Genetic Research Into Schizophrenia-associated Genes

Schizophrenia is a serious mental disorder with a lifetime prevalence rate of 1% in the general population worldwide. 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. 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 susceptibility gene having only a modest individual effect. The number of its predisposing genes decides susceptibility to a disease of every individual. Now, the main challenge to medical genetics is how to screen the human predisposing genes of common and momentous illness throughout the entire human genome.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. The major purpose of the Human Genome Project (HGP) is to solve the molecular genetic problems regarding human serious diseases. Most human serious diseases are all associated with genetic factors. However, it is difficult to make a big progress in searching for susceptibility genes for complex diseases. One of the important reasons is the polygenic feature of complex diseases. The construction of human genome maps, including genetic map, physical map, DNA sequence map and single nucleotide polymorphisms (SNPs) map, has paved a way direct to search for susceptibility genes for complex diseases throughout the entire human genome. Genome scan and linkage disequilibrium (LD) analysis make the study more powerful and reliable. The SNPs map combines linkage study with linkage disequilibrium, integrates the DNA sequence map, physical map and genetic map at the DNA level. It is beneficial to elucidate the difference of phenotype and susceptibility to disease of individual person. On the basis of high density and high polymorphisms SNPs map, genome scanning and association study are used to screen for more relative genes of complex diseases. It makes a great progress in mapping the susceptibility genes of polygenic disease. Genome-wide scanning shows the consistent linkage findings on some chromosome regions, such as 1q21-22, 5q22-23, 6p24-21, 8p22-21, 13q14-33 and 22q11-12.The present study had focused on identifying the candidate susceptibility genes on the 13q14.3 and 17p11.2 region using a family-based LD analysis and case-control analysis. A total of 270 family trios of Chinese Han descent, consisting of healthy fathers, healthy mothers and affected offspring with schizophrenia, and 537 unrelated patients with schizophrenia and 690 unrelated healthy individuals were recruited. The genetic loci associated with schizophrenia were chosed by using bioinformatics methods and molecular genetics techniques. These loci included 3 SNPs on the KPNA3 gene and 2 SNPs on the PEMT gene.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 genotype frequency 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 paired SNPs were tested by UNPHASED programs.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 case-control study was designed to check the results above-mentioned. The followings are the details of methodology and major results obtained in this study.1 DNA markersFive SNPs present in two genes on the 13q14.3 and 17p11.2 region were chosen by accessing the databases at http://www.ncbi.nlm.nih.gov/, http://www.ncbi.nlm.nih.gov/SNP and http://snp.cshl.org/ web sites. The candidate SNPs included SNP1, SNP2 and SNP3 present in the KPNA3 locus, SNP4 and SNP5 in the PEMT locus.2 The H-W equilibriumTheχ2 goodness-of-fit test showed that the genotypic distributions of all 5 SNPs were not deviated from the H-W equilibrium, and thus this sample pool was suitable for the genetic analysis.3 LD between paired SNPsThe estimated LD showed that SNP2 and SNP3 were in the same LD block.4 Association between SNPs and schizophrenia4.1 The TDT analysisThe TDT analysis showed that both SNP3 and SNP4 were associated with schizophrenia (P=0.016 and P=0.001, respectively). The SNP3 is a G to A base change and heterozygous parents have excessively transmitted allele G to their affected offspring, suggesting that the haplotype containing SNP3 (A) may carry a disease-resistant variant for schizophrenia. The SNP4 is a C to T base change and heterozygous parents have excessively transmitted allele C to their affected offspring, suggesting that the haplotype containing SNP4 (T) may carry a disease-resistant variant for schizophrenia.4.2 The HHRR analysisThe HHRR analysis revealed allelic association between two SNPs and schizophrenia, one the SNP3 and the other one the SNP4. These findings were consistent with TDT results.4.3 Analysis for clinical subgroupsEach SNP may have its own genetic heritage with a different haplotype and non-random association when these SNPs occurred differs. 