【作者】 李恒芬；

【导师】 谢光荣；

【作者基本信息】 中南大学 ， 精神病与精神卫生学， 2008， 博士

【摘要】 第一部分5-HT_2A及其信号转导SNP_s与心境障碍的关联分析目的:探讨5-HT_2A相关转导通路上G蛋白、腺苷酸环化酶、磷酸二酯酶和cAMP反应元件结合蛋白（CREB）相关基因SNPs与心境障碍关系,分析相关基因突变合并存在时是否增加心境障碍发病风险。方法:1.根据美国精神障碍诊断与统计手册第四版（DSM-Ⅳ）和《中国精神障碍分类方案及诊断标准（第三版）》（CCMD-3）有关心境障碍的诊断标准,选择符合有关双相或抑郁症诊断标准的汉族患者417例（其中抑郁症326例,双相障碍91例）,在相同地区范围内选择293名汉族健康对照;分别比较病例组基因型、等位基因分布差异,并与心境障碍进行关联分析。2.采用连接酶反应技术（LDR）,检测5-HT_2Ars6311、GNB3rs5443、ADCY9rs2230739和PDE_1Ars1549870等5-HT_2A相关信号转导的5个SNPs的基因多态性。3.选用SPSS 11.5 for windows软件包进行数据处理。采用拟合优度卡方检验,分析基因型、等位基因频率是否符合Hardy-Weinderg平衡定律;病例和对照组的基因型和等位基因频度比较,比较采用X₂检验;选用多元回归分析、关联比（odds ratio,OR）和95%的置信区间（confidence in teval,CI）评价患病风险;显著性水平为p=0.05。结果:1.5个SNPs位点的检测结果:对710个汉族人DNA样本的rs6311、rs5443、rs2230739、rs 2551638和rs1549870等5个SNPs位点的检测发现,rs6311、rs5443、rs2230739和rs1549870在汉族人群中存在多态性,而CREB1rs2551638则在汉族人中不存在多态性。2.病例组与对照组SNPs基因型频率和等位基因频率比较:417例心境障碍患者rs6311的TT、TC和CC三种基因型频率分别为0.316、0.494和0.19,其T等位基因频率为0.563,C等位基因频率为0.437;293名健康对照rs6311的TT、TC和CC三种基因型频率为0.433、0.430和0.137,其T等位基因频率为0.648,C等位基因频率为0.352;三种基因型分布存在显著性差异,病例组TI型明显减少,而CC型增多（x²=11.003,p=0.004）;病例组T等位基因频率降低,c等位基因频率增高（OR=1.434,95%CI=1.153～1.783;x²=10.523,p=0.001）。417例心境障碍患者rs5443的TT、TC和CC三种基因型频率为0.18、0.472和0.348,其T等位基因频率为0.416,C等位基因频率为0.584;293名健康对照rs5443的TT、TC和CC三种基因型频率为0.181、0.505和0.314,其T等位基因频率为0.433,C等位基因频率为0.567;两组基因型和等位基因比较差异均无显著性意义（p＞0.05）。415例心境障碍患者rs2230739的GG、GA、AA三种基因型频率分别为0.176、0.498和0.335,其G等位基因频率为0.42,A等位基因频率为0.58;293名对照组rs2230739的GG、GA、AA三种基因型频率分别为0.119、0.515和0.365,其G等位基因频率为0.377,A等位基因频率为0.632;两组基因型和等位基因比较差异均无显著性意义（p＞0.05）。408例心境障碍患者rs1549870的GG、GA、AA三种基因型频率分别为0.49、0.426和0.083,其G等位基因频率为0.703,A等位基因频率为0.297;291名对照组rs1549870的GG、GA、AA三种基因型频率分别为0.557、0.361和0.082,其G等位基因频率为0.737,A等位基因频率为0.263;两组基因型和等位基因比较差异均无显著性意义（p＞0.05）。3.不同亚型间SNPs基因型和等位基因分布比较:326例抑郁症患者中共有男134例,女192例,比较抑郁组与对照组比较发现,rs6311三种基因型分布存在组间显著性差异,抑郁症组TI型明显减少,而GG型增多（x²=6.460,p=0.04）,抑郁症组的T等位基因频率低于对照组,C等位基因频率则明显高于对照组（OR=1.360,95%CI 1.080～1.712;x²=6.865,p=0.009）;rs5443、rs2230739和rs1549870的基因型分布频率和等位基因频率则未见两组间存在明显差异（p＞0.05）。抑郁症组男性患者与女性患者4个SNPs基因型和等位基因分布比较,均未见明显组间差异（p＞0.05）。91例双相障碍患者与117健康对照比较发现,rs6311三种基因型分布存在组间显著性差异,双相组TT型明显减少,而GG型增多（x²=11.031,p=0.004、）;其他位点的基因型分布频率则未见两组间存在明显差异（p＞0.05）。双相组rs6311T等位基因频率低于对照组,C等位基因频率则明显高于对照组（OR=1.735,95%CI=1.168～2.578;X²=7.504,p=0.006）;其他SNPs的两组差异未见显著性意义（p＞0.05）。4.SNPs基因型与心境障碍的关联分析:将病例组与对照组rs6311、rs5443、rs2230739和rs1549870等4个SNPs的基因型进行关联分析发现,rs6311CC基因型患病的相对危险度为（OR=1.487,95%CI=0.983～2.249;x²=3.561,p=0.059）,TT基因型患病的相对危险度为（OR=1.659,95%CI=1.216～2.262;x²=10.287,p=0.001）。rs5443 TF、rs5443 TC、rs2230739AA、rs2230739GA、rs1549870 GG则均未见组间差异（p＞0.05）。采用logistic回归分析,4个SNPs各基因型心境障碍关系,结果发现rs6311的TT和TC基因型进入方程,回归系数为分别为-0.618和-0.161。5.rs6311与其他SNPs患病的联合风险:将rs6311各基因型与其他SNPs基因型的联合风险进行比较发现,rs6311的CC与rs5443TT或CC合并存在时,患病风险增加[（OR=4.838,95%CI=1.091～21.450;x²=5.237,p=0.022）或（OR=3.329,95%CI=1.703-6.510;x²=13.662,p=0.000）];rs6311的CC与rs2230739的GG合并时患病风险增加[（OR=3.375,95%CI=0.961～11.851,x²=4.046,p=0.044）];rs6311 CT与rs1549870 AG合并患病风险增加[（OR=1.645,95%CI=1.107～2.444,x²=6.313,p=0.013）]。结论:1.5-HT_2Ars6311在汉族心境障碍患者存在C等位基因频率增高,T位基因频率降低现象,且C等位基因频率增高在抑郁和双相中普遍存在,rs6311C等位基因频率升高导致患病风险增加,提示rs6311可能是5-HT_2A基因的高功能位点,建议将该位点作为心境障碍的标志性位点进一步研究。