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Association of AKT1 haplotype with the risk of schizophrenia in Iranian population.

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American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 141B:383 –386 (2006)
Association of AKT1 Haplotype With the Risk of
Schizophrenia in Iranian Population
Sepideh N. Bajestan,1 Amir H. Sabouri,1 Masayuki Nakamura,2 Hiroshi Takashima,1 Mohammad R. Keikhaee,3
Fatemeh Behdani,4 Mohammad R. Fayyazi,4 Mohammad R. Sargolzaee,4 Mahboobeh N. Bajestan,5
Zahra Sabouri,5 Esmaeil Khayami,6 Sima Haghighi,6 Susan B. Hashemi,3 Nobutaka Eiraku,7
Hamid Tufani,4 Hossein Najmabadi,3 Kimiyoshi Arimura,1 Akira Sano,2 and Mitsuhiro Osame1*
1
Departments of Neurology and Geriatrics, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
Department of Neuropsychiatry, Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima, Japan
3
Genetics Research Center, Social Welfare and Rehabilitation Sciences University, Tehran, Iran
4
Department of Psychiatry, Mashhad University of Medical Sciences, Mashhad, Iran
5
Deputy of Research, Mashhad University of Medical Sciences, Mashhad, Iran
6
Khorasan Blood Transfusion Center, Mashhad, Iran
7
Kagoshima University Health Service Center, Kagoshima, Japan
2
AKT-glycogen synthase kinase 3b (GSK3b) signaling is a target of lithium and has been implicated
in the pathogenesis of mood disorders and schizophrenia. AKT1 protein level is decreased in the
peripheral lymphocytes and brains of schizophrenic patients. The SNP2/3/4 TCG haplotype of AKT1
was associated with schizophrenia in patients
with Northern European origin. In the present
study, we genotyped five single nucleotide polymorphisms (SNP1–5) of AKT1 gene according to
the original study in Iranians comprising of 321
schizophrenic patients and 383 controls, all residing in Mashhad city, Northeastern Iran. Haplotype analysis showed that the frequency of a fiveSNP haplotype (AGCAG) was significantly higher
in schizophrenic patients (0.068) than that of
controls (0.034) (P ¼ 0.03 after Bonferroni correction, OR ¼ 2.04, CI ¼ 1.2–3.4). In stratified analysis
by schizophrenia subtypes, the frequency of the
same haplotype was significantly higher in disorganized subtype (n ¼ 78, frequency of haplotype¼0.081) when compared with normal
controls (P ¼ 0.04 after Bonferroni correction,
OR ¼ 2.59, CI ¼ 1.3–5.2). Our findings did not confirm the association of AKT1 SNP2/3/4 TCG haplotype with the risk of schizophrenia as reported in
the original study but showed the evidence of
association with a different haplotype, AKT1 fiveSNP AGCAG haplotype, with the risk of schizophrenia in Iranian population.
ß 2006 Wiley-Liss, Inc.
KEY WORDS:
AKT1; schizophrenia; Iranians;
risk; haplotype
Please cite this article as follows: Bajestan SN, Sabouri
AH, Nakamura M, Takashima H, Keikhaee MR, Behdani
F, Fayyazi MR, Sargolzaee MR, Bajestan MN, Sabouri Z,
*Correspondence to: Mitsuhiro Osame, Departments of Neurology and Geriatrics, Kagoshima University Graduate School of
Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima
890-8520, Japan. E-mail: osame@m2.kufm.kagoshima-u.ac.jp
Received 15 May 2005; Accepted 6 December 2005
DOI 10.1002/ajmg.b.30291
ß 2006 Wiley-Liss, Inc.
Khayami E, Haghighi S, Hashemi SB, Eiraku N, Tufani
H, Najmabadi H, Arimura K, Sano A, Osame M. 2006.
Association of AKT1 Haplotype With the Risk of
Schizophrenia in Iranian Population. Am J Med Genet
Part B 141B:383–386.
INTRODUCTION
The AKT (murine thymoma viral (v-akt) oncogene homolog),
also termed protein kinase B (PKB) or RAC protein kinase, is a
serine and threonine protein kinase homologous to protein
kinase A and C [Datta et al., 1999; Vanhaesebroeck and Alessi,
2000]. AKT-glycogen synthase kinase 3b (GSK3b) signaling is
known as a target of lithium and has been implicated in the
pathogenesis of dopamine-related disorders like schizophrenia
[Beaulieu et al., 2004]. Three mammalian isoforms (AKT1–3)
have been cloned. AKT1 is a downstream target of phosphatidylinositol-3-kinase pathway [Franke et al., 1995]. Modulation of AKT1 activity plays a key role in the mechanism of
glutamate excitotoxicity and in lithium and olanzapine
neuroprotection [Chalecka-Franaszek and Chuang, 1999; Lu
et al., 2004].
