCL, Saionz JR, Hawes NL,Chang B, Zheng
QY, Smith RS, Bronson RT, Nelson RJ, et al. 2002. Fierce: A new mouse
deletion of Nr2e1; violent behaviour and ocular abnormalities are
background-dependent. Behav Brain Res 132(2):145–158.
Zhang CL, Zou Y, Yu RT, Gage FH, Evans RM. 2006. Nuclear receptor TLX
prevents retinal dystrophy and recruits the corepressor atrophin1.
Genes Dev 20(10):1308–1320.
Association of NR2E1 With Bipolar Disorder889