2.Gβ3的rs5443和ADCY₉的rs2230739两个位点在汉族中均属高频位点,病例组与对照比较没有发现与心境障碍存在关联的证据,但当rs6311CC与rs5443TT或CC型,以及与rs2230739的GG合并存在时患病风险有明显增加趋势,提示Gβ₃和ADCY₉可能是心境障碍的微效基因之一。3.没有发现PDE1Ars1549870与心境障碍关联的直接证据。4.CREB1rs2551638位点在汉族患者中不存在多态性,无法依赖该位点推断CREB与心境障碍的关系。第二部分5-HT_2A信号转导相关SNP_s与抑郁症表型特征的关系目的:探讨5-HT_2A信号传导通路的rs6311、rs5443、rs2230739、rs1549870等4个SNPs与抑郁症表型特征的关系,进一步论证这些SNPs与心境障碍的关系。方法:参照DSM-Ⅳ和CCMD-3对194例抑郁症患者临床症状,发作倾向和是否伴有精神病性症状进行分级评定,采用开放性临床治疗观察了194抑郁症患者对SSRIS的疗效,并对174例进行了为期1年以上的随访,观察其长期疗效;采用最优尺度检验分析SNPs基因型抑郁症表型特征与疗效的关系。结果1.四个位点与基本症状的关系:四个SNP_s基因型与基本症状的多重对应分析图显示:rs6311CC、rs5443CC与rs1549870GG与兴趣减少（基本症状2）在一个区域内,相对距离较近,其中rs5443CC与rs1549870GG距离最为接近;rs5443CT、rs1549870AG、rs2230739AG在一个区内,距离相对较近,没有发现与心境低落或可疑心境低落相关的基因型存在。2.4个位点与附加症状的关系:4个SNPS基因型与附加症状的多重对应分析图显示:rs6311CC与自杀观念及行为在同一单位阵内,与自杀观念距离最近,rs6311TT则与无自杀者同在同一单位阵,且相距紧密;rs6311CC还与失眠症状、认识症状在一个单位阵内,rs5443CT,rs1549870AG与疲劳症状群在同一单位阵。3.其他临床特征的关系:4个SNPS基因型与其他临床特征的多重对应分析图显示:rs6311CC与伴有精神病性症状和多次复发同在一个单位阵内,rs6311TT和少复发同在一个单位阵内;rs1549870GG与不伴精神症状同在一个单位阵内,距离较近。4.4个位点与SSRI类疗效及长期疗效的关系:rs5443、rs2230739和SSRI疗效有关,其回归系数分别为0.181和0.283;rs2230739和长期疗效有关,其回归系数为0.400。结论:1.据本研究发现rs6311CC型与抑郁症的部分症状包括兴趣减少、自杀、失眠、精神病性症状和复发倾向关系密切,推测rs6311可能是5-HT_2A的功能位点,建议进一步探讨5-HT_2Ars6311T/C多态性是否属于抑郁症的生物学内表型。2.Gβ3rs5443TT和ACDY₉rs2230739GG患者对SSRI类的短期疗效相对较好,Gβ3和ACDY₉可能是不同患者SSRIs疗效差异的分子遗传学基础。3.ACDY₉rs2230739GG型长期抗抑郁治疗效果较好,推测ACDY₉rs2230739基因多态性可能是个体间长期疗效差异的生物学基础。更多还原

【Abstract】 PARTⅠAssociation between the 5-HT_2Apathway related genes and mood disordersObjective:This research aims at the investigating the association between 5-HT2A pathway related genes,including G protein,adenylcyclase, phosphodiesterase or cAMP responsive element binding protein,and mood disorders.Also,the effects of the gene x gene interaction on the risk of mood disorders will be investigated.Methods:（1）With the case-control design,417 mood disorders and depressive disorder patients（326 depressive disorder patients,91 mood disorder patients）,met the criteria of DSM-Ⅳand CCMD-3,and 293 healthy Chinese Han were recruited in Henan Province,and respectively compared distributional difference of genotype and allele between two groups,and analyzed association between SNP_s and mood disorders.（2）Five SNPs of 5-HT2A rs6311、GNβ3rs5443、ADCY9rs2230739, PDE1Ars1549870,which were located at the genes of 5-HT_2Apathway, were discriminated by using the assay of LDR.（3）Performing SPSS 11.5 package,the Hardy-Weinberg equilibrium, the distribution of the allelic and genotype frequencies among the cases and controls were analyzed by x² test.The morbidity risk was evaluated using 95%confidence interval,odds ratio and confidence interval.The level of significance was p=0.05.Results:1.The polymorphism of the five SNPs::There were polymorphism of rs6311,rs5443,rs2230739 and rs 1549870,and no polymorphism of CREB1 rs255163 among these 710 Chinese Han（417 patients with mood disorders and 293 healthy controls）.2.Comparison of genotypic/allele frequency between patients and controls:Genotypic frequencies of the wild-type homozygote（TT）,TC and the mutation homozygote（CC）of rs6311 among 417 patients with mood disorders were 0.316,0.494 and 0.19 respectively,T allel frequency was 0.563,and C allel frequency was 0.437.Correspondingly in 293 healthy controls,genotypic