Association Of Glutamate Transporter Network Genes Variants With Attention-deficit/hyperactivity Disorder

Attention-deficit/hyperactivity disorder(ADHD)is one of the most common neuro-developmental disorders among school children,which seriously affects the daily life,school performance,peer relationship and family harmony of the ADHD children.The etiology of ADHD is still unclear,and it is widely considered to be the consequence of interactions between multiple genes and complex environmental factors.It has been shown that glutamate is involved in the pathogenesis of ADHD as a vital excitatory neurotransmitter in the central nervous system,and glutamate transporter network is likely to be potentially related with ADHD,according to the association studies.Therefore,we selected glutamate transporter network genes(SLC1A3,SLC1A2,SLC1A6,SLC6A1,GAD1,GAD2,and STX1A)based on pathway analysis,and finally28 potential functional SNPs was determined by using bioinformatics analysis.We adopted the strategy of multi-center,two stage of case-control study to comprehensively analyze the association of glutamate transporter network genes with ADHD,and we also investigated the effects of gene-gene,gene-environment interactions on susceptibility to ADHD.Furthermore,we performed molecular biology tests and attempt to figure out the biological mechanism of the disorder.This research will provide novel evidence and theoretical basis for the early screening,prognosis surveillance and medication targets of ADHD.Part 1 Screening of susceptibility genes for ADHD based on glutamate transporter networkObjectives:Screening genetic mutations associated with susceptibility to ADHD in glutamate transporter network genes.Methods:A multi-center,two-stage case-control study strategy was used in this research.All of the 346 cases and 359 controls were recruited from Changsha in the discovery stage,and 406 cases and 359 controls were recruited from Wuhan in the validation stage.The cases were new medication-free cases diagnosed by Swanson,Nolan,and Pelham-IV Questionnaire(SNAP-IV),and the controls were healthy children conducted physical checkups during the same period.A Chinese Wechsler Intelligence Scale for children was administered to all participants,and the IQs were above 70.SNAP-IV,Parent Symptom Questionnaire(PSQ),and Integrated Visual and Auditory Continuous Performance Test(IVA-CPT)were conducted to measure the clinical characteristics of all participants.Genotyping of candidate genes was performed using the Sequenom MassARRAY.The t test and Pearson?~2 test were performed to examine the difference between cases and controls in the distribution of demographic characteristic and SNPs.The risk of clinical symptoms with SNPs was explored by ANOVA.Multivariate logistic regression models were conducted to analyze the association of SNPs with ADHD.FDR and Bonferroni correction were performed for multiple comparison corrections foe association analysis.Results:1.With the FDR correction,the results of association analysis in the combination stage showed that SLC1A3 rs1049522 was significantly associated with ADHD(OR=0.705,95%CI=0.574-0.867)in the dominant model;SLC1A2rs1042113 was associated with ADHD(OR=1.279,95%CI=1.030-1.590),and SLC6A1 rs1170695(OR=1.259,95%CI=1.090-1.453),GAD1 rs3749034(OR=1.351,95%CI=1.146-1.591),STX1A rs3793243(OR=0.778,95%CI=0.670-0.904),and rs875342(OR=1.225,95%CI=1.046-1.434)were all relative to ADHD in the addictive model.2.SLC1A3 rs1049522,SLC6A1 rs1042113,GAD1 rs3749034,and STX1A rs3793243were in connection with attention problems,while SLC6A1 rs1170695 and STX1A rs875342 were associated with hyperactivity/impulsion.3.SLC1A2 rs1042113,SLC6A1 rs1170695,STX1A rs3793243 and rs875342 were in relationship with ADHD-I;SLC1A3 rs1049522 and GAD1 rs3749034 were correlative to ADHD-H;SLC6A1 rs1170695,STX1A rs3749034 and rs875342 were associated with ADHD-C.Conclusions:SLC1A3(rs1049522),SLC1A2(rs1042113),SLC6A1(rs1170695),GAD1(rs3749034),and STX1A(rs3793243,rs875342)variations