Am. J. Hum. Genet. 75:624–638, 2004

Polymorphisms in the Trace Amine Receptor 4 (TRAR4) Geneon Chromosome 6q23.2 Are Associated with Susceptibility
to Schizophrenia
Jubao Duan,1 Maria Martinez,2 Alan R. Sanders,1 Cuiping Hou,1 Naruya Saitou,3
Takashi Kitano,3 Bryan J. Mowry,4,5 Raymond R. Crowe,6 Jeremy M. Silverman,7
Douglas F. Levinson,8 and Pablo V. Gejman1
1

Center for Psychiatric Genetics, Department of Psychiatry and Behavioral Sciences, Evanston Northwestern Healthcare and Feinberg Schoolof Medicine, Northwestern University, Evanston, IL; 2Méthodologie Statistique et Epidémiologie Génétique des Maladies Multifactorielles,
INSERM, Evry, France; 3Division of Population Genetics, National Institute of Genetics, Mishima, Japan; 4Queensland Centre for
Schizophrenia Research, The Park, Centre for Mental Health, Wacol, Australia; 5Department of Psychiatry, University of Queensland,
Brisbane, Australia; 6Mental Health Clinical Research Center and Department of Psychiatry, University of Iowa College of Medicine, Iowa
City; 7Department of Psychiatry, Mount Sinai School of Medicine, New York; and 8Department of Psychiatry, University of Pennsylvania,
Philadelphia

Several linkage studies across multiple population groups provide convergent support for a susceptibility locus forschizophrenia—and, more recently, for bipolar disorder—on chromosome 6q13-q26. We genotyped 192 Europeanancestry and African American (AA) pedigrees with schizophrenia from samples that previously showed linkage
evidence to 6q13-q26, focusing on the MOXD1-STX7-TRARs gene cluster at 6q23.2, which contains a number ofprime candidate genes for schizophrenia. Thirty-one screening single-nucleotide polymorphisms (SNPs) were selected, providing a minimum coverage of at least 1 SNP/20 kb. The association observed with rs4305745 (P p.0014) within the TRAR4 (trace amine receptor 4) gene remained significant after correction for multiple testing.
Evidence for association was proportionally stronger in the smaller AA sample. We performed database searches
and sequenced genomic DNA in a 30-proband subsample to obtain a high-density map of 23 SNPs spanning 21.6
kb of this gene. Single-SNP analyses and also haplotype analyses revealed that rs4305745 and/or two other polymorphisms in perfect linkage disequilibrium (LD) with rs4305745 appear to be the most likely variants underlying
the association of the TRAR4 region with schizophrenia. Comparative genomic analyses further revealed thatrs4305745 and/or the associated polymorphisms in complete LD with rs4305745 could potentially affect gene
expression. Moreover, RT-PCR studies of various human tissues, including brain, confirm that TRAR4 is preferentially expressed in those brain regions that have been implicated in the pathophysiology of schizophrenia. These
data provide strong preliminary evidence that TRAR4 is a candidate gene for schizophrenia; replication is currently
being attempted in additional clinical samples.

Introduction
Schizophrenia is a frequently chronic and devastating
brain disorder that affects ∼1% of the population worldwide (Jablensky et al. 1992). Typically, it presents inadolescence or young adulthood and is characterized bymajor disruptions of thinking (delusions and/or disorganization), perception (hallucinations), mood, and behavior (Gottesman and Shields 1982). Schizophrenia isstrongly familial, with a heritability of ∼80%, but its
etiology is hypothesized to involve both genetic and enReceived May 28, 2004; accepted for publication July 23, 2004;
electronically published August 24, 2004.
Address for correspondence and reprints: Dr. Pablo V. Gejman, Evanston Northwestern Healthcare Research Institute, 1001 University
Place, Evanston, IL 60201-3137. E-mail: pgejman@northwestern.edu
䉷 2004 by The American Society of Human Genetics. All rights reserved.
0002-9297/2004/7504-0009$15.00

624

vironmental factors (Sanders and Gejman 2001). Recently, encouraging evidence for several genes potentiallyinvolved in the etiology of schizophrenia has been reported—namely, dysbindin (DTNBP1) (Straub et al.
2002; Schwab et al. 2003), neuregulin 1 (NRG1) (Stefansson et al. 2002, 2003; Williams et al. 2003), proline
dehydrogenase (oxidase) 1 (PRODH) (Jacquet et al.
2002; Liu et al. 2002), catechol-O-methyltransferase(COMT) (Li et al. 2000; Egan et al. 2001; Shifman etal. 2002), regulator of G-protein signaling 4 (RGS4)(Chowdari et al. 2002; Morris et al. 2004; Williams etal. 2004), D-amino-acid oxidase activator (DAOA [previously called G72]) (Chumakov et al. 2002; Schumacher et al. 2004), and D-amino-acid oxidase (DAO)(Chumakov et al. 2002; Schumacher et al. 2004). Additionally, Brzustowicz et al. (2004) reported linkage disequilibrium between C-terminal PDZ domain ligand of
neuronal nitric oxide synthase (CAPON) and schizo-Duan et al.: TRAR4 Association with Schizophrenia

phrenia, perhaps related to a previous association signal
at D1S1679, which is ∼25 kb distal to CAPON (Rosaet al. 2002). Most of the aforementioned genes are positional candidates with likely involvement in dopaminergic or N-methyl D-aspartate (NMDA) brain
mechanisms.
We previously reported linkage of schizophrenia tochromosome 6q13-q26 (SCZD5 [MIM 603175]) (Caoet al. 1997), which has accumulated support from anumber of studies (Kaufmann et al. 1998; Martinez etal. 1999; Bailer et al. 2000; Levinson et al. 2000; Lindholm et al. 2001; Lerer et al. 2003; Lewis et al. 2003).
In the first report of linkage to 6q (Cao et al. 1997),
support for linkage was observed in the region from
D6S301 (located at 111 cM) to D6S305 (located at 170cM). Linkage to this region was confirmed in subsequent studies (Kaufmann et al. 1998; Martinez et al.
1999; Levinson et al. 2000). All the families used in thisstudy are from three data sets, which we call “NIMHIRP” (National Institute of Mental Health Intramural
Research Program), “NIMH-GI” (NIMH Genetics Initiative), and “AU/US” (Australia/United States), that
previously have been shown to yield evidence for linkage to chromosome 6q13-q26 (SCZD5) (Cao et al.
1997; Martinez et al. 1999; Levinson et al. 2000). An
analysis of 12 microsatellite markers in a 50-cM 6q
region was performed by Martinez et al. (1999); nonparametric affected sibling pair methods yielded P values of .00018, .00095, and .013 for the NIMH-IRP,
NIMH-GI, and AU/US data sets, respectively. In a Palestinian/Israeli pedigree sample (Lerer et al. 2003), thelinkage peak with a nonparametric linkage (NPL) scoreof 4.61 was at D6S292 (∼137 cM), and the region from∼131 cM to ∼144 cM contained the 1-NPL decrease
portion of the linkage peak. Furthermore, anothergroup found evidence for linkage in a Swedish pedigree
to the region from ∼170 cM to ∼180 cM (Lindholm etal. 2001). Whether these different results are better explained by the presence of more than one schizophrenia
susceptibility gene in 6q or reflect typical peak variability in complex disorders (Hauser and Boehnke 1997;
Hsueh et al. 2001) is currently unknown. Recently, bipolar disorder was reported to map to 6q, with one
study yielding a maximum LOD score of 2.2 at 113 cM
near D6S1021 (Dick et al. 2003) and another study
reporting a maximum LOD score of 3.56 at ∼124–126
cM near D6S1639 (Middleton et al. 2004), raising thepossibility that a common gene for schizophrenia andbipolar disorder may be located in 6q.
We were interested in a MOXD1-STX7-TRARs genecluster at 6q23.2 (132.8 cM) that harbors prime candidates for schizophrenia (fig. 1): MOXD1 (monooxygenase, dopamine-b-hydroxylase-like 1) (Chambers etal. 1998), STX7 (syntaxin 7) (Wang et al. 1997), andall known human trace amine receptor (TRAR) genes—

