Association Between Genetic Variations Of Kv Channels And Their Accessory Proteins And Attention-deficit/Hyperactivity Disorder

Attention-deficit/hyperactivity disorder(ADHD)is one of the most common childhood neurodevelopmental disorders,which seriously affects children’s physical and mental health.At present,the etiology of ADHD remains unclear,and most of the studies suggest that ADHD is a complex disease under genetic and environmental interactions.In genome-wide association studies(GWASs)Kv channels and their accessory proteins genes have been identified to be potentially associated with ADHD.Biological studies also show that Kv channels and their accessory proteins may be involved in the pathogenesis of ADHD.Thus.we conducted a two-stage association study to comprehensively explore the relationship between Kv channels and their accessory proteins and the risk of ADHD,especially the roles of gene-gene and gene-environment interactions.Furthermore,we used molecular biology technology to illuminate the biological mechanism of positive genetic variations.This study will provide evidence for the early screening,prognosis monitoring and new therapeutic targets of ADHD in Chinese population.Part 1 Study on the genetic susceptibility in Kv channels and their accessory proteins to ADHDObjectives:To explore the associations of the genetic variations in Kv channel genes with the risk of ADHD and its clinical characteristics,and to evaluate the effect of gene-gene interactions on ADHD susceptibility.Methods:We conducted two stages of case-control study.All cases were newly diagnosed ADHD children by DSM-Ⅳ,and the controls were the healthy children for physical examination during the same period.All subjects were tested by Chinese Wechsler Intelligence Scale for Children(C-WISC).Parent Symptom Questionnaire(PSQ)and Continuous Performance Test(CPT).The genotyping was performed by Sequenom MassArray both in the two stages.256 cases and 372 controls were recruited from Wuhan area in the first stage,and SNPs in Kv channel genes associated with ADHD risk and its clinical features were identified.The candidate SNPs were validated in the second Changsha stage(328 cases and 431 controls).Chi-square test and t test were applied to compare the basic characteristics of the cases and the controls.Logistic regression model was used to analyze the risk OR and P value of each SNPs,and the correlation between SNPs and ADHD symptom scores was analyzed by ANOVA.Bonferroni’s correction method was used for multiple corrections.The multiplicative and addictive gene-gene interactions were analyzed by Logistic regression model and Excel tables compiled by Anderson,respectively.Results:1.Corrected by Bonferroni’s correction,KChIP1 rs1541665(OR=1.665,95%CI=1.201-2.319)and FHIT rs3772475(OR=1.572,95%CI=1.128-2.306)were nominally associated with ADHD risk under dominant model in the first stage.In the second stage,we successfully demonstrated a nominal association between KChIP1 rs1541665 and the risk of ADHD(OR=1.543,95%CI=1.165-2.069).In combined samples.KChIP1 rs1541665(OR=1.559,95%CI=1.259-1.976)was significantly associated with ADHD,and FHIT rs3772475 was nominally associated ADHD risk(OR=1.476,95%CI=1.043-2.089).2.No significant interactions between KChIPl rs1541665 and FHIT rs3772475 was found in gene-gene interaction analysis(P>0.05).3.For the associations of genetic variations with ADHD clinical features,KChIP1 rs1541665 has been found nominally associations with ADHD-I(OR=1.878.95%CI=1.298-2.773;OR=1.678,95%CI=1.198-2.473).hyperactive index in PSQ scores(P=0.035;P=0.016)and attention deficit in CPT(OR=1.509,95%CI=1.011-2.431;OR=1.532,95%CI= 1.095-2.149)both in the stage one and the stage two.In two stages FHIT rs3772475 was associated with hyperactive index in PSQ scores(P=0.016;P=0.034).Conclusions:1.KChIP1 rs1541665 and FHIT rs3772475 were associated with the risk of ADHD.2.KChIP1 rs1541665 was associated with ADHD subtypes,ADHD symptoms and attention deficit.FHIT rs3772475 was associated with ADHD symptoms.Part 2 Study on the roles of gene-environment interactions of Kv channels and their accessory proteins in ADHDObjectives:To explore environmental factors associated with ADHD,and to evaluate the roles of gene-environment interactions of Kv channels and their accessory proteins in ADHD.Methods:A self-designed questionnaire was used to investigate the general situation and related environmental factors of all the subjects.Atomic absorption spectrometry was used to measure trace elements(blood lead,magnesium,calcium,zinc and iron).In the merged data of two stages.Logistic regression model was used to analyze environmental factors associated with ADHD,and gene-environment interactions were evaluated by classification and regression tree(CART)and multifactor dimensionality reduction(MDR).Results:Univariate and multivariate Logistic regression analysis showed that a lower educational level of mother,an uneasy parental relationship,lower family income,a noisy family environment,prenatal stress,high blood lead and low birth weight were risk factors for ADHD;while high blood magnesium was a protective factor for ADHD(P<0.05).The results of CART and MDR showed that KCNIP1 rsl541665 interacted with prenatal mood,family income and blood lead in the course of ADHD.Logistic regression showed that KChIP1 rs1541665 had a multiplicative interaction with prenatal mood and blood lead(Pmul=0.046;Pmul=0.017).The interactions of KChIP1 rs 1541665(CT+CC)genotype with bad mood during pregnancy and high blood lead can increase the risk of ADHD(OR =2.695,95%CI = 1.858-2.271;OR=1.937,95%CI = 1.444-2.598).Conclusions:A lower educational level of mother,an uneasy parental relationship,lower family income,a noisy family environment,prenatal stress,high blood lead and low birth weight were risk factors for ADHD;while high blood magnesium was a protective factor for ADHD.KChIP1 rs1541665 may interact with prenatal mood and blood lead in the course of ADHD.Part 3 Study on the biological mechanism of genetic variations of Kv channels and their accessory proteins gene in ADHDObjectives:To investigate the potential functional genetic variation of Kv channels and their accessory proteins gene and its mechanism in ADHD.Methods:Several bioinformatics tools,such as SNP Info,RegulomeDB,rSNPBase,Haploreg and GWAVA were used to annotate and screen out the potentially functional variants in the tight LD region of significant associated SNPs rs1541665 and rs3772475.Dual luciferase reporter assay was performed to analyze the effects of different alleles on transcription efficiency in vitro.Results:We selected the most potentially functional SNP rs1363713(possible miRNA hsa-miR-3126-5p binding site)from the tight LD regions of the significant SNP rs1541665 through multiple bioinformatics analyses.Dual luciferase reporter assay indicated that the fragment with rs1363713 C allele significantly reduced luciferase activity with rs1363713 T allele(P<0.05).Conclusions:The positive association site rs1541665 may be regulated by miRNA through its tightly linked 3 ’UTR rs1363713,resulting in a decrease in KChIP1 gene expression,down regulation of type A potassium channels,and increased excitation of local neurons.Further study is needed to explore its biological mechanism.