were involved in ADHD.Part 2 Effects of gene-gene,gene-environment interaction on susceptibility to ADHD in genetic variation of glutamate transporter network genesObjectives:To investigate the effect of the interactions between glutamate transporter network genes and the interaction between genes and environmental factors in the occurrence of ADHD.Methods:Self-designed questionnaires were used to investigate the demographic characteristics and related environmental factors of all participants.Atomic absorption spectrometry was used to measure of serum metal elements(lead,magnesium,calcium,iron,zinc)levels in all subjects.In the combination stage,gene-gene and gene-environment interactions between the positive association SNPs in the former part and risk factors were investigated by categorical regression tree(CART)and multiple factor dimensionality reduction(MDR).Moreover,logistic regression models were used to estimate the effects of the interplays.Results:Multiple logistic regression analysis showed that maternal stress during pregnancy(OR=1.466(1.032,2.082))and high blood lead levels(BLLs)in children(OR=1.370(1.112,1.689)were risk factors for ADHD,while breastfeeding(OR=0.332(0.157,0.702))was protected factor for ADHD.CART and MDR analysis showed that SLC1A3 rs1049522 interplayed with SLC6A1 rs1170695,and children carry both rs1049522 AA and rs1170695 GA/GG genotypes had 1.108 times more risk to suffer ADHD.There were also interactions between rs1049522 and breastfeeding(OR=0.605(0.448,0.818)),and between rs117095 and BLLs(OR=2.426(1.808,3.256)),maternal stress during pregnancy(OR=2.784(1.786,4.384)),and breastfeeding(OR=1.367(1.028,1.819)).Conclusions:1.Maternal stress during pregnancy and high BLLs in children were risk factors for ADHD,while breastfeeding was protected factor for ADHD.2.SLC1A3rs1049522 interacted with SLC6A1 rs1170695.There was an interaction between rs1049522 and breastfeeding,and rs1170695 interacted with maternal stress during pregnancy,high BLLs in children,and breastfeeding,respectively.Part 3 Biological function identification of genetic variants associated with susceptibility to ADHDObjectives:To explore the potential functional variation of association SNPs in the development of ADHD and its biological mechanism.Methods:Several informatics databases such as HaploReg,MirSNP,AliBaba etc.were used to perform further functional prediction of significant SNPs in the former two-stage association study,and eQTL analysis was applied to investigate the effect of different SNP genotypes on the expression of target genes.Dual luciferase reporter assays and electrophoresis mobility shift assays(EMSA)were used to validate the predictions,and investigating the biological mechanism of genetic variation in posttranscriptional regulation of ADHD in vitro.Results:1.All 8 different transcripts of rs1049522 CC genotype were mainly related to the increased expression of SLC1A3 in the cerebellum(CRBL).The rs1049522 CT type was associated with increased expression of SLC1A2 in the hippocampus(HIPP).The rs1170695 GG genotype increased the expression of SLC6A1 in the thalamus(THAL)and putamen(PUTM).All 28 transcripts of GAD1 rs3749034 were associated with quantitative traits in several brain areas,which were the substantia nigra(SNIG),PUTM,and medulla medulla(MEDU).The 10 transcripts of rs3793243 GG-type were mainly elevated STX1A in white matter.2.Double luciferase reporter assay showed that the rs1049522 A allele was combined to hsa-miR-3171,and EMSA showed that the rs1049522 A allele also bound to nuclear proteins.Conclusions:1.The variation of rs1049522,rs1042113,rs1170695,rs3749034,and rs3793243 were associated with the expression of SLC1A3,SLC1A2,SLC6A1,GAD1,and STX1A in the brain,respectively.2.The rs1049522 A allele undergone posttranscriptional regulation of SLC1A3 gene expression by binding to hsa-miR-3171and nuclear proteins.The above evidences indicate that the decreased expression of SLC1A3 leads to an increased risk of ADHD.