625

namely, TRAR1, TRAR3, TRAR4, TRAR5, PNR (putative neurotransmitter receptor gene) (Zeng et al. 1998;
Borowsky et al. 2001; Bunzow et al. 2001; Lee et al.
2001), and three TRAR pseudogenes (TRAR2 , GPR57,
and GPR58 [GPR57 and GPR58 are G-protein–coupled receptor pseudogenes]) (Liu et al. 1998; Borowsky
et al. 2001; Bunzow et al. 2001; Lee et al. 2001).
Trace amines (TAs) are endogenous amine compounds that are chemically similar to classic biogenic
amines like dopamine (DA), norepinephrine, serotonin,and histamine. Abnormalities that involve the classic
biogenic amines are the basis for biological hypotheses
for a wide variety of disorders, including dystonias, Parkinson disease, schizophrenia, drug addiction, andmood disorders. In mammals, TAs are present at low
levels, with no apparent dedicated synapses, but ablockage of amine degradation leads to significant accumulations of TAs, suggesting high synthesis and turnover, as reviewed elsewhere (Premont et al. 2001). TAs
in mammals include tyramine (TYR), tryptamine, bphenylethylamine (b-PEA), and octopamine (OCT)(Branchek and Blackburn 2003), and all are synthesizedfrom amino acid precursors by the aromatic amino aciddecarboxylase.
TAs were thought to be “false transmitters,” whichdisplace classic biogenic amines from their storage andact on transporters in a fashion similar to the amphetamine (Parker and Cubeddu 1986), but the identification of brain receptors specific to TAs indicates that theyalso have effects of their own (Borowsky et al. 2001).
This might explain the fact that, although TYR, b-PEA,
OCT, and amphetamine would require the integrity ofvesicular stores of DA if displacement of DA were their
only mechanism of action, they (except OCT) are still
active when DA is depleted (Baud et al. 1985). TRARs
bind amphetamine, MDMA (3,4-methylenedioxymethamphetamine, known as “ecstasy”), and LSD (D-lysergic acid diethylamide) with high affinity. This suggests
a direct link between TRARs and mechanisms of psychosis, because the administration of amphetamine can
induce a schizophrenia-like psychosis (Connell 1958;
Snyder et al. 1967; Angrist et al. 1974; Laruelle and
Abi-Dargham 1999). In addition, psychedelic experiences induced by LSD can have a remarkable similarity
to schizophrenia (Vardy and Kay 1983; Gouzoulis et al.
1994). Furthermore, LSD can induce habituation deficits (the normal decrease in response magnitude to repeated stimuli over time) that are similar to those exhibited by patients with schizophrenia (Geyer and Braff
1987; Braff et al. 1992).
MOXD1 is a homologue of dopamine-b-hydroxylasethat is potentially involved in the biosynthesis of norepinephrine from DA (Chambers et al. 1998). Syntaxin
7 (STX7) is a critical component of the synaptic protein
complex SNARE (receptor for soluble N-ethylmaleim-

626

Am. J. Hum. Genet. 75:624–638, 2004

Figure 1

Genomic structure of the 6q23.2 gene cluster and association mapping of the initial screening. The genomic positions are basedon the UCSC July 2003 assembly of the human genome (see the UCSC Genome Bioinformatics Web site). a, The relative position of the 6q23.2
gene cluster to the peak markers from various linkage studies: D6S424 (Cao et al. 1997; Martinez et al. 1999), D6S416 (Cao et al. 1997),
D6S292 (Lerer et al. 2003), and D6S264 (Lindholm et al. 2001). b, Genes in the 6q23.2 gene cluster. c, The ⫺log transformation of the FBATP value for the 31 SNP markers analyzed in the initial association screening. Each data point of the markers points to its relative position in
the gene cluster shown in panel b. The most significant marker is rs4305745, with a P value of .0014 (see table A5 [online only], for FBAT P
values and other detailed information for all the initially selected markers, and table 1, for the single-marker association results for all the
additional TRAR4 markers examined in the dense mapping effort).

ide-sensitive factor attachment proteins), which is involved in NMDA receptor and dopaminergic receptor
function (Pei et al. 2004); the dysfunction of SNARE
has been suggested in schizophrenia (Honer et al. 2002).
Specifically, syntaxins mediate vesicle fusion in vesiculartransport processes (Teng et al. 2001). We investigatedDNA polymorphisms in the MOXD1-STX7-TRARs
cluster by use of family-based association methods, andhere we present preliminary evidence of association between TRAR4 and schizophrenia.
Material and Methods
Subjects and Phenotypes
Three samples were studied, which we call the“NIMH-IRP,” “NIMH-GI,” and “AU/US” collections.

