| Background and purpose:Schizophrenia is a severely mental illness and the etiology is not clear. In most cases, the illness has occurred slowly or subacutely in the young adults and easy to relapse and be chronicity. They exhibit a range of clinical manifestations,, involving the incongruity of perception, thinking, feeling and behavior disorders and various spiritual activities. Global Burden of Disease reported the rank of the disease is 7. Prevalence of the disease is 0.6% to 1.2% in China, accounting for more than 50% of the total inpatient psychiatric disease.Now, Schizophrenia is recognized as a class of multi-gene genetic diseases, and caused by genetic factors and environmental factors. Heritability in schizophrenia is 70%-90%.Research has shown that infection during pregnancy, perinatal brain injury, lactation malnutrition, adolescent marijuana, living environment and various environmental factors can affect the occurrence of schizophrenia. In term of genetic factors, the latest statistics showed that the two married patients with schizophrenia have children with prevalence rate is 68.1%, which is 100 times as high as the normal ones. At present, for the aspects of the pathophysiology of schizophrenia,research mainly focused on dopamine hypothesis, the serotoninhypothesis and glutamate hypothesis. The serotonin neurons and glutamate neurons has both distributed of endogenous cannabinoid receptors, which plays presynaptic inhibition or presynaptic excitability in the neurons. Endogenous cannabinoids may also indirectly impacted the dopaminergic neurons with the help of the two neurotransmitters. A study also found the lever of endocannabinoids in the blood and cerebrospinal fluid were changed in schizophrenia, compared with the normal. Therefore, some scholars believe that the ECS(Endogenous cannabinoid system) has involved in schizophrenia. ECS is a regulation system of the central nervous system(CNS), widely distributed in the brain, including the amygdala, the frontal cortex, hippocampus, cerebellum, thalamus, mainly comprises the receptors for the cannabinoids, thus termed cannabinoid type-1 and type-2 receptors(CB1-R and CB2-R); their endogenous ligands, anandamide(AEA), and 2-arachydonoyl glycerol(2-AG). NAPE-PLD(N-acyl phosphatidylethanolamine phospholipase) and FAAH( fatty acid amide hydrolase) are the enzymes responsible for AEA synthesis and catabolism, respectively.In order to study the association between NAPE-PLD and FAAH gene polymorphism(SNP) and schiziphrenia, this reseach selected NAPE-PLD gene rs12540583 locus and FAAH gene rs2295633, rs324420, rs6429600 in Henan Han population, using restriction fragment length polymorphism polymerase chain reaction(PCR-RFLP), to detect the genotype and gene frequencies distributions, genetic models and haplotype of the 4 SNPs, and at the same time analyze the interaction of the two gene,which aims to enrich the molecular etiology of schizophrenia database and help to establish clinical molecular diagnosis or treatment.Materials and Methods:Total 345 patients(196 men and 149 women, mean age 37.5 ±12.35 years) were recruited from Zhengzhou psychiatric hospital and Luoyang Rongkang hospital. They were diagnosed as schizophrenia according to the US DSM-IV criteria,and the PANSS scores were greater than 45. The exclusion criteria were unknown diagnosis,brain organic disease, mental retardation, epilepsy, liver and kidney dysfunction, alcohol dependence and other psychoactive substance abuse. The 403 healthy individuals(225 men and 178 women, mean age 35.6±10.0 years)were recruited from Han population the healthy check-up random individuals,removing the one with history of family psychosis. There were no significant difference of age and gender between case and control(t = 0.61, P>0.05; χ2= 1.77,P > 0.05, respectively). All subjects were informed of the purpose and procedures involved in this study.1. Genomic DNA Extraction: peripheral blood samples were collected from the paticipants in 1.5 ml EDTA vacuum tube. Genomic DNA was extracted from peripheral blood according to standard phenol-chloroform method, dissolved in sterile, double distilled water and stored at-20 °C until genotyping.2. PCR: design and synthesize the primers and the PCR of the target gene.3. Restriction Enzyme Digestion: use the specific restriction enzyme to digest of the PCR product.4. Agarose Gel Electrophoresis: The PCR products and enzyme fragments were electrophoresed on 2% agarose gels at 110 V for an hour. The results were photographed and recorded with a UV imaging system. Statistical Analysis: Using the online platform(http://ihg.gsf.de/cgi-bin/hw/hwa1.pl) to test The Hardy–Weinberg equilibrium and Genetic Models analysis. Using SPSS17.0 to analyze the allele frequency distribution. The haplotype analyses w carried out by the online platform(http://analysis.bio-x.cn/my Analysis.php). Gene-gene Interaction were investigated by MDR. For multiple test correction,the Bonferroni correction was applied and the significance level α was set at 0.0125(0.05/4).Result:1. The distribution frequency of rs12540583,rs2295633,rs324420,rs6429600 for the case and control groups were consistent with Hardy–Weinberg equilibrium(P Value were 0.84 and 0.14; 0.33 and 0.23; 0.07 and 0.06; 0.54 and 0.76, respectively). 2. NAPE-PLD gene rs12540583 was significant differences in the genotypefrequencies between the case and control groups in the dominant model and the additive model..(χ2=19.77, χ2=19.87, respectively, P<0.0125). 3. There was a significant difference in the allele frequency between the two groups in NAPE-PLD gene rs12540583,C allele frequency of the case group were higher than the control.The C allele was positively correlated with the risk of schizophrenia and was deemed a risk factor(OR=1.54, 95 % CI:1.27-1.88). 4. There was a significant difference in the genotype frequencies between the case and control in NAPE-PLD gene rs12540583,AC genotype frequency of the case group were lower than the control. Using the wild genotype AA as reference, Relative risk analysis showed that AC is positively correlated with schizophrenia,as risk genotypes(χ2=17.18,P<0.0125). 5. There was no difference in the haplotype distribution between the case group and the control group of FAAH 3 loci(rs2295633, rs324420, rs6429600). 6. All of the models had statistical significances(P=0.001), in which Cross-validation consistency of one-locus model(rs324420)and four-locus model(rs12540583, rs2295633, rs324420, rs6429600) were 10/10.The four-locus model as the best model had a maximum testing accuracy of 76.49%. Interaction Dendrogram also showed there was Interaction among the four SNP.Conclusion:1. NAPE-PLD rs12540583 C allele and AC genotype were risk factors for schizophrenia in Henan Han population. 2. NAPE-PLD gene rs12540583 and FAAH gene rs2295633, rs324420, rs6429600 were found to interact in cases of schizophrenia. |
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