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Progress in Neuro-Psychopharmacology & Biological Psychiatry 34 (2010) 1412–1418
Contents lists available at ScienceDirect
Progress in Neuro-Psychop
Psychi
j ourna l homepage: www.e1. Introduction
Schizophrenia (SCZ) is a severe disabling neuropsychiatric disorder
characterized by a collection of symptoms that may include positive
symptoms such as paranoia and auditory hallucinations, negative
symptoms including thought poverty, anhedonia, and social withdraw-
al, as well as cognitive impairment. It affects approximately 1% of the
general population. Findings from family, twin, and adoption studies
support a genetic basis for SCZ (reviewed in McGuffin et al., 1984), but
its etiology is still unclear. Dopamine hypothesis of SCZ arose from the
observations that dopamine-mimetic agents including amphetamine,
induce SCZ associated symptoms, and that all antipsychoticmedications
have a certain degree of dopamine-blocking capacity.
D3 is a G-protein coupled receptor (GPCR) that, upon binding of
dopamine, transduces signal via inhibition of cyclic Adenosine Mono-
Phosphate (cAMP) synthesis. D3 is encoded by the DRD3 gene, which
is located on chromosomal region 3q13.3. It is primarily expressed in
the striatum, with the highest expression in the ventral portion. These
brain regions have been implicated in SCZ pathophysiology. Ser9Gly is
the only polymorphism that has been studied for its effects on D3
function. The Gly allele has been associated with increased binding
affinity of dopamine (Lundstrom and Turpin, 1996), as well as
increased dopamine-induced stimulation of Extracellular signal
Regulated Kinase (ERK), an important intracellular signaling molecule
involved in cell growth and differentiation, and inhibition of cAMP
synthesis (Jeanneteau et al., 2006).
Researchers found decreased DRD3 (Sokoloff et al., 1990) mRNA
expression in postmortem brain samples (Schmauss et al., 1993) and
peripheral blood lymphocytes (Vogel et al., 2004) of SCZ patients
compared to those of controls. The decrease could be due to increasedAbbreviations: SCZ, schizophrenia; DRD3, dopamine
derived neurotrophic factor gene; SNP, single-nucleo
protein coupled receptor; ERK, extracellular signal r
Adenosine Mono-Phosphate; FBAT, family based associati
⁎ Corresponding author. Neurogenetics Section, Cen
Health, Toronto, Ontario, Canada M5T 1R8.
E-mail address: james_kennedy@camh.net (J.L. Kenn
0278-5846/$ – see front matter © 2010 Elsevier Inc. Al
doi:10.1016/j.pnpbp.2010.07.019© 2010 Elsevier Inc. All rights reserved.Article history:
Received 7 May 2010
Received in revised form 12 July 2010
Accepted 19 July 2010
Available online 24 July 2010
Keywords:
Schizophrenia
Dopamine receptor DRD3
Brain-derived neurotrophic factor BDNF
Candidate gene association
Family based association studySchizophrenia (SCZ) is a severe neuropsychiatric disorder with prominent genetic etiologic factors. The
dopamine receptor DRD3 gene is a strong candidate in genetic studies of SCZ because of the dopamine
hypothesis of SCZ and the selective expression of D3 in areas of the limbic system implicated in the disease.
We examined 15 single-nucleotide polymorphisms (SNPs) in DRD3 in our sample of European origin
consisting of 95 small nuclear SCZ families and 167 case–control pairs. We also examined four BDNF SNPs in
our samples because of evidence for BDNF regulation of DRD3 expression (Guillin et al., 2001). We found a
nominally significant genotypic association with rs7633291 and allelic association with rs1025398 alleles.
However, these observations did not survive correction for multiple testing. We did not find a statistically
significant association with the other DRD3 and BDNF polymorphisms. Taken together, the results from the
present study suggest that BDNF and DRD3 may not be involved in SCZ susceptibility.splicing of the 3
leading to an in
mRNA. In contra
levels in SCZ pat
mixed results c
(Ilani et al., 20
influence of anti
have accounted
D3 receptor gene; BDNF, brain-
tide polymorphism; GPCR, G-
egulated kinase; cAMP, cyclic
on test; CI, confidence interval.
tre for Addiction and Mental
edy).
l rights reserved.a r t i c l e i n f o a b s t r a c tAssociation study of BDNF and DRD3 genes
case–control and family based study desi
Clement C. Zai a,b, Mirko Manchia a,c, Vincenzo De Lu
Gwyneth C. Zai a, John Strauss a, Sajid A. Shaikh a, Nata
Bernard Le Foll b,f, James L. Kennedy a,b,⁎
a Neurogenetics Section, Centre for Addiction and Mental Health, Toronto, Ontario, Canada
b Department of Psychiatry, University of Toronto, Toronto, Ontario, Canada M5T 1R8
c Laboratory of Molecular Genetic, Department of Neurosciences “B.B.Brodie”, University o
d Psychiatric Hospital at Vanderbilt University, Nashville, TN, USA
e New York State Psychiatric Institute, Columbia University Medical Centre, New York City
f Translational Addiction Research Laboratory, Addiction Program, Centre for Addiction ann schizophrenia diagnosis using matched
s
a,b, Arun K. Tiwari a, Alessio Squassina c,
Freeman a, Herbert Y. Meltzer d, Jeffrey Lieberman e,
T 1R8
gliari, Cagliari, Italy
, USA
ental Health, Toronto, Canada
harmacology & Biological
atry
l sev ie r.com/ locate /pnp′ region of the DRD3 pre-mRNA (Schmauss, 1996),
creased ratio of truncated (D3nf) to full-length (D3)
st, Ilani et al. (2001) found increased DRD3 mRNA
ients' blood lymphocytes compared to controls. The
ould be due to different housekeeping genes used
01; Vogel et al., 2004). It is noteworthy that the
psychotic medication at the time of death could also
for these discrepancies. In this regard, Gurevich et al.

