Whole Exome Sequencing Identified Novel Susceptibility Genes Responsible For Mood Disorder In A Chinese Pedigree And Validated In Vitro And Vivo

Objective: Mood disorders are a group of serious,complex mental illnesses.Although their etiology remains largely unknown,a lot of studies have shown that mood disorders have a strong genetic component with heritabilities estimated to be as high as 80%-85%.Despite of the success of genome-wide association studies,the identified and validated causal or associated genetic variants so far can explain only a small portion of the heritability.Possible reasons for the missing inheritance include genetic heterogeneity,rare variants,and genetic-environment interaction.Availability of next-generation whole genome or exome sequencing now permits the study of rare single nucleotide variants(SNVs)and small insertions/deletions(in/dels)in a systematic genome-wide manner.Although whole genome sequencing has many advantages,such as allowing examination of both coding and non-coding regions(especially regulatory regions),WES is more cost effective,has much less computational burdens,and can quickly and effectively identify common and rare variants.The aim of this study is to examine the role that genetic variation confers on disease risk in a severely affected pedigree.We conducted in vivo and in vitro validations of the top candidate gene to explore the pathogenesis of mood disorder.We expect that our study will advance the understanding of mood disorders and provide new insight into their pathogenesis,early diagnosis and treatment and prevention.Methods: 1.Whole exon sequencing study of a pedigree To find susceptibility genes or loci of mood disorder,exome sequencing was performed in a multigenerational,severely affected pedigree of the mood disorders.High-quality reads were retrieved from raw reads by filtering out the low quality reads and adaptor sequences using the Solexa QA package and the cutadapt program,respectively.Following the strategy proposed in the program package Mendel Scan,we prioritized potential causal variants within the pedigree based on segregation with mood disorder,predicted functional effects,and prevalence in human populations.2.In vivo studies(1)Differential expression analysis Using case-control and forward-looking tracking study design,differential expression analysis was conducted for the candidate gene MAPKAP1 in peripheral blood leukocytes for 22 MDD patients vs.22 healthy controls,and for the 22 MDD patients before vs.after antidepressant treatment for 8 weeks were detected.(2)Genetic association analysis Genetic association tests were performed between the top candidate variant and quantitative metrics characterizing mood disorders using data from 307 Han Chinese patients with MDD and 504 ESA(including 103 CHB and 105 CHS)healthy controls from the Hap Map project.The mutation allele frequency of the top candidate variant in the MDD patients was contrasted with that in the general population.The statistical significance of the comparison of the mutation allele frequency for the top candidate variant in the patient group and the general population was evaluated using ?2 and Fisher's exact probability tests.3.In vitro studies Validation of cellular level function of MAPKAP1 We used SH SY5 Y cell line,transfected with N.C.siRNA and MAPKAP1-siRNA virus to elucidate the biological function of MAPKAP1 at the cellular level.We detected key MAPKAP1 and its targets and signaling pathways genes gene expression using Real-Time qPCR and western blot test.4.Validation of mood disorder pathway of MAPKAP1 in mood disords of human and animal model We validated the pathway of MAPKAP1 in relation to mood disorders we identified,using data from high-throughput gene expression and hybrid microarray experiment from the GEO database.We extracted data from mouse brain tissue sample(GDS5307),the peripheral blood samples(GPL10558)of the human population and human population brain tissue samples(GSE53987).We conducted key MAPKAP1 and its targets and signaling pathways gene expression using t-test.Results: 1.Whole exon sequencing study of a pedigree Our whole exon sequencing study identified 26 candidate rare variants for the familial mood disorder.The top-ranked variant is located in the intronic region of the MAPKAP1 gene,a key component of mTORC2 signaling complex necessary for AKT phosphorylation.Our team have previously reported that the key genes,AKT1 and GSK3 B,of mTORC2 signaling appear to be associated with MDD in the Han Chinese population.We thus hypothesize that MAPKAP1 play an important role in the development of mood disorders through proliferation,differentiation and apoptosis of neurons..2.In vivo studies(1)Genetic association analysis The mutation allele(G)frequency of the candidate variant rs78809014 is 6.1% in 307 MDD patients,marginally significantly higher than in the CHB population in the Hap Map project(4.4%),significantly higher than in CHB+CHS 3.1%(?2=4.57,P=0.03)and significantly higher than ESA 2.8%(?2=10.50,P=0.001),We also found that rs78809014 was significantly associated with cognitive impairment factor,feelings of guilt and suicide attempts(p<0.05).Patients with the rs78809014-G allele had a significantly more severe illness compared to patients with the rs78809014-C allele.(2)Differential expression analysis 1)The expression level of MAPKAP1 in MDD patients was significantly higher than that in control subjects(t=-3.654,P=0.001),and significantly decreased after treatment(t=-2.105,P=0.048).2)The expression level of MAPKAP1 was positively correlated with the score of retardation factor of HAMD-17(r=-0.444,P=0.038),and positively correlated with the score of depression of retardation factor(r=-0.470,P=0.027).3)The expression of MAPKAP1 was negatively correlated with immediate memory(r=-0.531,P<0.001),visual span(r=-0.456,P=0.002).The expression of MAPKAP1 was positively correlated with attachment A time(r=0.425,P=0.004).3.In vitro studies The m RNA expression level of MAPKAP1 in MAPKAP1-siRNA cells was significantly lower than that in N.C.siRNA cells.The m RNA expression level of GSK3B??-catenin in MAPKAP1-siRNA cells was also significantly lower than that in N.C.siRNA cells.The protein expression level of p AKT1 in MAPKAP1-siRNA cells was also significantly lower than that in N.C.siRNA cells.Decreasing of expression of MAPKAP1 reduced the expression of related genes of the mTOR signal pathway in SH-SY5 Y cell.4.Validation of mood disorder pathway of MAPKAP1 in mood disords of human and animal model(1)The expression level of MAPKAP1 in MDD animals models(CUMS)was significantly higher than that in controls(t=-3.216,P=0.005),and the expression level of MAPKAP1 in antidepressant response group was significantly higher than that in controls(t=4.946,P<0.001),and the expression level of MAPKAP1 in antidepressant response group was marginally significantly higher than that in antidepressant resistant group(t=-2.055,P=0.07).(2)The expression level of MAPKAP1 in autopsy hippocampus of mood disorders was higher than that in healthy controls(t=-1.874,P=0.067).The expression level of AKT1 in autopsy hippocampus of mood disorders was higher than that in healthy controls(Z=-2.592,P=0.010).The expression level of MAPKAP1 was positively correlated with AKT1 both in peripheral blood(r=0.427,P=0.001)and autopsy prefrontal lobe of mood disorders(r=0.364,P=0.007).Conclusion: 1.MAPKAP1 may be a susceptibility gene for mood disorders,and may be involved in the etiology of mood disorders by the neuroplasticity function pathway(PI3K/Akt/mTORC2).2.The candidate variant rs78809014 in the MAPKAP1 gene appears to be associated with cognitive impairment factor,feelings of guilt and suicide attempts in patients with depressive disorder.3.The expression level of MAPKAP1 in MDD patients was significantly higher than health controls,and decreased significantly after antidepressant treatment.The expression of MAPKAP1 was positive correlated with the score of retardation factor of HAMD-17,and positively correlated with the score of depression of retardation factor.The expression of MAPKAP1 was negatively correlated with immediate memory,and a higher expression of MAPKAP1 has poorer immediate memory.4.The expression level of MAPKAP1 in the CUMS mice ventral prefrontal cortex was correlated with antidepressant response and maybe a target of antidepressant treatment.