Ascertainment of the NIMH-IRP sample was initiallydescribed by Gershon et al. (1988). The full sample—
from which the present sample of 67 pedigrees was
drawn—was described later (Cao et al. 1997; Gejman
et al. 2001). The collection of the NIMH-GI sample wasdescribed in a report of a genome scan of 71 pedigrees(Cloninger et al. 1998), and additional NIMH-GI families were subsequently included in the repository-based
data set (see the “Electronic-Database Information” section). Of these NIMH-GI pedigrees, 69 were used in thepresent analysis and for two previous ones (Cao et al.
1997; Martinez et al. 1999). The AU/US sample wasdescribed initially in a report of a genome scan of 43
pedigrees (Levinson et al. 1998); full or partial trios forthe present study were from 56 of the 71 pedigrees in627

Duan et al.: TRAR4 Association with Schizophrenia

the expanded sample used in linkage fine-mapping studies (Mowry et al. 2000) and in additional analyses ofthis data set (Martinez et al. 1999; Levinson et al. 2000).
For the present study, we genotyped a total of 827 individuals from 192 families (67 NIMH-IRP families, 69
NIMH-GI families, and 56 AU/US families). Details areprovided in table A1 (online only) and in the “ElectronicDatabase Information” section. Schizophrenia and schizoaffective disorder were diagnosed using the criteria of
the DSM-IIIR (American Psychiatric Association 1987).
The institutional review board of the Evanston Northwestern Healthcare Research Institute approved thisstudy.
SNP Selection and Genotyping
SNPs were selected from public databases with thehelp of a bioinformatics tool, SNPper (Riva and Kohane2002), and novel TRAR4 SNPs were identified by direct
sequencing. The DNA samples were genotyped using
one of two methods: (1) template-directed dye-terminator incorporation with fluorescence-polarization detection (FP-TDI) (Chen et al. 1999) or (2) the TaqMan
assay developed by Applied Biosystems. For the FP-TDI
assays, after PCR amplification of genomic DNA, theAcycloPrime-FP SNP detection kit (PerkinElmer) wasused for post-PCR cleanup and the single-base extension
reaction. We detected FP by use of either an Analyst
fluorescence reader (LJL Biosystems) or a Wallac Victor3(PerkinElmer), and FP data were converted to genotypes
with the assistance of an automated genotype-callingspreadsheet (Akula et al. 2002). PCR primers and probes
for the FP-TDI assays were designed using Primer3(Rozen and Skaletsky 2000). For the TaqMan assays,
the genomic sequence flanking the SNP was submitted
to Applied Biosystems for development of an assay-bydesign. Each unique TaqMan minor-groove–binding(MGB) allele-specific probe was labeled with either a 5FAM or a 5-VIC reporter dye. PCR amplification of
genomic DNA was performed in a 384-well plate in an
ABI Prism 7900 or a DNA Engine Tetrad 2 (MJ Research). After PCR, the allele discrimination was performed on an ABI Prism 7900 Sequence Detection System by use of Sequence Detector Software (SDS), version
2.0. Standard genotype calling was converted by a customized spreadsheet. Nucleotide sequences for the PCRprimers, the FP-TDI and TaqMan probes, and related
information for each marker can be found in table A2(online only).
The average completion rate of our experiments was
96%. To empirically check for errors in the genotyping
method, we compared the results for marker rs4305745
from both methods; the difference rate between FP-TDI
and the TaqMan assay was ∼0.25% (data not shown).
We used MERLIN (Multipoint Engine for Rapid Like-

lihood Inference) (Abecasis et al. 2002) (with all the
SNPs at once) to check for Mendelian inconsistencies,
blanked them as described below (sometimes for individuals and sometimes for the family, when the error
could not be traced to a particular individual), and thenaddressed all unlikely recombinants. Genotyping errors
were detected for 0.17% of genotypes (MERLIN) (95errors/54,611 nonzero genotypes), including 26 Mendelian inconsistencies (0.047%) and 69 unlikely recombinants (0.12%). We did not change genotypes for unlikely recombinants unless (1) MERLIN estimated a highprobability of an individual genotype error, comparedwith other possible errors (see MERLIN documentation
for details), and/or (2) manual rereading of each genotype tracing or other raw genotyping output for the family/marker in question pinpointed a specific error for aparticular individual. All genotype errors (Mendelian inconsistencies or specific errors that resulted in unlikely
recombinants) were blanked (zeroed) for the involved
individuals, and, given our high genotyping completion
rate and low genotyping error, we did not perform asecond-pass genotyping procedure for these individuals.
Genotypes were read blind to psychiatric status. We
checked Hardy-Weinberg equilibrium (HWE) for fam
ily founders (at least 200 individuals) for all 55 SNPs.
Three MOXD1 SNPs were found not to be in HWE(rs2206064, rs1981187, and rs2275394 [although the
HWE P value for rs2206064 did not remain significant
after the number of markers examined for HWE wastaken into account]). The minor-allele frequency ofrs2206064 was only 2%, which might explain the lackof HWE. The frequencies of homozygotes for both
rs1981187 and rs2275394 were higher than expected(this was not a bias introduced by cleaning—only ahandful genotypes were blanked, and they were not primarily heterozygous genotypes). We suspected that our
mixed-pedigree sample might have contributed to theabsence of HWE, and, indeed, we found that these
MOXD1 SNPs were all in HWE when the Europeanancestry (EA) founders and the African American (AA)founders were analyzed separately (data not shown).

Intermarker Linkage Disequilibrium (LD) Analysis
LD between the SNPs was estimated with the program
ldmax from the GOLD (Graphical Overview of Linkage
Disequilibrium) package (Abecasis and Cookson 2000)
by use of the genotypes from unrelated founders. The
program ldmax estimates haplotype frequencies fromgenotype data by employing an expectation-maximization algorithm (Excoffier and Slatkin 1995). The standard and normalized Lewontin’s disequilibrium coefficients (D, D) are derived. Association significance isassessed from a x2 distribution with (n 1 ⫺ 1)(n 2 ⫺ 1) de-628
grees of freedom, where n1 and n2 are the number ofalleles at each marker locus.
Association Analysis
To detect LD with illness, we used the transmission/
disequilibrium test (TDT), as implemented in the FamilyBased Association Test (FBAT) program, version 1.5(Laird et al. 2000; Rabinowitz and Laird 2000). The
null hypothesis of interest is the absence of associationin the presence of linkage. We thus employed the empirical-variance estimator (the ⫺e flag option in the
FBAT program) to account for SNP-genotype correlations among affected siblings that result from linkage.
The FBAT test statistic uses a score function, Z p
Sj ⫺ E(Sj)/ Var (Sj), where Sj is the observed number oftransmitted marker alleles, j, to affected offspring, andE(Sj) and Var(Sj) are the expected and variance valuesof Sj under the null hypothesis. Asymptotically, Z is assumed to follow a normal distribution, with a mean anda variance equal to 0 and 1, respectively. The test statistic
can also be expressed as Z2, which follows a x2 distribution, with 1 df. The FBAT program is able to deal
with the transmission of multilocus haplotypes, evenwhen phase is unknown and parental genotypes may be
missing. It can use pedigrees as well as nuclear families,
but pedigrees are broken down into all individual nuclear families; only informative families (i.e., those contributing to the test statistic) are included. For the analyses of the screening SNPs, alleles and haplotypes were
tested for association if they were present in at least 10
informative families; in our data, this corresponds to not
testing alleles and haplotypes rarer than 3%. This restriction, however, was not used when the investigation
was limited to specific subsets of families in the secondary analyses. For multilocus association analyses, FBAT
provides global P values, which assess the significanceof transmission distortion for all the tested haplotypes.
In the present analyses, we limited the number of multilocus systems tested by use of a stepwise procedure andby restriction of multilocus tests to combinations that
included the SNP with highest single Z score value, as
further detailed in the “Results” section below. For the
FBAT analyses, we assumed an additive model for each
SNP, and only one affection status model was used (affected subjects had schizophrenia or schizoaffective disorder). The additive model is expected to perform welleven when the true model is nonadditive.
Linkage Analysis
Model-free linkage analyses with the MOXD1-STX7TRARs gene cluster were performed using the LOD
score test from the affecteds-only sharing method (Kongand Cox 1997), as implemented in the MERLIN program (Abecasis et al. 2002). The likelihood of the observed marker information among affected relatives is