s of
e w
1413C.C. Zai et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 34 (2010) 1412–1418(1997) found increased D3 levels in the ventral striatum of
antipsychotic-free SCZ patients at the time of death, but similar D3
levels in SCZ patients taking antipsychotics up to the time of death.
These results suggest that while D3 is up-regulated in SCZ,
antipsychotics down-regulate it.
In the original genetic study of DRD3 in SCZ, Crocq et al. (1992)
found Ser9Gly to be associated with SCZ using two matched case–
control samples from France and the UK. Mant et al. (1994) from the
same research group updated the positive findings with additional
subjects, with an excess of the lower functioning Ser allele and Ser/Ser
genotype in SCZ cases compared to controls. Our laboratory replicated
the original positive findings using two independent samples from
North America and Italy (Kennedy et al., 1995). The positive findings
were also replicated in other independent samples (Shaikh et al.,
1996; Spurlock et al., 1998; Ishiguro et al., 2000). However, they were
challenged by results from many other studies, including the most
Fig. 1. a. Schematic diagram of the DRD3 gene with its exons and introns. The position
Supplementary Table 1 for more details.66.85 kb. b. Schematic diagram of the BDNF gen
study are indicated within the gene. See Supplementary Table 1 for more details.recent meta-analyses (Ma et al., 2008; Utsunomiya et al., 2008).
Despite having accumulated genotype data from over 10,000 SCZ
patients across different studies, with most studies having tested only
the Ser9Gly polymorphism within this 50-kb gene, the role of DRD3 in
SCZ remains elusive. Recently, Talkowski et al. (2006) investigated 11
polymorphisms evenly distributed within and around DRD3 for
association with SCZ in two family samples (US and Indian) and a
case–control sample. They found consistent association between
Ser9Gly and SCZ in both family samples, but only against a common
haplotype background (Talkowski et al., 2006). Dominguez and cow-
orkers (2007) genotyped 17 polymorphisms spanning DRD3 and found
haplotypes in the3′portion of thegene to be significantly associated in a
Galician isolate population. These latest findings suggest that Ser9Gly
may be a marker for other polymorphism(s) that confer risk to SCZ and
encourage further more comprehensive examination of DRD3 in SCZ.
The brain-derived neurotrophic factor (BDNF) plays a critical role in
dopaminergic neuronal establishment (Baquet et al., 2005). The number
of tyrosine hydroxylase-expressing dopaminergic neuronswas reduced
in themidbrain–hindbrain regionswhere theBDNFgenewas selectively
deleted (Baquet et al., 2005). BDNF also specifically regulates the in-vivo
expression of DRD3 in thenucleus accumbens both duringdevelopment
and in adulthood (Guillin et al., 2001). Postmortem studies in the brains
of SCZ patients showed decreases in BDNF protein (Iritani et al., 2003;
Weickert et al., 2003) and mRNA levels (Weickert et al., 2003), while
Buckley et al. (2007) and Palomino et al. (2006) recently founddecreased plasma BDNF levels in first-episode patients. However,
Takahashi et al. (2000) found increased BDNF protein levels in the
hippocampus and nucleus accumbens in SCZ subjects. SCZ patients who
were chronically on antipsychotic medications showed increased
(Gama et al., 2007) or decreased (Grillo et al., 2007; Toyooka et al.,
2002) serum BDNF levels compared to healthy controls. Similar BDNF
levels between the chronically treated SCZ and controls were also
reported (Shimizu et al., 2003). The mixed results could be due to a
number of factors, including insufficient sample sizes, different patient
populations, different brain regions examined, or different antipsycho-
tics at different doses. Genetically, carriers of theBDNFMet66 allele have
lower cognitive abilities of learning and memory associated with lower
hippocampal activation compared to ValVal carriers (Egan et al., 2003).
The mechanism of action could be reduced Met66-BDNF protein
packaging and secretion from neurons compared to Val66-BDNF
(Egan et al., 2003; Chen et al., 2004). The Met66 allele has also been
the 15 polymorphisms used for the present study are indicated within the gene. See
ith its exons and introns. The positions of the four polymorphisms used for the presentassociated with hippocampal volume (Pezawas et al., 2004). Five recent
meta-analyses on Val66Met did not find a significant association with
SCZ (Naoe et al., 2007;Qian et al., 2007; Xuet al., 2007a; Kanazawa et al.,
2007; Zintzaras, 2007). The C270T polymorphism has also been
analyzed in two meta-analyses, which found weak (Zintzaras, 2007)
or no (Xu et al., 2007a) association with SCZ. However, the role of the
BDNF gene could not be excluded as Qian et al. (2007) found a four-
marker haplotype to be protective against SCZ in their Chinese sample.
These results suggest that polymorphisms other than Val66Met and
C207T may be involved in SCZ.
In the present study, we tested for the possibility of an association
of tag polymorphisms in the DRD3 and BDNF genes with SCZ. We
analyzed individual polymorphisms, as well as two-marker and three-
marker haplotypes in a sliding window approach on our Caucasian
paired case–control and small nuclear-family samples. In view of the
substantive evidence that suggests a functional relationship between
DRD3 and BDNF, we also examined their single-marker interactions
for association with SCZ.
2. Patients and methods
2.1. Subjects
The research subjects were recruited at the Centre for Addiction and
Mental Health, except for eight triads that were recruited from the