Recently, several lines of evidence have suggested that
AKT1 has important roles in neurodevelopment [Wang et al.,
2003] and in working memory formation [Mizuno et al., 2003],
abnormalities of which are hypothesized to be susceptibility
factors in schizophrenia. GSK3b mRNA levels were reduced in
the dorsolateral prefrontal cortex of schizophrenic patients
[Kozlovsky et al., 2004]. Emamian et al. [2004] also showed
lower levels of AKT1 protein and phosphorylation of GSK3b at
Ser9 in the peripheral lymphocytes and brains of schizophrenic
patients. Based on typing of five single nucleotide polymorphisms (SNP1–5), AKT1 SNP2/3/4 TCG haplotype showed the
highest significant association with schizophrenia. AKT1 core
risk haplotype (SNP2/3 TC) was associated with lower AKT1
protein levels. A Japanese study found the association between
schizophrenia and SNP5 of AKT1 gene [Ikeda et al., 2004].
However, another Japanese replication study failed to confirm
any association of AKT1 SNPs and haplotypes with schizophrenia [Ohtsuki et al., 2004].
The aim of our study was to investigate the association of
AKT1 gene polymorphisms and haplotypes with schizophrenia
in Iranian schizophrenic patients and normal controls residing
in Mashhad city, in Northeastern Iran. We genotyped five
SNPs (SNP1–5) located on AKT1 gene in order to replicate
the original study [Emamian et al., 2004] and studied the
association of AKT1 gene SNPs and haplotypes with schizophrenia and schizophrenia subtypes in Iranian population.
384
Bajestan et al.
TABLE I. Primers and Restriction Enzymes Used for Restriction Fragment Length Polymorphism Analysis of AKT1 Gene Single
Nucleotide Polymorphisms
SNP namea
(dbSNP ID)
SNP1
(rs3803300)
SNP2
(rs1130214)
SNP3
(rs3730358)
SNP4
(rs2498799)
SNP5
(rs2494732)
Primer ID
Primer sequence
Product size
(bp)
Restriction
enzyme
Enzyme-digested
fragments (bp)
640
Bsal
431,209
248
XcmI
157,91
107
HaeIII
66,41
599
AflIII
415,184
600
PvuII
421,179
50 -CCTGGGGCACAGTGAGTG-30
50 -GTTCGAGGAGCGTCAGTCTC-30
50 -CTGACGGACTTGTCTGAACC-30
50 -GCAAAGAAATTCAAACATGAGG-30
50 -GCAGGATGTGGACCAACG-30
50 -AGAGGGCTCCAGCCAACC-30
50 -TACTCTTTCCAGACCCACGA-30
50 -TCTCCAGCTAGGGGAAAGGT-30
50 -GACAGAGGCCCAACTGACC-30
50 -CGGCACACCTGAGTACCTG-30
SNP1-F
SNP1-R
SNP2-F
SNP2-R
SNP3-F
SNP3-R
SNP4-F
SNP4-R
SNP5-F
SNP5-R
F, forward; R, reverse.
a
SNP names are according to Emamian et al. [2004] study.
MATERIALS AND METHODS
Subjects
Study population consisted of 321 unrelated schizophrenic
patients (mean age: 42.5 years) and 383 unrelated healthy
controls (mean age: 35.8 years) all residing in Mashhad city, in
Northeastern Iran. Diagnoses were determined independently
by two psychiatrists according to DSM-IV schizophrenia
criteria. Schizophrenic patients were grouped according to
the following DSM-IV subtypes of schizophrenia: paranoid
(n ¼ 130), disorganized (n ¼ 78), residual (n ¼ 74), undifferentiated (n ¼ 38), and catatonic (n ¼ 1). Written informed consent
was obtained from all subjects. This study was approved by the
Ethics Committees of Ibne-Sina Hospital and Mashhad
University of Medical Sciences, Mashhad, Iran.
In order to eliminate the genotyping errors, all raw genotyping
data were independently read by two researchers and the
suspect genotypes were retyped. Furthermore, for each SNP, a
random group of samples were also re-genotyped by direct
sequencing to confirm the genotyping results of restriction
fragment enzyme method.