Am. J. Hum. Genet. 75:624–638, 2004

maximized as a function of the marker-allele–sharing
parameter and is compared, using a likelihood-ratio test,with the likelihood of the marker data under the null
hypothesis of no linkage. The resulting distribution of
the allele-sharing test (T) is x2, with 1 df, and the statistic
can also be reported as a LOD score of T/2 ln (10).
We performed additional analyses, by race, to accountfor putative genetic and/or allelic heterogeneity within
our family samples. Association and linkage tests were
evaluated separately in AA and EA families.
Mutation Detection
Sequencing of TRAR4 was performed on an ABI 3100
Genetic Analyzer. Purified PCR products from various
amplicons of relevant genomic DNA fragments wereused as templates in sequencing reactions with the chemistry of BigDye 3.1 (Applied Biosystems). PCR primers
were designed by Primer3 (Rozen and Skaletsky 2000)
and were also used as sequencing primers for forward
and reverse sequencing. The primer sequences and product sizes are given in table A3 (online only). We used
the software SeqScape, version 2.1 (Applied Biosystems),
to assist in mutation detection, and we visually verified
each mutation. The reference sequence of TRAR4 usedin the analysis was from the University of California–
Santa Cruz (UCSC) human genome draft, July 2003
freeze (see the UCSC Genome Bioinformatics Web site).
For the nonhuman primates, DNAs were extracted
from peripheral blood samples of two different chimpanzees (PTR-S109 and PTR-S286) from West Africa andfrom tissue samples of two different lowland gorillas(GGO-S110 and GGO-S249). The forward primer for
amplicon 1 and the reverse primer for amplicon 7 wereused to PCR-amplify the entire DNA segment by standard
methods, with an annealing temperature of 60⬚C. This
product was then sequenced bidirectionally with the sevenprimer pairs that are detailed in table A3 (online only).
PCR products were confirmed by 1.5% agarose gel electrophoresis and were purified using MicroSpin Columns(Amersham Biosciences). The purified PCR products weresequenced using the BigDye Terminator Cycle Sequencing
Kit (Applied Biosystems) on an ABI Prism 377/3100 DNA
sequencer. Sequence data were assembled by the Phred/
Phrap program (Ewing et al. 1998) and also were checked
manually using the Consed program (Gordon et al. 1998).
Sequence data with reads for both strands and/or withhigh quality (130 quality value) were used and deposited into the DDBJ/EMBL/GenBank International Nucleotide Sequence Database (accession numbers AB180397–
AB180400).
RT-PCR and Real-Time PCR
Total mRNAs from various brain tissues were purchased from either BD Biosciences or Ambion. Gene expression of TRAR4 was first confirmed with general RT-

629

Duan et al.: TRAR4 Association with Schizophrenia

Table 1
Single-Marker Association Results for TRAR4
TRAR4
MARKERdbSNP
ACCESSION
NO.a

DISTANCE(bp)b

POSITION
(bp)c

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23

rs2840837
rs1361280
rs4473885
rs4085406
rs6907909
ss28447860
ss28447862
ss28447876
ss28447863
ss28447865
rs8192624
rs8192625d
ss28447866
rs7772821
ss28447871
rs4305745d
rs7745308
rs6912930
rs6903874
rs7765655
rs6937506
rs4129284
rs9321354

1,753
1,196
813
3,403
437
385
383
25
165
110
79
104
70
434
772
716
819
1,663
948
1,541
3,016
2,772
NA

132,864,153
132,865,906
132,867,102
132,867,915
132,871,318
132,871,755
132,872,140
132,872,523
132,872,548
132,872,713
132,872,823
132,872,902
132,873,006
132,873,076
132,873,510
132,874,282
132,874,998
132,875,817
132,877,480
132,878,428
132,879,969
132,882,985
132,885,757

NUCLEOTIDE AT

TRANSMISSIONSASSOCIATED
ALLELE
Allele 1 Allele 2
ALLELEFREQUENCY Observed Expected Variance
A
A
C
A
A
C
T
G
A
C
A
A
G
G
G
G
T
A
T
G
G
C
A

G
G
T
G
G
G
C
A
G
T
G
G
A
T
A
A
G
C
C
A
A
T
C

1
1
1
1
1
1
2
1
2
2
2
2
1
1
2
2
2
1
1
1
1
1
1

.281
.549
.548
.545
.636
.848
.009
.992
.008
.030
.933
.912
.989
.264
.698
.526
.071
.390
.757
.245
.736
.413
.165

95
186
205
189
193
143
8
5
10
20
86
101
12
102
185
168
30
124
204
73
199
140
88

93.60
175.93193.60178.60181.03132.20
6.50
4.17
8.17
17.63
83.12
91.53
9.30
96.30
179.67143.42
26.87
119.78180.5771.65
177.07131.98
83.43

46.41
72.91
82.47
80.13
80.69
37.38
3.25
.36
3.36
21.73
20.67
21.15
2.69
45.90
48.99
59.01
16.66
60.11
60.40
39.31
61.65
62.72
37.66

Z

P

.21
1.18
1.26
1.16
1.33
1.77
.83
1.39
1.00
.51
.63
2.06
1.65
.84
.76
3.20
.77
.54
3.02
.22
2.79
1.01
.74

.84
.24
.21
.25
.18
.08
.41
.17
.32
.61
.53
.0396
.10
.40
.45
.0014
.44
.59
.0026
.83
.0052
.31
.46

a

Previously known SNPs are indicated by an rs number; novel SNPs are indicated by an NCBI Assay ID number (ss number) that we obtained(data to be released in the next build of dbSNP).
b
Distance is the number of bp to the next SNP. NA p not applicable.
c
Position in bp was derived from UCSC July 2003 freeze of chromosome 6 (see the UCSC Genome Bioinformatics Web site).
d
TRAR4 screening SNPs.