frequencies of TT,TC and CC were 0.433, 0.430 and 0.137 respectively,T allel frequency was 0.648,and C allel frequency was 0.352.There was significant difference of genotypes distribution of rs6311 between patients and controls.The mutation homozygote（CC）frequency is significantly higher in patients than in controls,but the wild-type homozygote（TT）frequency is lower （x²=11.003,p=0.004）.Also the T allele frequency was lower and the C allelic frequency is higher in the patients（OR=1.434,95%CI=1.153～1.783;x²=10.523,p=0.001）.As for rs5443,its genotypic frequencies of TT,TC and CC in 417 patients were 0.18,0.472 and 0.348 respectively,T allel frequency was 0.416,and C allel frequency was 0.581. Correspondingly in healthy controls,its genotypic frequencies of TT, TC and CC were 0.119,0.515 and 0.365 respectively,T allel frequency was 0.433,and C allel frequency was 0.567.There were significant difference in neither genotype nor allel distribution between patients and controls（p＞0.05）.Genotypic frequencies of GG,GA and AA of rs 2230739 among 415 patients with mood disorders were 0.176,0.498 and 0.335 respectively,G allel frequency was 0.563,and A allel frequency was 0.437.Correspondingly in 293 healthy controls,genotypic frequencies of GG,GA and AA of rs2230739 were 0.119,0.515 and 0.365 respectively,G allel frequency was 0.377,and A allel frequency was 0.632.There was no significant difference of genotype\allel distribution between patients and controls（p＞0.05）.As for rs1549870,its genotypic frequency of GG,GA and AA among 408 patients were 0.49,0.426 and 0.083 respectively,G allel frequency was 0.703,and A allel frequency was 0.297.Correspondingly in healthy controls,its genotypic frequencies of GG,GA and AA were 0.557,0.361 and 0.082 respectively,G allel frequency was 0.737,and A allel frequency was 0.263.There were significant difference in neither genotype nor allel distribution between patients and controls（p＞0.05）3.Comparison of genotypic and allele distribution of SNPs in different subtypes:There were significant difference in three genotype interclasses of rs6311 by comparing between control group and depressive group（326 cases,134 males,192 females）.In depressive group, not only TT obviously decreased,while CC mcreased（x²=6.460,p-0.04）, but also T allele frequency of depressive group was lower,while C allele frequency was higher in depressive group（OR=1.360,95%CI=1.080～1.712;x²= 6.865,p=0.009）;There were no significant difference of genotypic \allele distribution of rs5443,rs2230739 and rs1549870 among interclasses（p＞0.05）,and no significant difference genotypic\allele distribution of four SNPs in interclasses between male group and female group also（p＞0.05）However,there were significant difference distribution of rs6311 in three genotype interclasses between 117 healthy controls and 91 patients with bipolar mood disorders.TT frequency is significantly higher in patients than in controls,but GG frequency is lower（x²= 11.031,p=0.004）,and there were no significant difference of genotype frequencies in other gene locuses between two groups（p＞0.05）. T allele frequency of bipolar mood disorder group was