| The human genome is the whole set of DNA arrayed in 24 distinct chromosomes, which consists of 3.2 billions of base pairs (bp). The genomic DNA is 99.9% identical to that of other humans and 0.1% is variable. One of the fruits of the Human Genome Project is the discovery of millions of DNA sequence variants in the human genome. The use of these genetic markers has been playing an increasing part in genetics studies. Most variation of the human genome is attributable to single nucleotide polymorphisms (SNPs), which contributes to the differences between individuals in physical appearances, susceptibility to a disease and response to medication with drugs. SNPs have been widely used. A dense set of SNP markers opens up the possibility of studying the genetic basis of complex disease. In recent years, SNPs have been used as new DNA markers to replace microsatellites in mapping of disease-related genes in humans. There is no doubt that SNPs will play a key role in identifying and cloning of disease-related genes, investigating the mechanism of the genome-environment interaction and gene-gene interaction.Schizophrenia is a serious mental disorder with a lifetime prevalence rate of 1% in the general population worldwide. It is characterized by the abnormal mental functions and disturbed behaviors, which characteristically appears as a series of clinical features, such as positive and negative symptoms, and disturbances in basic cognitive functions. Because the illness causes heavy economical and social burdens to families and societies, it is very important to establish a procedure of treating and preventing schizophrenia. While the cause for schizophrenia remains unknown, several lines of evidence from family, twin and adoption suggest that genetic factors are likely to play an essential role in the developing of schizophrenia and influence susceptibility to schizophrenia. Epidemiological data have demonstrated that schizophrenia is not a simple Mendelian disease but looks like a complex disease involving several genes with each susceptiblity gene having only a modest individual effect. Genome-wide scan has suggested that the following chromosomal regions may contain several susceptibility genes contributing to schizophrenia, including 1q21-22, 5q31-33, 6p21-24, 6q25-26, 8p21-22, 10p11-15, 13q14-33 and 22q11-13. To validate these initial findings, the present study focused on identification of the candidate susceptibility genes on the 6q21.3 region using a family-based linkage disequilibrium (LD).The family trio consists of healthy fathers, healthy mothers and affected offspring with schizophrenia. Seven SNPs were chosen totally, 5 of which are located in the NOTCH4 locus on the 6p21.3 region including rs387071, rs367398, rs915894, rs520688 and rs422951. Meanwhile, 2 SNPs were chosen from the PPARγlocus including rs2972162 and rs 2938395 on the 3p25 region.SNPs were genotyped using PCR-based RFLP analysis. Genotyping data were put into the SPSS database. The Hardy-Weinberg (H-W) equilibrium was tested for the genotypic distributions of SNPs using the goodness-of-fit test. The LD between paired SNPs was estimated with UNPHASED programs. The haplotype-based haplotype relative risk (HHRR) test and the transmission disequilibrium test (TDT) were applied to detect allelic association between SNPs and schizophrenia. To elucidate genetic heterogeneity, in addition, schizophrenic patients were sub-grouped based on their clinical symptoms and the genetic association between SNPs and clinical subgroups was then analyzed. Combined effects of multiple SNPs were tested by the UNPHASED program.The patients were divided into two groups according to the clinical psychotic symptoms. We analyzed psychotic symptoms versus allelic and genotypic frequency for each SNP.The details of methodology and major results obtained in this study are as follows:1 The H-W equilibrium The goodness-of-fit test showed that the genotypic distributions of all subjects were not deviated from H-W equilibrium, and thus these samples were suitable for the genetic analysis.2 LD between paired SNPsThe estimated LD showed that rs2972162 and rs2938395 were in the same LD block.3 Association between SNPs and schizophrenia3.1 The TDT analysis :The TDT analysis showed that rs520688 was associated with schizophrenia (P = 0.002). The rs520688 present in the NOTCH4 locus was a A to G base change and heterozygous parents have excessively transmitted allele A to their affected offspring, suggesting that the haplotype containing rs520688(A)may carry disease-resistant variant for schizophrenia.3.2 The HHRR analysis:The HHRR analysis revealed an allelic association between rs520688 and schizophrenia. This finding was consistent with TDT result.3.3 Analysis for clinical subgroups:The results showed that rs2972162, rs367398, rs915894, rs520688 and rs422951 were associated with schizophrenia. Since the time at which SNPs occurred differs, each SNP may have its own genetic heritage with a different haplotype and non-random association. They may affect each other in allele frequency distribution, so as to reduce the power to detect their association with the illness. The advantage of clinical subgroup analysis is to limit the interference from different LD signals to reduce the false negative results and validate the hypothesis of genetic heterogeneity.3.4 Analysis for haplotype transmissionThe analysis for multi-SNP hapoltype transmission is essential to look for a specific haplotype or chromosome possibly carrying a gene for schizophrenia.3.4.1 A number of haplotype systems were constructed with these 7 SNPs studied, including 6 two-SNP haplotype systems and 5 three-SNP haplotype systems. The globalχ~2 test of the two-SNP haplotype systems showed that the