PCR with primer pairs used previously for amplificationof segment 4 of TRAR4 (table A3 [online only]). In brief,
total mRNA was reverse-transcribed with TaqMan Reverse Transcription Reagents (Applied Biosystems), andthe synthesized first-strand cDNAs were then used as
templates to amplify TRAR4 with HotStarTaq polymerase (Qiagen). b-actin was used as an internal control
in the RT-PCR.
Reverse-transcribed cDNAs were also used in realtime PCR on an ABI Prism 7900 Sequence Detection
System, in accordance with the manufacturer’s protocol.
The TaqMan MGB probes and PCR primer pairs forthe gene-expression assay of TRAR4, GAPD, (glyceraldehyde-3-phosphate dehydrogenase), and/or TRAR1
were purchased as an Assay-On-Demand from Applied
Biosystems. The relative gene expression in different
brain tissues was normalized to GAPD expression byuse of the standard-curve method, as described by Applied Biosystems.
Bioinformatics Tools for Prediction of Functional Effectsof Genetic Polymorphisms
We used SIFT and PolyPhen to predict the potentialfunctional effect of missense polymorphisms (Ramensky
et al. 2002; Ng and Henikoff 2003). We used VISTA,

which defines the conserved region among genomic sequences from different species (Couronne et al. 2003),
to predict the potential regulatory sequences.
Results
We have studied 192 families with previous evidence of
linkage. Of the 33 SNPs initially selected for study, 31
were accepted for analysis in the screening experiment,
since 2 of the MOXD1 SNPs, rs2206064 and rs7751860,
had minor-allele frequencies resulting in !3% informative
families. The screening SNPs spanned ∼500 kb of the
MOXD1-STX7-TRARs gene cluster, which contains aprime set of positional and pathophysiological candidates
for schizophrenia susceptibility. We selected at least one
common SNP for each gene, with a minimum coverageof ⭓1 SNP/20 kb (the screening set of SNPs). Linkage
analyses confirmed the presence of excess allele sharing
in this region with individual SNPs from the MOXD1STX7-TRARs gene cluster. Nine SNPs showed linkage P
values !.05 (table A4 [online only]), with the most significant one being rs6937506 (TRAR4), with a LOD scoreof 1.76 (P p .002).
Association results are presented in table 1 and canalso be seen in table A5 (online only). Of 31 screening

630

Am. J. Hum. Genet. 75:624–638, 2004

SNPs, 4 SNPs spanning 106 kb, 2 located in TRAR4
and 1 each in STX7 and GPR57, yielded a P value !.05.
The most significant one, rs4305745, was found in
TRAR4 (P p .0014) (fig. 1c). This SNP was the only
one that remained significant after Bonferroni correction for 31 tests (it should be noted that the questionof whether Bonferroni corrections are appropriate or
overly conservative is open to different interpretations;however, given that LD is likely to be present over shortphysical distances, the tests cannot be considered independent). The SNP rs4305745 is located 1,214 bp
downstream from the stop codon of TRAR4. We concentrated further laboratory efforts on TRAR4. We
aimed for a high-density map of 11 SNP/2 kb by searching public SNP databases and sequencing genomic
DNA. We sequenced the TRAR4 gene (∼1 kb of the 5
region, the 1,038-bp CDS (coding DNA sequence), and∼1.5 kb of the 3 UTR, which spans rs4305745) in 30
probands selected from the NIMH-GI families: 16 EA
families and 14 AA families. We found 10 coding variants (26 total variants [table 2 and online-only tableA6]) by sequencing TRAR4. Three of these variants had
previously been found in 96 healthy EA individuals(Freudenberg-Hua et al. 2003). Of the seven novel variants, five are missense and are present only in AA individuals, as shown in table 2. From this combined
bioinformatics and sequencing effort, 20 SNPs spanning21.6 kb of the TRAR4 gene (11 SNP/2 kb) were identified for genotyping. Two additional markers 3 to
rs4305745 showed association with schizophrenia in

the whole sample (table 1 and online-only table A7):
rs6903874 (P p .0026) and rs6937506 (P p .0052).
Two other additional markers (ss28447873 [an insertion/deletion polymorphism] and rs7452939) were
found to be in perfect LD with rs4305745 (they span
53 bp) and, hence, were also associated with illness tothe same degree as rs4305745.
Although similar association trends were observed in
EA and AA individuals, table A7 (online only) showsthat the evidence for association was proportionallystronger in the AA sample. The evidence for association
with rs4305745 and rs6937506 was significant in both
samples (P p .035 in the AA sample and P p .015 in
the EA sample for rs4305745; P p .025 in the AA sample and P p .035 in the EA sample for rs6937506).
However, a cluster of three SNPs in the promoter regionof TRAR4 yielded significant association in the AA sample—rs4473885 (P p .032), rs4085406 (P p .047),
and rs6907909 (P p .019)—but not in the EA sample(table A7 [online only]). These differences raise the possibility of allelic heterogeneity specific to the AA population. All new missense mutations detected by sequencing were also exclusive to the AA sample (table2). Two mutations, A518G (Tyr173Cys) and C683T(Ala228Val), were predicted by either PolyPhen or SIFT
(Ramensky et al. 2002; Couronne et al. 2003; Ng andHenikoff 2003) to be nonconservative (table 2). However, after genotyping the whole sample, we noted thatnone of the newly found missense SNPs cosegregatedwith disease in a specific manner (data not shown). Fur-

Table 2
Coding SNPs Captured by Resequencing TRAR4 in 30 Probands with Schizophrenia (14 AA and 16 EA Individuals)

dbSNP
ACCESSION NO.ars8192622
ss28447862
ss28447876
ss28447863
ss28447864
ss28447865
ss28447867
rs8192624
rs8192625
ss28447866
a

POSITION
(bp)b

SNPc

AMINO
ACID
CHANGE132,872,108
132,872,140
132,872,523
132,872,548
132,872,660
132,872,713
132,872,774
132,872,823
132,872,902
132,873,006

C78T
T110C
G493A
A518G
C630G
C683T
A744G
G793A
G872A
G976A

Pro26Pro
Ile37Thr
Gly165Ser
Tyr173Cys
Thr210Thr
Ala228Val
Arg248Arg
Val265Ile
Cys291Tyr
Val326Ile