lower,but C allele frequency was higher than control group（OR=1.735,95%CI = 1.168-2.578;x²=7.504,p =0.006）.There were no significant difference of other SNPs between two groups（p＞0.05）4.Association analysis between SNPs genotypes and mood disorders:The relative risk of rs6311 CC genotype was（OR=1.487, 95%CI=0.983～2.249,x²=3.561,p=0.059）,and that of TT was（OR =1.659,95%CI=1.216～2.262;x²=10.287,p=0.001）.The genotype frequencies of rs5443 TT、rs 5443 TC、rs 2230739 AA、rs 2230739GA and rs 1549870 GG is trendily higher in patients.However,there were no significant difference（p＞0.05）.Regression analysis was performed to inVestigate the effects of the four SNPs genotypes on mood disorders.We found that TT and TC of rs 6311 entered equations,and their regression coefficient were -0.618 and -0.161 respectively.5.The sick combination risk of RS6311 with other SNPs genetypeWhen TT of rs6311 combined with CC of rs5443 or CC of rs6311, the sick risk would be increased,especially CC of rs6311 with TT or CC of rs5443[（OR=4.838,95%CI=1.091～21.450;x²=5.237,p=0.022） or（OR=3.329,95%CI= 1.703～6.510;x²=13.662,p=0.000）];When CC of rs63 combined with GG of rs 2230739,the sick risk would be increased（OR=3.375,95%CI=0.961～11.851,x²=4.046,p=0.044）; When CT of rs6311 combined with AG of rs1549870,the sick risk was increased（OR=1.645,95%CI=1.107～2.444,x²=6.313,p=0.013）,too.Conclusions:1.The SNP of rs6311,located in 5-HT2A,associates with the mood disorders in Chinese Han.It also has the association with the depressive disorder and the bipolar affective disorder.The gene mutation of rs6311 would lead to mood disorder directly perhaps or there were virulence gene near the genetic locus possibly.We could reach the conclusion that the CC genotype carrier suffer from higher risk of the onset of the disorders,while T allele carrier lower risk.On the other hand,since there is no evidence that the rs6311 affects the expression of the 5-HT_2Agene, the results on rs 6311 might suggest that there is another functional locus near it.Therefore,it is worthy to undergo the detail investigation.2.Both rs 5443 in GNB3 and rs 2230739 in ADCY9 are high allelic frequency SNPs in Chinese Han.When CC of rs6311 combined with Tr or CC and with GG of rs 2230739,the sick risk is increased obviously, although there is no evidence for association between them and mood disorders.The two SNPs allele located at coding region,so GNβ3 and ADCY9 may be the minor genes on mood disorders.3.No proofs were found that there were association between PDE1Ars1549870 and mood disorders.4.No polymorphism of rs 2551638 in CREB1 among Chinese Han was found,so we can not infer whether CREB is related to mood disorders. PARTⅡAssociation between Polymorphisms of the Genes Related to 5-HT_2APathway and Phenotypes of Major Depression DisorderObjective:To investigate the relationship between four SNPs located in the genes related to 5-HT_2Apathways and symptoms of major depression disorder（MDD）and the association of the SNPs with MDD.Methods:A total of 194 patients with major depression disorder（MDD）were recruited in this study.According to DSM-Ⅳand CCMD-Ⅲcriteria,the symptoms,episode of