| Objective:(1) To explore the association of Neuregulin-1 (Nrg-1) gene polymorphism with schizophrenia by analyzing alleles' transmissions in scizophrenic central families.(2) To get to know the relationship of the Nrg-1mRNA expression between center and periphery in schizophrenic animal models.(3) To explore initially the change of Nrg-1 gene functions and the relationship between the changs and antipsychotics' efficacy by analysising the changes of Nrg-1 gene mRNA and NRG-1 protein in schizophrenics' peripheral leucocyte.To demonstrate the involvement of the gene in schizophrenia at gene level, transcription level and protein level for infering the effects of the gene on schizophrenic pathology and providing with cue to elucidating the pathomechanism of schizophrenia.Method:(1) Selected four SNP—rs221533(C/T), rs7820838(C/T), 433E1006 (A/G) and rs3924999(C/T)—localed 5' terminal of Nrg-1 gene and genetyped by using real time quantitative PCR in 258 Chinese Han schizophrenic central families. Analyzed the transmissions of the alleles and haplotypes under TDT programe by applying genehunter2.0 software to explore the association of the gene with schizophrenia.(2) Schizophrenic animal models were found in male SD rats by being injected 0.6 mg/kg MK-801(100μl/20g). and two control groups were set up. Control group 1 were treated without any factors, but isotonic Na chloride with equal volume to MK-801 were given to control group2. Detected the Nrg-1mRNA expression by using semi-quantitative RT-PCR in brain tissue and peripheral blood leucocytes, and the amount of NRG-1protein was measured in the brains with immunohistochemistry. Took the average of percentage in some area as an index to evaluate the expression of NRG-1protein. The comparison of Nrg-1mRNA was performed among three groups(model group, control group 1 and control group2) by using one-way ANOVA.(3) The amount of Nrg-1mRNA in peripheral blood leucocytes was measured by using semi-quantitative RT-PCR in 31 first-episode positive subtype schizophrenics. The patients were followed for 4 weeks with taking antipsychotics. The reduction rate of PANSS was used to evaluate the efficacy. The comparison of Nrg-1mRNA was performed among schizophrenics, 16 siblings of the schizophrenics and 33 healthy no-sibship controls by using one-way ANOVA and GLM-General Factorial programes. (4) NRG-1proteinγcounts were detected in 21 patients and 33 controls who came from the subjects mentioned in (3) by using double antibody sandwich radio-immunoassays, and followed the patients for 4 weeks. Comparisons ofγcounts between patient group and control group and among patient groups at different time were performed by using independent samples t-test and GLM-General Factorial programes, respectively.(5) TDT was performed by using software Genehunter2.0. SPSS 12.0 was used to analyze other data.Results:(1) For all subjects, four SNP genetypes were in Hard-Weinberge equilibrium, x~2 value was 2.494, 3.800, 5.454 and 4.912, respectively,and P value was 0.287, 0.150, 0.065 and 0.086, respectively.(2) In 258 central families, the transmission disequilibrium exhibited in the transmissions of allele C, A and T from rs221533,433E1006 and rs3924999, respectively (the x~2 value was 27.45, 56.08 and 10.53, and the P value was 0.000, 0.000 and 0.001, respectively), but not in the transmission of alleles from rs7820838(x~2=3.31, P=0.081). Haplotypes which frequency exceeded 5% were analyzed. C/C/G and C/C/A (markers ordering: rs221533, rs7820838,433E1006) transmitted predominantly(the x~2 value was 5.26 and 45.08, and the P value was 0.026 and 0.000, respectively). In four-marker-haplotypes (markers ordering: rs221533,rs7820838,433E1006,rs3924999), C/C/G/T exhbited transimission disequilibrium statistically (x~2=10.71, P=0.001). To emphasized that there were 27, 27 transmissions of C/C/A/C, C/C/A/T to the affected offspring 6, 0 nontransmissions, respectively, though their sequency was lower 5%.(3) In 213 positive subtype schizophrenic families out of 258 central families, the transmission disequilibrium exhibited in the transmissions of allele C, A and T from rs221533,433E1006 and rs3924999, respectively (the x~2 value was 20.83,60.48 and 9.32, and the P value was 0.000, 0.000 and 0.003, respectively), but not in the transmission of alleles from rs7820838(x~2=0.96, P=0.369). Haplotypes which frequency exceeded 5% were analyzed. There were 39 transmissions of C/C/A (markers ordering: rs221533, rs7820838, 433E1006) to the affected offspring and 0 nontransmissions (x~2=39, P=0.000). 14 haplotypes were found in four linked markers(rs221533, rs7820838,433E1006,rs3924999). C/C/G/T transimitted predominantly (x~2=7.00, P=0.011). For two haplotypes C/C/A/C and C/C/A/T, there were 18, 18 transmissions respectively to affected offspring and 0 nontransmissions.(4) Significant differences of Nrg-1mRNA existed among control.group 1, control group2 and model group not only in brain but also in peripheral blood leucocyte, and the amount of Nrg-1mRNA in model group was more than that in control groups. No significant difference existed in two control groups; Positive correlation existed between Nrg-1mRNA in brain and in peripheral blood leucocyte (r=0.597, P=0.000). The amount of NRG-1 protein, detected by immunohistochemistry, in the brains of model group was more than that in the brains of two control groups.(5) The amount of Nrg-1 mRNA was significantly different among the patient group before treatment, sibling group and no-sibship control group (F=6.722 P=0.002). Before patients' taking psychotics, The amount of Nrg-1 mRNA in the patient group was lower than that in sibling group and in no-sibship control group. No significant difference existed between no-sibship control group and sibling one. In the patient group, there were significant differences between 0w and 2nd w, 0w and 3rd w, 0w and 4th w, 1stw and 3rdw, 1stw and 4thw. The amount of Nrg-1 mRNA increased gradully with taking antipsychotics. Divided patients into two groups according to antipsychotics-taken, the amount of Nrg-1 mRNA was compared between the two groups at 0w, 1st w, 2ndw, 3rdw, and 4th w, respectively. No statistical significance existed.(6) The correlation between the amount of Nrg-1mRNA and the value of PANSS at 0w was analyzed. No significant correlation was shown (r=-0.251, P=0.172). After 4 weeks, the correlation between the reduction rate of the total PANSS score and the change of Nrg-1mRNA was analyzed. Coefficient correlation was 0.332, but no statistical significance existed (P=0.068).(7) Theγcounts of NRG-1 protein in patient group were lower than that in no-sibship control group (t=5.325,P=0.000). After 4 weeks, no significant difference existed between them (t=1.382, P=0.173). theγcounts of NRG-1 protein in patient group at different time (0w, 1st w, 2nd w, 3rd w and 4th w) were significantly different(F=6.3666,P=0.000). It increased gradully with taking antipsychotics. The counts at 4th w were higher than that at 0w, 1stw, 2ndw and 3rdw.Conelussion:(1)Association of Nrg-1 gene polymorphism with Chinese Han schizophrenia, especially with positive subtype schizophrenia, exists.(2) For SD rats, the expression of Nrg-1 mRNA in peripheral blood leucocytes changes at equal pace in brains. The change of Nrg-1 mRNA in peripheral blood leucocyte can reflect that in center to some extent.(3) The function of Nrg-1(at trancription or protein level) decreases in schizophrenia, and it improves with taking antipsychotics. The improvement is caused by different antipschotics.(4) It is very possile that Nrg-1 gene contributs to schizophrenic pathology by interacting with glutamategic nerve system, especialy NMDA receptors. Its' function deficiency meybe one of susceptibility factors of schizophrenia, especially of positive subtype schizophrenia.
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