MINOR-ALLELE
FREQUENCY

TYPE OFCHANGEFUNCTIONd

IN PROTEIN

LOCATION

Average

EAf

AAg

Synonymous
Conservative
Nonconservative
Nonconservative
Synonymous
Conservative
Synonymous
Conservative
Nonconservative
ConservativeNA
Tolerated
Tolerated
Damaging
NA
Damaging
NA
Tolerated
Tolerated
Tolerated

Extracellular
TMD 1
TMD 4
Extracellular
TMD 5
Cytoplasmic
Cytoplasmic
TMD 6
TMD 7
Cytoplasmic

.05
.03
.02
.03
.03
.10
.02
.10
.13
.03

.09
.00
.00
.00
.03
.00
.00
.16
.16
.00

.00
.07
.04
.07
.04
.21
.04
.04
.11
.07

e

Previously known SNPs are indicated by an rs number; novel SNPs are indicated by an NCBI Assay ID number (ss number) that
we obtained (data to be released in the next build of dbSNP).
b
Position in bp was derived from the UCSC July 2003 freeze of chromosome 6 (see the UCSC Genome Bioinformatics Web site).
c
SNPs were named according to their relative position to the first letter of start codon ATG and with the first base as the major
allele.
d
Function determined by SIFT. NA p not applicable.
e
The position in the protein was predicted on the basis of 7-transmembrane GPCR structures, as depicted in the GPCR database(see GPCRDB Web site). TMD p transmembrane domain.
f
EA p European ancestry.
g
AA p African American.

Duan et al.: TRAR4 Association with Schizophrenia

thermore, all the missense variants, except for A518G(Tyr173Cys), were also found in a set of 48 AA subjects
from the Coriell Human Variation AA DNA panel (table
A8 [online only]). Additionally, some of these missense
variants were homozygous in some control individuals.
It is interesting to note that the ratio of missense tosynonymous mutations (9:3) is close to what is predicted under neutral expectations (i.e., a pseudogenethat has an expected ratio close to 4:1). Indeed, in thegorilla, TRAR4 has already become a pseudogene (witha nonsense mutation at codon 15: Tyr in chimpanzeeand human and STOP in gorilla), although the humanand chimpanzee TRAR4 versions have not yet become
pseudogenes.
The TRAR4 region was found to have two LD blocks,
which are depicted in figure 2. The SNP rs4305745(marker 16 in fig. 2) is in the LD block constituted by
3-flanking SNPs. The pattern suggests that association
for TRAR4 originates from rs4305745. None of the 5flanking SNPs are in LD with rs4305745, which instead

Figure 2631

is in strong LD with markers 19 (rs6903874) and 21(rs6937506) from the 3 LD block (and also shows atrend with marker 12 [rs8192625]). The LD patterngenerated from the 31 initial screening markers indicated that the whole region of the MOXD1-STX7TRARs gene cluster is separated into four major strongLD blocks, whereas the TRAR4 region represented by
rs4305745 is not in strong LD with any of the major
LD blocks (data not shown).
We conducted haplotype association analyses with all
TRAR4 two-locus systems (n p 17, after exclusion of
5 markers with minor-allele frequencies !3%) derived
from rs4305745, which was chosen as the anchor since
it had the most significant single-locus association(P p .0014). For each two-locus system, we derived theglobal x2 value by use of only those haplotypes withfrequencies 13% (table A9 [online only]). All 17 of these
two-locus systems exhibited P values !.05 (not corrected by multiple testing), and all harbored the “A”
allele of rs4305745. None of the two-locus systems

Pairwise LD in 192 founders for the TRAR4 region. a, Relative physical position of 23 markers in the TRAR4 region (shownin table 1). The SNPs ss28447862, ss28447876, ss28447865, rs8192624, and ss28447866 were excluded in the following LD measurements
because of their low minor-allele frequencies. b, LD pattern in AA subjects (left panel) and EA subjects (right panel). The graph was generated
by GOLD (Abecasis and Cookson 2000). In each LD pattern, the D values (upper left diagonal) and the P values (lower right diagonal,
converted to log P values) are calculated from the program ldmax of the GOLD package (Abecasis and Cookson 2000).

632

showed association stronger than that of rs4305745
alone (table A9 [online only]), and the same results wereobtained with up to five multilocus systems, with eachhaplotype system extended stepwise to contain the mostsignificant previous smaller haplotype (data not shown).
This suggests that rs4305745 and/or the other two
nearby polymorphisms, ss28447873 and rs7452939, in
perfect LD (further confirmed by genotyping the wholesample) with rs4305745 (table A6 [online only]) are themost likely mutations underlying the association of theTRAR4 region with schizophrenia susceptibility.
To explore the possible functional effects of associated SNPs and their haplotypes, we first defined theconserved noncoding sequence (considered as a potential functional region) by comparative genomic analysisof TRAR4 genomic sequences of human, mouse, andrat by use of VISTA (Couronne et al. 2003). The clusterof three polymorphisms (rs4305745, ss28447873, andrs7452939—all equally implicated as candidates by the
association analysis) that exhibit the most significant
association is very close to two conserved regions rightafter the stop codon (sequence identity 170% among
human, mouse, and rat genomes). The sequence identity
immediately around this SNP (rs4305745) is ∼50% (fig.
A1 [online only]), suggesting that this SNP and/or otherpolymorphisms in perfect LD with rs4305745 may ultimately affect gene expression, a hypothesis we are currently testing. RT-PCR from various brain tissues also
confirmed that rs4305745 was flanked by the 3 UTR
of TRAR4 (data not shown), suggesting that rs4305745
or one of its haplotypes may affect gene expression at
the posttranscriptional level. Another significant SNP,
rs6903874, is also within a conserved region (fig. A1[online only]), and it is possible that this SNP and/or
other SNPs nearby might be functional, although thisSNP is much farther than rs4305745 from the stop codon of TRAR4.
A comprehensive gene-expression analysis of TRAR4
will help to elucidate its potential functional roles in thepathophysiology and pharmacology of schizophrenia.
We investigated TRAR4 expression in various humantissues by RT-PCR and found that TRAR4 was expressed at low abundance in various human brain tissues as well as in human fetal liver but not in the cerebellum or placenta (fig. 3a). A quantitative real-time
PCR, shown in figure 3b, further revealed that TRAR4
has comparable levels of expression in basal ganglia,
frontal cortex, substantia nigra, amygdala, and hippocampus, with the highest expression in hippocampus
and the lowest expression in basal ganglia. These results
are consistent with a previous expression study that included TRAR4 (Borowsky et al. 2001). These regions
have been implicated in the pathophysiology and pharmacology of schizophrenia (Grossberg 2000; Freedman
2003). The tissue distribution of TRAR4 gene expres-Am. J. Hum. Genet. 75:624–638, 2004