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 was to limit the interference from different LD signals to reduce the false negative results and validate the hypothesis of genetic heterogeneity. The results showed that SNP2, SNP3, SNP4 and SNP5 were associated with schizophrenia.4.4 The association between SNPs and sex of schizophreniaThe HHRR analysis showed that the parents have excessively transmitted SNP2 allele G (P=0.039), SNP3 allele G (P=0.006) and SNP4 allele C (P=0.003) to their male affected offspring. These results suggested that SNP2, SNP3 and SNP4 were associated with male schizophrenia. 5 Analysis for combined effects of multiple locus5.1 Analysis for haplotype transmission5.1.1 A number of haplotype systems were constructed with these 5 SNPs studied, including 4 haplotype systems, such as SNP1-SNP2, SNP2-SNP3, SNP1-SNP2-SNP3 and SNP4-SNP5. The globalχ2 test showed that the SNP4-SNP5 haplotype system was associated with schizophrenia (P<0.05).5.1.2 The 1-df test for individual haplotype showed that the SNP2 (G) -SNP3 (G) haplotype, SNP1 (C) -SNP2 (G) -SNP3 (G) haplotype and SNP1 (T) -SNP2 (C) -SNP3 (G) haplotype were excessively transmitted (P<0.05). The individual haplotype mentioned above contains the SNP3 (G) allele that shows a positive association with schizophrenia. They may be derived originally from the same haplotype system and carry the same susceptibility for schizophrenia.5.1.3 The 1-df test for individual haplotype also showed that the SNP4 (C) -SNP5 (G) haplotype was excessively transmitted (P<0.05) and this haplotype may contain a susceptibility allele for schizophrenia. The SNP4 (T) -SNP5 (G) haplotype was excessively non-transmitted (P<0.05). And it may be carry the disease resistant allele for schizophrenia.6 Analysis for conditional testThe conditional test was used to test the combined effect of distinct loci on the disease by conditioning on allele (COA) or by conditioning on genotype (COG) .The COA test showed that the SNP4 and SNP1, SNP2 or SNP3 combinations, SNP4-SNP5 and SNP1, SNP2 or SNP3 combinations, SNP1-SNP3 and SNP5 combinations associated with the schizophrenia. The COG test also showed an association for SNP4 and SNP1 or SNP2 combinations SNP4-SNP5 and SNP1, SNP2 or SNP3 combinations.7 The association between SNPs and psychotic symptoms of schizophreniaBecause the genotypic distributions of all 5 SNPs were in the H-W equilibrium, we were able to analyze psychotic symptoms versus allelic and genotypic frequency of each SNP using a case-control design. The results were shown as follows:7.1 The distribution of allele frequencies of SNP3 was significantly different between the patients with incoherence of thinking and illogic thought and those without the symptom (P=0.017 and P=0.043, respectively). It suggested that the SNP3 was associated with these positive symptoms of schizophrenia, incoherence of thinking and illogic thought.7.2 The distribution of allele and genotype frequencies of SNP4 was significantly different between the patients bizarre behaviour and those without the symptom (P=0.002, P=0.006, respectively). It suggested that the SNP4 was associated with bizarre behaviour, one of the positive symptoms of schizophrenia.7.3 The distribution of genotype frequencies of SNP3 was significantly different between the patients with incoherence of thinking and those without the symptom (P=0.035). It suggested that the SNP3 was associated with the positive symptoms of schizophrenia, incoherence of thinking.7.4 The distribution of frequencies of SNP5 was significantly different between the patients with delusion of grandeur and those without the symptom (P=0.022). It suggested that the SNP5 was associated with the positive symptoms of schizophrenia, delusion of grandeur.8 Analysis for case-controlThe SNP4 was associated with schizophrenia. There was a significant difference in frequency of allele and genotype between the patient group and the control group. The frequency of allele C was greater in the patient group than that in the control group.Taken together, the present study demonstrated that there may be one or more disease susceptibility genes located on the 13q14.3 and 17p11.2 region. The KPNA3 gene and the PEMT gene may be associated with schizophrenia. SNP2, SNP3 and SNP4 may be associated with schizophrenia in male. The 3 SNPs, including SNP3, SNP4 and SNP5, were associated with some positive 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.