tendency,and psychotic symptoms were scaled.Among 194 patients with MDD,the SSRIs treated outcome was evaluated in an open-trail study.174 out of them were followed up longer than one year while the optimal scaling was used to investigate the association between different genotypes of the SNPs and phenotypes, such as the symptoms and the outcome with SSRIs treatment.Results:1.Relationship between four SNPs and fundamental symptomsThe Multiple Correspondence Analysis shows the strong association between the four SNPs and the symptoms of MDD.The genotypes of rs6311CC,rs5443CC,and rs1549870GG are relatively closed to the symptom 2 in the same block.The distance from rs5443CC and rs1549870GG to the symptom 2 is the shortest.The genotypes of rs5443CT,rs1549870AG and rs2230739AG are in the same zone. Although the distances between the three SNPs are short,no association between them and the fundamental symptoms of MDD or suspected depressive mood was found.2.Relationship between four SNPs and accessory symptomsThe Multiple Correspondence Analysis also shows the association between the four SNPs and the accessory symptoms.The genotype of rs6311CC and accessory symptoms 7（suicidal thoughts and acts）are in the same zone,because the distance between them is the shortest. However,the genotype rs6311TT and the non-suicide are in the same block and stay very close.The distance between rs6311CC and symptoms, including insomnia and cognitive symptoms,is relative short.The rs5443CT and the rs1549870AG associate with fatigue symptoms.3.Relationship between four SNPs and other clinical charactersThe Multiple Correspondence Analysis shows the association between the four SNPs and other clinical characters.The rs6311CC and depressive episode with psychotic symptoms and relapse are in the same block.The genotype rs6311Tr predicts less relapses of MDD because both of them are in the same block.The rs1549870GG and depression without psychotic symptoms are in the same zone and near.4.Relationship between the four SNPs and the long-term outcome with SSRIs treatment.Two SNPs of rs5443 and rs2230739 associated with short-term outcome with SSRIs treatment.The coefficients of regression are 0.181 and 0.283,respectively.The rs2230739GG predicts the good long-term outcome with SSRIs treatment.The coefficient of regression is 0.400.Conclusion:1.The rs6311CC strong associates with depressive symptoms, including loss of interest and pleasurable feeling,suicide,insomnia, psychotic symptoms and relapse tendency.It suggests that rs6311 may be a functional SNP affecting the expression of the 5-HT_2Agene.It remains unclear whether the polymorphism of rs6311 is the genotype of depression or not.2.The rs5443TT（Gβ₃）and rs2230739GG（ACDY₉）carriers have better short-term response to SSRIs.It is presumed that different genotypes of rs5443（Gβ₃）and rs223073（ADCY₉）can modulate the second message signal conduction pathways,and therefore lead to different response.3.The rs2230739GG（ADCY₉）carriers have better long-term respponse to SSRIs.We may conclude the polymorphism of rs2230739 （ADCY₉）may be the biological basis for the inter-individual different response.更多还原