rs915894-rs520688 haplotype system, the rs520688-rs422951 haplotype system and the SNP4-SNP5 haplotype system were associated with schizophrenia.3.4.2 The globalχ~2 test for the three-SNP haplotype systems showed that the rs387071- rs 367398-rs915894 haplotype system and the rs367398-rs915894-rs520688 haplotype system and rs 915894-rs520688-rs422951 were associated with schizophrenia.3.4.3 The 1-df test for individual haplotypes showed that the rs915894(A)-rs520688(A) haplotype and the rs367398(C)-rs915894(A)-rs520688(A) haplotype were excessively transmitted (P < 0.05). They may be derived originally from the same haplotype system showing a positive association with schizophrenia and carry the same disease-susceptible allele. The rs367398(C)-rs915894(A)-rs520688(G) haplotype was excessively non-transmitted (P < 0.05) and this haplotype may contain a resistant allele for schizophrenia.3.4.4 The 1-df test for individual haplotypes also showed that the rs520688 (G)-rs422951(A) haplotype was excessively non-transmitted (P < 0.05), suggesting that it may contain the disease-resistant allele. The rs915894(A)-rs520688(A)-rs422951(A) haplotype was excessively transmitted and this haplotype may contain a susceptible allele for schizophrenia.3.4.5 The 1-df test for individual haplotypes also showed that the rs387071 (A)-rs367398 (C)-rs915894(A) and rs387071(G)-rs367398(T)-rs915894(A) haplotype were excessively transmitted, and he rs387071(A)-rs367398(C)- rs915894(C) haplotype was excessively non-transmitted suggesting that rs387071-rs367398- rs915894 may contain susceptible and resistant alleles for schizophrenia.4 The association between SNPs and psychotic symptoms of schizophreniaTo investigate the association between SNPs and positive and negative symptoms of schizophrenia, the SPSS program and the UNPHASED program were applied for the statistical analysis. The results were shown as follows:4.1 The association between rs2972162 and psychotic symptoms of schizophreniaThe distribution of allele and genotype frequencies was significantly different between the patients with incoherentce of thinking and those without the symptom (P=0.021; P=0.029 respectively). It suggested that the 2972162 was associated with incoherentce of thinking,one of positive symptoms of schizophrenia.4.2 The association between rs2938395 and psychotic symptoms of schizophreniaThe distribution of allele frequencies was significantly different between the patients with delusion of being revealed and those without the symptom (P = 0.034). It suggested that the rs2938395 was associated with the delusion of being revealed, one of positive symptoms of schizophrenia.4.3 The association between rs367398 and psychotic symptoms of schizophreniaThe distribution of allele frequencies was significantly different between the patients with delusion of influence and those without the symptoms (P = 0.026). It suggested that the rs36739 was associated with delusion of influence of positive symptoms of schizophrenia.4.4 The association between rs520688 and psychotic symptoms of schizophreniaThe distribution of allele frequencies was significantly different between the patients with genuine auditory hallucination, delusion of influence, delusion of sin and delusion of grandeur and those without the symptom (P = 0.003, P = 0.048, P = 0.035, P = 0.024) . It suggested that the rs520688 was associated with genuine auditory hallucination, delusion of influence, delusion of sin and delusion of grandeur, the positive symptoms of schizophrenia. The distribution of genotype frequencies was significantly different between the patients with genuine auditory hallucination, delusion of sin, delusion of being revealed, delusion of grandeur and incoherence of thinking and those without the symptom (P = 0.007, P = 0.000, P = 0.025, P = 0.018, P = 0.047). It suggested that the rs520688 was associated with genuine auditory hallucination, delusion of sin, delusion of being revealed, delusion of grandeur and incoherence of thinking, the positive symptoms of schizophrenia.4.5 The association between rs422951 and psychotic symptoms of schizophreniaThe distribution of allele frequencies was significantly different between the patients with genuine auditory hallucination and those without the symptom (P = 0.040). It suggested that the rs422951 was associated with genuine auditory hallucination,one of positive symptoms of schizophrenia.4.6 The association between rs915894 and psychotic symptoms of schizophreniaThe quantitative trait analysis showed that the rs915894 was associated with poverty of thought and abulia (P = 0.035; P = 0.008).5 The association between SNPs and sex of schizophreniaThe HHRR analysis showed that the parents have excessively transmitted rs520688 allele A to their male affected offspring (P = 0.001).The result suggested that rs520688 was associated with male schizophrenia.6 Analysis for case-controlThere was not a significant difference in frequency of allele and genotype of rs520688 between the patient group and the control group.Taken together, the present study demonstrated that NOTCH4 might play an important role in the developing of schizophrenia, but couldn't rule out the small effect of other genes. Four SNPs, including rs2972162, rs2938395, rs367398 and rs422951, were associated with some positive symptoms of schizophrenia. The rs915894 was found to be associated with poverty of thought and abulia that were classified as three negative symptoms of schizophrenia. These findings are very important for elucidating the genetic mechanisms of schizophrenia at a molecular level, and also for the development of genetic diagnosis, new drugs for the treatment of the illness and prediction of schizophrenia risk.
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