Figure 3
Expression pattern of TRAR4 in human tissues. a,
TRAR4 expression pattern in various human brain regions. Lane 1 is
a 100-bp molecular weight standard ladder (Promega). Lanes 2–13
are human brain, human fetal brain, cerebellum, fetal liver, placental,
spinal cord, control (no reverse transcriptase added), basal ganglia,
frontal cortex, substantia nigra, amygdala, and hippocampus. RT-PCR
products from total RNAs are displayed in the photograph of theethidium bromide–stained agarose gel; b-actin was used as internal
control. b, Quantitative real-time PCR determined the relative abundance of the TRAR4 transcript in various human brain regions. c,Comparison of gene expression of TRAR4 with TRAR1. Samples S1–
S6 in panels b and c are basal ganglia, frontal cortex, substantia nigra,
amygdala, hippocampus, and cerebellum.

sion is similar to that of the only well-characterized
TRAR, TRAR1 (Borowsky et al. 2001; Bunzow et al.
2001). However, further comparison of gene expressionof TRAR4 and TRAR1 indicated that TRAR4 is, overall, more abundant than TRAR1, particularly in basal
ganglia (∼14-fold), frontal cortex (∼21-fold), and substantia nigria (∼14-fold) (fig. 3c), which suggests that

Duan et al.: TRAR4 Association with Schizophrenia

TRAR4 may play a more important role than TRAR1
in those regions.
Discussion
TA receptor genes have been proposed as candidate
genes for schizophrenia on the basis of highly provocative pathophysiological evidence (Boulton 1980; Premont et al. 2001; Branchek and Blackburn 2003) as wellas linkage mapping data (Cao et al. 1997). Here, we
present preliminary evidence that TRAR4, a gene thatbelongs to the TRAR family, contributes to susceptibility
to schizophrenia in three data sets, with evidence of genetic linkage to 6q. Furthermore, the TRARs gene cluster
at chromosome 6q23 is contained within a wide area oflinkage detected in several other clinical samples (Bailer
et al. 2000; Levinson et al. 2000; Lindholm et al. 2001;
Lerer et al. 2003; Lewis et al. 2003). The linkage evidence for schizophrenia in 6q is not population specific—it has been gathered from multiple populationgroups: AA individuals, EA individuals, and Jews andArabs from Israel. However, the evidence for associationof TRAR4 in our samples, although present in both EA
and AA individuals, appears higher in AA subjects.
The most significantly associated SNPs within
TRAR4 are located in the 3 UTR of TRAR4, and thesepolymorphisms may affect the gene expression at the
posttranscriptional level. There are several possible
mechanisms by which TRAR4 could contribute to thepathogenesis of schizophrenia. First, the to-be-determined ligand(s) of TRAR4 may be neurotransmitter(s)specifically implicated—by a new signaling pathway—inschizophrenia. Second, TRAR4 may be activated strongly
by b-PEA, an endogenous chemical analogue of amphetamine—or perhaps by amphetamine itself. It has
been documented that amphetamine can produce a paranoid schizophrenia syndrome in humans by inducing
DA release in the striatum, and the DA release inducedby amphetamine (or b-PEA) is increased in patients withschizophrenia (Laruelle et al. 2003). Pharmacological
profiling has shown that b-PEA and amphetamine candirectly activate TRAR1 (Borowsky et al. 2001; Bunzow
et al. 2001). Finally, TRAR4 may crosstalk with the
COMT system, which has been implicated in the pathogenesis and etiology of schizophrenia (Li et al. 2000;
Egan et al. 2001; Shifman et al. 2002). It has been shownthat rat TRAR1 can be activated with higher potency
and efficacy by 3-methyltyramine, a major metabolite of
DA produced by COMT, than by the precursor catecholamines (Bunzow et al. 2001). If TRAR4 were shown to
act like TRAR1 in this regard, some “inactive” catecholamine metabolites might act as endogenous agonists of
TRAR4, which could predispose to psychosis. Moreover,
we have shown that TRAR4 is more abundant than
TRAR1 (fig. 3), which suggests that TRAR4 may play a633

more important role in areas of the brain that are relevantto the dopaminergic system. It can be seen that the plausible contributions of TRAR4 to schizophrenia susceptibility are closely related to the DA hypothesis of schizophrenia, a major hypothesis that has been intensively
researched over the past 50 years. Given the increased
understanding about the interactions of the DA systemwith the glutamate system in the pathogenesis of schizophrenia (Laruelle et al. 2003), our finding about TRAR4
may prompt new investigations of the interactions among
these neurotransmitter systems.
Regulatory sequence disruption can affect protein expression and cause disease (Mitchison 2001). The associated SNPs in the 3 UTR of TRAR4 may contributeto the susceptibility for schizophrenia by affecting thegene expression at the posttranscriptional level. Our RTPCR experiment indicated that the TRAR4 3 UTR
spanned the most associated SNP, rs4305745; therefore,
it is possible that TRAR4 gene expression was affected
at the posttranscriptional level by these 3 UTR SNPs
(rs4305745 and/or ss28447873 and rs7452939 [twoSNPs in perfect LD with rs4305745]). The chimpanzeeand gorilla sequencing results indicated that the ancestralallele for rs4305745 is A (table A6 [online only]). Becauseof the perfect LD among rs4305745, ss28447873, andrs7452939, the associated allele A of rs4305745 actually represents a human haplotype of A-A-A, spanning
rs4305745, ss28447873, and rs7452939 (fig. A1c [online
only]). (Also note that the ancestral haplotype appears
to be A-A-G, as seen in table A6 [online only].) It isinteresting that the predicted TRAR4 mRNA structure
exhibited a significant change for the overtransmitted
haplotype A-A-A, compared with haplotype G-del-G; thesame structure change can be generated by allele A of
rs4305745 alone (data not shown), suggesting thatrs4305745 is most likely the causative SNP.
We have found that the mutation rate in the coding
region for TRAR4 (1 mutation/100 bp) is well above
the average (1 mutation/346 bp) (Cargill et al. 1999).
In addition, there are more missense mutations in
TRAR4 than synonymous mutations (nine vs. three)(table 2 and online-only table A8), suggesting that
TRAR4 may be becoming a pseudogene. However, an
expressed pseudogene sometimes regulates the mRNAstability of a homologous gene (Hirotsune et al. 2003).
We did not find evidence of association with schizophrenia for missense SNPs; however, some missense mutations—particularly A518G (Tyr173Cys)—may bepharmacologically important. A518G is located in theputative extracellular domain of the receptor and,hence, may affect ligand binding (table 2). Besides thechanges in protein structure, these missense mutations
may also alter the gene expression by affecting mRNAfolding structures, as described for dopamine D2 receptor (DRD2) (Duan et al. 2003). Actually, A518G

