Association of AKT1 haplotype with the risk of schizophrenia in Iranian population.код для вставкиСкачать
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: firstname.lastname@example.org 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.  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.  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.  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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