An association study of ADSS gene polymorphisms with schizophrenia

Behavioral and Brain Functions An association study of ADSS gene polymorphisms with schizophrenia Fuquan Zhang 2 Pak C Sham 1 Hua Fan 0 Yong Xu 2 Xuezhu Huang 2 Honcheong So 1 Yuqing Song 1 Pozi Liu 2 0 Department of Psychiatry, Beijing Anding Hospital, Capital Medical University , Bejing , PR China 1 Department of Psychiatry, University of Hong Kong , Hong Kong , PR China 2 Institute of Neurological disorders, Tsinghua University; Department of Psychiatry, Yuquan Hospital, Tsinghua University , Bejing, 100049 , PR China Background: Adenylosuccinate synthase (ADSS) catalyzes the first committed step of AMP synthesis. It was suggested that the blood-derived RNA of ADSS was down-regulated in schizophrenia (SZ) and one of the eight putative biomarker genes to discriminate SZ from normal controls. However, it remains unclear whether the reduction of ADSS RNA is due to the polymorphisms of the gene or not. Methods: We attempted to examine the association of ADSS gene with schizophrenia in a Chinese population of 480 schizophrenics and 502 normal controls. Genotyping was performed by the Sequenom platform. Results: The 6 marker SNPs (rs3102460, rs3127459, rs3127460, rs3127465, rs3006001, and rs3003211) were genotyped. The frequencies of alleles, genotypes, and haplotypes were tested between cases and controls. There was no significant difference of genotypic, allelic, or haplotypic distributions of the 6 SNPs between the two groups. Conclusion: Our data did not support ADSS gene as a susceptibility gene for SZ in Chinese Han population. Large sample size study is needed to validate or replicate our association study, especially from other ethnic populations. - Background Schizophrenia is a complex genetic disorder characterized by profound disturbances of cognition, emotion and social functioning. The median lifetime prevalence of SZ is 0.7%0.8% [1]. The public health importance of SZ is clear. Numerous family, twin, and adoption studies showed conclusively that the risk of schizophrenia was increased among the relatives of affected individuals and that it was the result largely of genes rather than shared environment [2]. It's believed that the disease tends to run in families, with an estimated heritability of 8085% [3]. In the children and siblings of individuals with schizophrenia, the increase in risk is approximately 10-fold. Recent decades, many candidate genes have been implicated in the susceptibility of SZ with independent replicative evidences from multiple populations [2,4,5]. Following the searching of genetic basis for the mechanism of SZ, some lines of evidences have also emerged from other kinds of biomarkers, such as level of gene expression. Recent advances have facilitated the use of circulating blood to conduct genomic analyses of human disease [6,7]. Vawter et al. [8] identified nine genes that were differentially expressed between SZ patients and controls. Thereafter, by analyzing the blood-derived RNA from 74 samples, linear and non-linear combinations of eight putative biomarker genes (APOBEC3B, ADSS, ATM, CLC, CTBP1, DIDO1, CXCL1, and S100A9) were able to discriminate among schizophrenia, bipolar disorder, and control samples [9], with an overall accuracy of 95% 97%. None of these genes have yet been investigated for their association or linkage disequilibrium with SZ. Among them, ADSS is down-regulated in the SZ patients. The de novo biosynthesis of AMP from IMP involves two steps, with the first step catalyzed by ADSS followed by adenylosuccinate lyase catalyzing the second step [10]. The gene encoding ADSS maps to 1cen-q12, the chromosomal loci previously linked to schizophrenia in metaanalysis [11,12]. The sequence of ADSS gene is 44 kb in length, with 13 extrons and 12 introns. However, it is unknown whether the alteration in ADSS expression is due to defect of the gene, or secondary to other disease-related factors. In order to address this issue, and to test the hypothesis that sequence variations of ADSS gene influence the risk for the disease, we conducted a case-control association study on 6 SNPs (rs3102460, rs3127459, rs3127460, rs3127465, rs3006001, and rs3003211) within the gene in a Chinese Han Population. Methods Subjects Subjects were 480 unrelated schizophrenics and 502 healthy controls. Cases (age: 41.8 10.3) were recruited from Hong Kong hospitals. All patients were interviewed using the Structured Clinical Interview for DSM-IV (SCID) and met the DSM-IV diagnostic criteria for schizophrenia. Healthy controls (age: 41.9 9.79) were recruited from blood donors who were not screened for psychiatric diseases. However, in Hong Kong an individual would be ineligible for blood donation if he is under doctor's care, taking medications, awaiting test results or suffering from any serious illness. All subjects were Han Chinese. Peripheral blood sample were obtained from the subjects. All participants provided written informed consent. Genotyping In view of information from dbSNP [13], according to the location and the heterozygosity of SNP, we selected 6 SNPs from intron-11 (rs3102460), intron-6 (rs3127459), intron-4 (rs3127460), intron-1 (rs3127465, rs3006001 and rs3003211) to check the allelic and haplotypic association of ADSS with SZ. The average estimated heterozygosity of these SNPs is 0.37, and they span 36 Kb nucleotides in the 44 Kb pairs of ADSS. We employed a Sequenom platform (Sequenom MassARRAY system, Sequenom, San Diego CA) for assay design and genotyping. SNP sites were amplified by PCR in multiplex format in 384-microtiter plates by a pair of specifically designed forward and reverse PCR primers. The length of the amplicons for SNP capture ranged from 60 to 120 base pairs (bp). Following genomic amplification of the target regions, PCR products were treated with shrimp alkaline phosphatase for 20 minutes at 37C to dephosphorylate any residual nucleotides and to prevent their future incorporation and interference with the primer extension assay. Extension primers, DNA polymerase, and a cocktail mixture of deoxynucleotides (dNTPs) and dideoxynucleotide triphosphates (ddNTPs) were added to each mix. These were then followed by cycles of homogeneous MassEXTEND (hME) reaction probed by the extension primers for each SNP. The MassARRAY typer software version 3.1 was then used to read out the extended mass and assign the genotype call. Quality control criteria included genotype call rate of >80%, less than 1 duplicate errors (5 duplicates in each 96 well-plate), and significant Hardy-Weinberg disequilibrium. Statistical analyses Hardy-Weinberg equilibrium, genotype and allele frequencies between cases and controls for ADSS markers were tested using PLINK [14]. Linkage disequilibrium (LD) between markers was tested by Haploview [15]. Haplotype analyses were performed using UNPHASED [16] as well as SHEsis [17]. UNPHASED contains a suite of programs for association analysis of haplotype data, including COCAPHASE and QTP (...truncated)