634

was predicted to have a remarkable effect on TRAR4mRNA folding, as predicted in silico by Mfold (Zuker
et al. 1999) (data not shown). It is also notable that
this missense SNP and several others were only foundin AA subjects, and 173Cys was only detected in AA
probands with schizophrenia. It will be important toexamine the functional effects of some of these missense
SNPs and to test the association with schizophrenia in
additional samples.
Molecular genetics studies of schizophrenia havefound several replicated linkages to various chromosomal regions (see review by Owen et al. [2004]), andassociation studies have recently pointed to several
genes at some of those linkage regions with independentconfirmations, including NRG1 at 8p21-p12 (Stefansson et al. 2002, 2003; Williams et al. 2003), DTNBP1
at 6p22.3 (Straub et al. 2002; Schwab et al. 2003),
COMT at 22q11.21 (Li et al. 2000; Egan et al. 2001;
Shifman et al. 2002), RGS4 at 1q23.3 (Chowdari et al.
2002; Morris et al. 2004; Williams et al. 2004), and
DAOA at 13q33.2 (Chumakov et al. 2002; Schumacher
et al. 2004). Although nonreplications have been reported, it would be extremely unlikely that all the aforementioned results will end as false positives. We now
propose that TRAR4 is also a susceptibility gene forschizophrenia. There are also two other weak association peaks at the MOXD1-STX7-TRARs gene cluster,in addition to the TRAR4 peak shown in figure 1: one
is in STX7 and the other is in the pseudogene, GPR5.
Therefore, we cannot exclude the possibility that thoseloci may also contribute to susceptibility for schizophrenia. The identification of TRAR4 as a susceptibility
gene for schizophrenia, which is consistent with humanand animal models of toxic psychosis and is in agreement with the expression pattern of TRAR4 (expressedin frontal cortex, amygdala, and hippocampus), appears
to substantiate the dopaminergic hypothesis of schizophrenia, but the exact mechanisms of disease mediatedby TRAR4 remain to be elucidated. The two reportssuggesting linkage of the same 6q chromosomal area tobipolar disorder raise the possibility that TRAR4 might
be involved in the pathophysiology of both schizophrenia and bipolar disorder, and there is a precedent for agene potentially involved in both disorders (Chumakov
et al. 2002; Hattori et al. 2003; Chen et al. 2004; Schumacher et al. 2004). We would like to note a couple ofcaveats: first, our combined sample has a rather modest
size in the context of complex genetics, and, second,
additional gene(s) in the region might account for our
association signal. However, although it is certainly truethat gene-prediction programs have their limitations
and different methods sometimes produce different results, at least one of them has been able to pick up all
the TRARs genes and pseudogenes we know about (seethe UCSC Genome Bioinformatics Web site [July 2003

Am. J. Hum. Genet. 75:624–638, 2004

freeze]). Although it is possible that gene-prediction
programs have missed a G-protein–coupled receptor(GPCR) pseudogene, there is little chance they havemissed an actual GPCR gene in this cluster since the
GPCR motif is so robust. Other non-GPCR genes, however, might have been missed. Although the identification of TRAR4 as a susceptibility gene for schizophrenia
is encouraging, it is important that our observations
should be considered as preliminary until replication has
been completed in additional samples.

Acknowledgments
We thank the patients and families for their participation. We
thank Dr. Josep M. Comeron for helpful comments and suggestions regarding the evolutionary context of TRAR4. The data
and biomaterials for the NIMH-GI families were collected inthree projects. From 1991 to 1997, the Principal Investigators
and Co-Investigators were as follows: Ming T. Tsuang, M.D.,
Ph.D., D.Sc., Stephen Faraone, Ph.D., and John Pepple, Ph.D.
(Harvard University, Boston [U01 MH46318]); C. Robert Cloninger, M.D., Theodore Reich, M.D., and Dragan Svrakic, M.D.
(Washington University, St. Louis [U01 MH46276]); Charles
Kaufmann, M.D., Dolores Malaspina, M.D., and Jill Harkavy Friedman, Ph.D. (Columbia University, New York [U01MH46289]). We thank Dr. Wendy Cao for providing the b-actin
primers. We also acknowledge Drs. Elliot S. Gershon and Lynn
E. DeLisi for work on recruitment of part of the sample. We
thank Kumamoto Primate Park and Ueno Zoo in Tokyo for
chimpanzee and gorilla samples, respectively. Research Career
Development Awards (to J.D. and A.R.S.) at the Evanston
Northwestern Healthcare Research Institute (Evanston, IL) supported this work.

Electronic-Database Information
Accession numbers and URLs for data presented herein are
as follows:
dbSNP Home Page, http://www.ncbi.nlm.nih.gov/SNP/ (forthe 18 novel SNPs [accession numbers ss28447859–
ss28447876]; data will become available to the public when
the National Center for Biotechnology Information releases
the latest dbSNP build, and, at that time, the accession numbers will be converted into reference cluster ID numbers)
GenBank, http://www.ncbi.nlm.nih.gov/Genbank/ (for the 18
novel SNPs [accession numbers BV154568–BV154585] andgorilla and chimpanzee TRAR4 region sequences [accession
numbers AB180397–AB180400])
G-protein–coupled receptors database (GPCRDB), http://www.gpcr.org/
Mfold, http://www.bioinfo.rpi.edu/applications/mfold/ (forRNA structure prediction)
National Institute of Mental Health (NIMH) Schizophrenia
Genetics Initiative Data Archive, http://zork.wustl.edu/nimh/sz.html (families used in this study were 30101, 30103,
30104, 30106, 30108, 30110, 30111, 30112, 30113,
30114, 30116, 30119, 30122, 30123, 30124, 30126,

Duan et al.: TRAR4 Association with Schizophrenia

30127, 30128, 30130, 30131, 30132, 30133, 30134,
30136, 30140, 30142, 30146, 31102, 31107, 31108,
31109, 31114, 31115, 31118, 31119, 31129, 31130,
31135, 31137, 31139, 31155, 32108, 32109, 32200,
32201, 32202, 32203, 32204, 32205, 32206, 32209,
32211, 32212, 32217, 32218, 32303, 32304, 32306,
32307, 32309, 32310, 32311, 32312, 32313, 32315,
32319, 32320, 32402, and 32403)
Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for SCZD5)PolyPhen, http://tux.embl-heidelberg.de/ramensky/
SIFT, http://blocks.fhcrc.org/sift/SIFT.html
SNPper, http://snpper.chip.org/
University of California at Santa Cruz (UCSC) Genome Bioinformatics, http://genome.ucsc.edu/ (for July 2003 assemblyof the human genome)
VISTA, http://www-gsd.lbl.gov/vista/

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