Data Analysis
DNA Preparation and Genotyping
Only haplotypes with a frequency >3% were retained in all
analysis. Bonferroni correction was applied for all significant P
values. Pairwise linkage disequilibrium (LD) was calculated
with Haploview program (http://www.broad.mit.edu/personal/
jcbarret/haploview/). Hardy–Weinberg equilibrium, global
tests, and multilocus haplotype analyses were performed by
Arlequin software (http://lgb.unige.ch/arlequin/). P values less
than 0.05 were considered statistically significant.
All blood samples were taken by vacuum tube pre-filled with
the anticoagulant EDTA. Genomic DNA of nucleated blood
cells was isolated from whole blood using the PureGene DNA
Purification kit (Gentra Systems, Inc., Minneapolis, MN). We
genotyped five SNPs (Table I) located on AKT1 gene as
described elsewhere [Emamian et al., 2004]. Primers were
designed using the Primer3 website (http://frodo.wi.mit.edu/
cgi-bin/primer3/primer3_www.cgi). Primer sequence, PCR
product size, and corresponding restriction fragment enzyme
for each SNP are shown in Table I. Annealing temperatures
were either 558C or 608C.
The genotype frequencies of all SNPs were in Hardy–
Weinberg equilibrium. All studied SNPs showed the frequencies of more than 10% for their minor alleles, which improves
their comparability to the common disease-susceptibility
polymorphisms [Dunning et al., 2000] and the power to detect
linkage disequilibrium [Terwilliger et al., 1998] (Table II).
Pairwise LD is shown in Table II. We found no association
between each SNP and schizophrenia (Table II). The global
RESULTS
TABLE II. Pairwise Linkage Disequilibrium and Association of AKT1 Gene Single Nucleotide Polymorphisms and Haplotypes With
Schizophrenia
LDb
SNP namea
(dbSNP ID)
Global P values
Inter-SNP
distance (bp)
D0
Delta
Allele
Allele frequencyc
(Scz/Cnt)
SNP1 (rs3803300)
—
—
—
SNP2 (rs1130214)
10045
0.52
0.02
G
A
G
T
C
T
G
A
A
G
0.779/0.80
0.221/0.20
0.804/0.789
0.196/0.211
0.832/0.830
0.168/0.170
0.738/0.740
0.262/0.260
0.537/0.535
0.463/0.465
SNP3 (rs3730358)
SNP4 (rs2498799)
SNP5 (rs2494732)
a
13327
6513
702
0.44
0.84
0.9
2
0.15
0.42
0.33
SNP names are according to Emamian et al. [2004] study.
LD: linkage disequilibrium of adjacent SNPs was shown by D0 and Delta2.
Allele frequencies were shown in schizophrenic patients (Scz) and controls (Cnt).
b
c
1SNP
2SNP
3SNP
4SNP
5SNP
0.33
0.56
0.48
0.87
0.64
0.94
0.15
0.74
0.28
0.94
0.15
0.98
0.94
0.05
0.51
AKT1 Haplotype and Schizophrenia in Iranians
TABLE III. Five-SNP Haplotypes and Their Frequencies
(if >0.03) in Iranian Schizophrenic Patients and Controls
Haplotypes
AGCAG
AGCGA
AGCGG
GGCGA
GGCGG
GGTAG
GTCGG
GTTAG
Schizophrenic
patients (n ¼ 321)
Controls
(n ¼ 383)
P values
0.068
0.063
0.058
0.412
0.091
0.052
0.047
0.076
0.034
0.08
0.063
0.403
0.089
0.058
0.06
0.096
0.004
0.21
0.7
0.77
0.92
0.62
0.27
0.17
tests of association taking into account all alleles were not
significant (Table II).
In multilocus analysis, the frequency of a five-SNP1/2/3/4/5
AGCAG haplotype was significantly higher in schizophrenic
patients (0.068) in comparison with controls (0.034) (w2 ¼ 8.2,
P ¼ 0.004, OR ¼ 2.04, CI ¼ 1.2–3.4). The association remained
significant even after Bonferroni correction for the number of
haplotypes (P ¼ 0.03). The five-SNP haplotypes and their
frequencies (if the frequency >3%) are shown in Table III. In
stratified analysis by schizophrenia subtypes, the frequency of
the same haplotype (5-SNP AGCAG) was significantly higher
in disorganized (frq¼ 0.081, w2 ¼ 7.8, P ¼ 0.0052, OR ¼ 2.59,
CI ¼ 1.3–5.2) and residual (frq¼0.08, w2 ¼ 6.9, P ¼ 0.0085,
OR ¼ 2.51, CI ¼ 1.2–5.1) subtypes, when compared with controls. However, after Bonferroni correction, the association
remained significant only in disorganized subtype (P ¼ 0.04).
