Association Study Of NEDD4 Polymorphisms With Schizophrenia In Chinese Han Population

ObjectiveSchizophrenia is one of complex diseases which heritability is 80%. It is widely accepted that with early neurodevelopment abnormalities, schizophrenia is affected by both multiple minor genes and environmental factors. Ubiquitin proteasome system (UPS) is one kind of post-translational modification mechanisms which related to the function of the nervous system tightly. Nowadays, many studies have suggested that the pathogenesis of schizophrenia is related to abnormal gene expression or protein function of UPS. Neural precursor cell expressed developmentally down-regulated 4 (NEDD4) belongs to ubiquitin ligase which could regulate the turnover of glutamate receptor 1 (GluR1) and involve in the differentiation process of central nervous system. Recently, NEDD4 may play a role in the pathological process of neurodegeneration have been found both in animal models and postmortem studies, while the association between NEDD4 and schizophrenia remains unknown. We hypothesized that NEDD4 gene polymorphisms related to schizophrenia. A case-control study was conducted to detect whether the five single nucleotide polymorphisms (SNPs) of NEDD4 correlated with schizophrenia and its symptoms. Furthermore, a preliminary functional study on the positively associated SNPs was designed to confirm our association study results and explain the possible molecular pathogenesis of schizophrenia that Nedd4 may be functionally involved in.MethodsBased on neurodevelopment hypothesis of schizophrenia, we selected NEDD4 as our candidate gene. To decide which SNP to analyze, we combined those functional SNPs located on 3’untranslated region (3’UTR),5’UTR or exon and those tagger SNPs selected by Haploview.5 SNPs (rs3088077, rs11550869, rs7162435, rs2303579 and rs62043855) were chosen for our study. To explore the relationship between NEDD4 and schizophrenia,5 SNPs of NEDD4 gene were genotyped by Taqman in 464 cases and 487 controls. The general information questionnaire was used to collect demographic information and the Positive and Negative Syndrome Scale (PANSS) was used to assess the severity of psychotic symptoms. For those positively associated SNPs, we used different molecular biology methods according to their location on gene aiming to find whether their mutations have impact on NEDD4 gene expression or protein function. On one hand, after enzyme digestion and fragment purification, the 3’UTR fragment from wild-type NEDD4 was fused in the firefly luciferase reporter in pmirGLO Dual-Luciferase miRNA Target Expression Vector. The mutants were constructed by polymerase chain reaction (PCR) mediated site-directed mutagenesis. Luciferase reporter gene assays were performed on wild type and mutant types after cell transfection. On the other hand, the wide type and mutant type of coding sequence of NEDD4 were constructed in pcDNA3.1 if the SNP located in exon. Using immunoblotting and immunoprecipitation, we detect the ubiquitinated level of GluRl which is one substrate protein of Nedd4.Results1. rs3088077 (allele:χ2=18.024, P<0.001, genotype:χ2= 16.634, P<0.001), rs7162435 (allele:χ2=6.771, P=0.009, genotype:χ2=7.352, P=0.025) and rs2303579 (alleles:χ2= 11.253, P=0.001, genotype:χ2= 12.248, P=0.002) both in allele and genotype distributions have significant difference between the patients and the controls, rs11550869 (allele:χ2=0.684, P=0.408, genotype:χ2=0.636, P=0.728), rs62043855 (allele:χ2=1.090, P=0.296, genotype:χ2=1.156, P=0.561) neither in allele nor in genotype distributions have significant difference between the two groups.2. The genotype of rs7162435 was found significantly correlated with excitement and hostile factor in the PANSS scores (F=3.551, P=0.029), especially the TT genotype (14.53±3.925). Multivariate stepwise regression equation also confirmed the result that rs7162435 was associated with excitement and hostile factor. The determination of coefficient R2 displayed that rs7162435 polymorphisms and age of onset could explain 3.5% variation of the dependent variable.3. The full length wild type 3’UTR of NEDD4 gene was constructed into the luciferase reporter vector PGLO. Mutation of rs3088077 or rs7162435 and mutanted both of those two were constructed as mutant recombinant plasmids. Compared with the empty vector, the full length 3’UTR of NEDD4 gene could inhibit the luciferase activity (P<0.001) no matter the 3’UTR got mutant or not. The mutantion of rs3088077 and rs7162435 could not affect the luciferase expression.4. Ubiquitination level of GluRl were performed on wild type coding sequence of NEDD4 and mutant NEDD4 rs2303579 constructions. The mutantion of rs2303579 could not alter the ubiquitination level of GluRl significantly (P= 0.476).Conclusions1. Our data suggested that the NEDD4 gene might association with schizophrenia for the rs3088077, rs7162435 and rs2303579 may increase the susceptibility to surfer schizophrenia and the TT genotype of rs7162435 may increase the severity of excitement and hostility. These 3 SNPs may be worked as molecular genetic markers.2. The whole length 3’UTR of NEDD4 gene would down regulated the activity of luciferase no matter rs3088077 and rs7162435 mutant or not. The ubiquitin level of GluRl caused by mutant NEDD4 rs2303579 constructions was consistent with the wild type. Further studies were needed to explore the function of these 3 SNPs.3. The ubiquitin proteasome system, the glutamate system and nervous system development may interact with each other and play role in the pathological process of schizophrenia.