Associations Of Metabotropic Glutamate Receptor Genetic Variations With Attention Deficit Hyperactivity Disorder

Objective:The purpose of this study is to identify attention deficit hyperactivity disorder（ADHD）susceptibility genetic variations of metabotropic glutamate receptor（mGluR）family,analyze the influence of the interactions between mGluR genetic variations and environmental factors on ADHD susceptibility,and explore the functions of the susceptibility locus and its potential role in the biological mechanism of ADHD,which provide new ideas for the discovery of ADHD biomarkers,and provide a theoretical basis for early screening and clinical precision treatment of people who are susceptible to ADHD.Methods:1.A two-stage case-control study was conducted to systematically analyze the associations between ADHD and genetic variations of the mGluR falimy.Based on the Diagnostic and Statistical Manual of Mental Disoeders,4^th Edition（DSM-Ⅳ）disgnostic criteria,in stage one,617 cases and 636 controls were recruited in Wuhan;in stage two,352 cases and 346 controls were recruited in Changsha.The Swanson,Nolan,and Pelham versionⅣ（SNAP-Ⅳ）symptom assessment scale and the Integrated Visual and Auditory Continuous Performance Test（IVA-CPT）were used to assess the severity of ADHD-related symptoms.Combined with the results of ADHD GWASs and the published literatures,bioinformatics databases（Mala Cards,STRING,NCBI-Gene,SNPinfo,HaploReg and Endembl genome browser）were used to screen candidate mGluR genes and SNPs.Genotyping was performed with the Mass ARRAY and iPlex systems of the Agena genotyping platform.Logistic regression model was used to analyze the associations between SNPs and the risk of ADHD under different genetic models.The distribution difference of ADHD clinical feature scores among different genotypes of SNPs was explored by ANOVA.2.The questionnaires was used to collect the relevant information of subjects,including general information,family status,maternal pre-pregnancy and pregnancy conditions,children’s sleep conditions and allergic diseases.Logistic regression model was used to analyze the associations between environmental factors and the risk of ADHD.Classification decision tree and multi-factor dimensionality reduction methods were used to analyze the multi-factor interactions between genetic variations and environmental factors.Finally,the Excel spreadsheet set up by Tomas Andersson and logistic regression model were used to analyze the effects of additive and multiplicative interaction between SNPs and environmental factors on the risk of ADHD,respectively.3.The study used bioinformatics tools to annotate the functions of SNPs that were associated with the risk of ADHD,including SNPinfo Web Server,HaploReg v4.1,Regulome DB,GWAVA and r SNPBase databases.The Mir SNP,Micro SNi Per and Polymi RTS Database 3.0 databases were used to predict the micro RNA that could bind to SNPs.We downloaded SNPs genotyping data and gene expression data in different brain tissues from the BRAINEAC database,and used these data for e-QTL analysis.Finally,the study further explored the functions and potential biological mechanisms of SNPs associated with ADHD through dual-luciferase reporter gene assay,electrophoretic mobility shift assay and real-time reverse transcription-PCR（RT-PCR）quantitative detection.Results:1.GRM4 rs1906953 and GRM7 rs9826579 were found to be associated with the risk of ADHD risk in the discovery,verification and combined stage.In the combined stage,rs1906953 T allele was associated with a decreased risk of ADHD under codominant,recessive and additive models（TT:CC,OR=0.54,95%CI=0.41-0.71,P<0.001;dominant model,OR=0.77,95%CI=0.64-0.94,P=0.008;recessive model,OR=0.60,95%CI=0.47-0.76,P<0.001;additive model,OR=0.76,95%CI=0.66-0.86,P<0.001）;compared with subjects with the rs1906953 CC genotype,subjects with the TT genotype had lower attention deficit scores and hyperactivity-impulsive scores,higher comprehensive attention quotients and visual attention quotients.rs9826579 C allele was associated with an increased risk of ADHD under codominant,dominant and additive models（CT:TT,OR=1.71,95%CI=1.39-2.11,P<0.001;dominant model,OR=1.64,95%CI=1.34-2.00,P<0.001;additive model,OR=1.46,95%CI=1.22-1.75,P<0.001）;compared with subjects with the rs9826579 TT genotype,CT genotype carriers had higher attention deficit scores.2.Logistic regression model analysis found that single-parent families,sleep disorders and allergic diseases were risk factors for ADHD.The risk to develop ADHD of children from single-parent families,with sleep disorders and with allergic diseases was 1.76 times（OR=1.76,95%CI=1.06-2.92）,1.27 times（OR=1.27,95%CI=1.03-1.58）,1.36 times（OR=1.36,95%CI=1.08-1.71）that of children from non-single-parent families,without sleep disorders and without allergic diseases,respectively.In multi-factor interaction analysis,the analysis results of classification decision tree and multi-factor dimensionality reduction showed that rs9826579 and sleep disorders were the best interaction model.In addition,the multiplication interaction between rs9826579 and sleep disorders was statistically significant（P=0.019）.Compared with children with rs9826579 TT-no sleep disorders,the risk of ADHD increased by 47%（OR=1.47,95%CI=1.13-1.92）,86%（OR=1.86,95%CI=1.47-2.35）,56%（OR=1.56,95%CI=1.09-2.22）in children with rs9826579TT-sleep disorders,with TC/CC-no sleep disorders and with TC/CC-sleep disorders,respectively.3.The results of function annotation showed that rs1906953 and rs9826579 were both regulatory SNPs.The results of e-QTL analysis showed that rs1906953 T was associated with the decreased expression of GRM4 in the intralobular white matter and occipital cortex（P<0.05）;rs9826579 T was associated with the increased expression of GRM7 in the medulla and intralobular white matter（P<0.05）.The results of the dual-luciderase reporter gene assay showed that the expression of luciferase in the rs1906953-T group was significantly reduced compared with that in the rs1906953-C group（P<0.001）,which indicated rs1906953 might affecte the expression of the reporter gene by interfering with the promoter activity.The results of the electrophoretic mobility shift assay showed that the rs1906953 C probe binded strongly to nucleoprotein but weakly for the rs1906953 T probe.Real-time RT-PCR quantitative detection results showed that the expression of GRM4 in the peripheral blood of children with ADHD was significantly higher than that of the control group（P<0.001）.Conclusions:1.GRM4 rs1906953 T allele was associated with a decreased risk of ADHD;compared with subjects with the 1906953 CC genotype,subjects with the TT genotype showed slighter attention deficit and hyperactivity/impulsive symptoms.GRM7 rs9826579 C allele was associated with an increased risk of ADHD;compared with subjects with the rs9826579 TT genotype,subjects with the CT genotype had more serious attention deficit symptom.2.Among many environmental factors,GRM7 rs9826579 has the significant interaction with sleep disorders.Compared with children with rs9826579 TT-no sleep disorders,children with rs9826579 TT-sleep disorders,children with TC/CC-no sleep disorders and children with TC/CC-sleep disorders had a significantly higher risk of ADHD.3.GRM4 rs1906953 T allele might reduce the expression of GRM4 by interfering with the binding of transcription factors to DNA sequences,thereby reducing the risk of ADHD.