The reported associations of SNP2/3 TC and SNP2/3/4 TCG
haplotypes with the increased risk of schizophrenia [Emamian
et al., 2004] were not observed in our population. The frequency
of TC and TCG haplotypes in our schizophrenic patients/
controls were 0.109/0.11 and 0.087/0.096, respectively. The
powers of our study to find the increased risk of schizophrenia
were 0.9 in TCG and 0.7 in TC haplotypes.
DISCUSSION
Recent evidences have suggested that AKT1-GSK3b signaling is impaired in schizophrenic patients [Emamian et al.,
2004; Kozlovsky et al., 2004] and modulation of AKT1 activity
is important in the mechanism of lithium and olanzapine
neuroprotection [Chalecka-Franaszek and Chuang, 1999; Lu
et al., 2004]. Other studies have also shown the association of
AKT1 polymorphisms and haplotypes with the risk of schizophrenia in families with Northern European origin and
Japanese population [Emamian et al., 2004; Ikeda et al.,
2004], although it was not confirmed in another Japanese
cohort [Ohtsuki et al., 2004].
In the present study, we have found that in multilocus
analysis of AKT1 gene, five-SNP AGCAG haplotype was
significantly associated with the risk of the disease in
schizophrenic patients. The association of AKT1 AGCAG
haplotype with the risk of schizophrenia have not been
reported in previous studies [Emamian et al., 2004; Ikeda
et al., 2004; Ohtsuki et al., 2004].
Current studies have also shown the association of some
candidate genes with the schizophrenia subtypes of residual
[Kaiser et al., 2001], paranoid [Kaiser et al., 2001; Nakata et al.,
2003], and catatonic [Stober et al., 2000]. Assuming the notion
that the etiology of schizophrenia is likely to be heterogeneous
and the genetic contribution to schizophrenia varies among its
subtypes [Tsuang and Faraone, 1995; Stober et al., 2000], we
also analyzed the schizophrenia subtypes for possible associations with AKT1 SNPs and haplotypes. We found that the same
five-SNP AGCAG haplotype was significantly associated with
385
the risk of the disease in disorganized subtype of schizophrenia. Although the disease-risk association was disappeared in
some comparisons after Bonferroni correction, small sample
size might be a reason for this observation especially in
schizophrenia subtypes. As genotyping errors are potential
sources to show false haplotype frequencies, we conducted
several procedures to prevent such errors during genotyping
(see Materials and Methods) and also checked the genotype
frequencies of each SNP to be in Hardy–Weinberg equilibrium.
In concordance with the studies on Japanese population
[Ikeda et al., 2004; Ohtsuki et al., 2004], our findings do not
confirm that SNP2/3 TC of AKT1 gene is a core at risk
haplotype of schizophrenia [Emamian et al., 2004]. We could
not also replicate the significant association of AKT1 SNP 5
with the risk of schizophrenia [Ikeda et al., 2004]. Contradictory associations of SNPs and haplotypes with the risk of
schizophrenia have been also reported for other molecules in
genetically different populations [Berry et al., 2003]. Different
population histories of the studied ethnics may preclude the
confirmation of the association for the same AKT1 SNPs or
haplotypes with schizophrenia [Reich et al., 2001].
In summary, although we could not directly replicate the
results of the previous studies on the association of AKT1 gene
with the risk of schizophrenia but we showed the association of
another AKT1 gene haplotype with the risk of schizophrenia.
Therefore, our results, along with other recent reports
[Emamian et al., 2004; Ikeda et al., 2004], suggest that AKT1
gene might either play a role in predisposing to schizophrenia
or be in linkage disequilibrium with a causal locus for
schizophrenia. Although our samples were from a fairly
homogenous ethnic population, the possibility of population
stratification should be considered in interpretation of our
results.
ACKNOWLEDGMENTS
We thank the staff of the Blood Transfusion Center in
Mashhad, the personnel of the Department of Psychiatry and
Faculty of Pharmacology in Mashhad University of Medical
Sciences, and Dr. Hoda Azizi for their cooperation.
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