【作者】 龚云国；

【导师】 朱军； 贺林；

【作者基本信息】 浙江大学 ， 作物学， 2009， 博士

【摘要】 精神分裂症是一种严重的精神疾病，在普通人群中发病率约为1％，多数人终身不愈。本文通过荟萃分析和基于microRNA预测软件的生物信息学手段，挖掘精神分裂症易感基因以及相关功能性多态性位点，这些位点可能通过调节某些重要基因mRNA的表达，从而在疾病中发挥重要功能。本研究结果将为深入研究特定多态性位点的功能提供有价值的指导。谷胺酸脱羧酶(GAD1)催化谷胺酸合成一种重要的神经递质——γ-氨基丁酸，许多研究均显示该通路与精神疾病有关。有关GAD1基因多态性与精神疾病的相关性研究试图从遗传学角度探讨相关精神疾病的发病机制，但结果很不一致。我们收集了2008年8月以前pubmed收录的所有GAD1基因多态性与精神分裂症及其相关疾病的文献进行荟萃分析。结果表明，在该基因中没有发现与疾病明显相关的SNPs；但是，文献报道阳性的haplotype block覆盖整个GAD1基因，这提示精神分裂症的易感性可能与GAD1的特定SNP组合有关，而不是单一的某个SNP位点。神经调节因子(NRG1)也是研究精神分裂症倍受关注的基因之一，对其与精神分裂症的关联已经有3次meta分析被报道，但互相矛盾。本文分析了2008年9月以前pubmed收录的所有case-control以及family-based的相关性研究，共8049例病人和8869例对照人群以及1515个家系。结果表明(1)对于已有的两种整合case-control研究和family研究的方法进行对比，发现Lohmueller方法与Kazeem方法在分析结果上没有统计差异，表明这两种整合方法没有明显差异；(2)case-control研究设计与包括case-control和family在内的研究设计其荟萃分析结果也没有统计差异，说明family与case-control研究其结果的一致性；整合两种设计的实验其结果将更加准确，统计效力增强；(3) NRG1的SNP8NRG221132，420M9-1395(0)，478B14-848(0)呈阳性结果，其余的包括研究最多的SNP8NRG221533都呈阴性。根据标记频率计算的共祖先系数显示人群分层非常明显；(4)构建的易感基因功能网络显示，这些易感基因相互联系很小，提示精神分裂症多致病因之间的独立性。越来越多研究证明，miRNA与精神分裂症等神经和精神性疾病密切相关。miRNA与靶基因3’UTR结合区域内部的SNPs(PolymiRTS)可能会影响miRNA与靶基因的结合。关于精神分裂症易感基因可能的PolymiRTS及其在精神分裂症中的作用，目前尚没有文献报道。我们通过miRNA靶基因预测软件(pictar，microinspector，rnahybrid，miRbase，miRanda)，对425个与精神分裂症相关联或落在关联区域的基因进行了分析。我们共定位到803个SNPs可能位于miRNA的结合域(PolymiRTS)，并计算了这些位点与miRNA结合的吉布斯自由能(△G)以及不同基因型对△G的改变(△△G)。我们接下来检索了Szdabase和Pubmed关于这803个SNPs与精神分裂症及其他疾病的关联研究，并基于“不同基因型——影响转录本表达——导致表型改变”的模式和△G、△△G的变化，我们对这些SNPs与精神分裂症可能的关联和功能进行了分析。结果如下：(1)部分PolymiRTS在3个以上的研究中被报道与精神分裂症阳性关联，包括rs10759 of RGS4，rs165599 of COMT以及rs372055 of PRODH；(2)被报道与其他疾病相关联的预测到的PolymiRTS可能与精神分裂症阳性关联；(3)目前尚没有关联研究报道的预测到的PolymiRTS可能是精神分裂症阳性关联位点。总的来说，本论文对GAD1和NRG1的荟萃分析在方法上有了新的改进和扩展，并强烈提示了易感基因的单个突变位点对疾病表型影响的弱效性和遗传因素在人群中的异质性；采用遗传学方法来处理复杂疾病，有待更新的遗传统计理论和算法来解决这些问题。另外，本研究中所识别到的可能具有功能的PolymiRTS将对今后设计精神分裂症的关联分析以及进行miRNA在精神分裂症中的实验性研究提供重要的依据。更多还原

【Abstract】 Schizophrenia is kind of serious psychiatric disease with the disease incidence being about 1% among common population. Most patients are incurable permanently. By meta-analysis and microRNA-based bioinformatics prediction software, we scooped some susceptible genes and some potentially functional SNPs. These SNPs can regulate the mRNA expression of some critical genes and thus play a very important role in schizophrenia. Our findings hold great significance for further experimental studies of the SNPs functions in schizophrenia.Theγ-amino butyric acid is synthesized by glutamate decarboxylase (GAD1) and is involved in a number of neural disorders. But the results of the the association between GAD1 and schizophrenia have been inconsistent. We therefore performed the meta-analysis to try to reconcile the inconsistency and to clarify the contribution of the GAD1 to schizophrenia. We collected all the published association studies up to August 2008 involving thirteen studies. The results showed no association for SNPs of GAD1 with schizophrenia and other psychic disorders. Interestingly, the risk attached to haplotypes in one block spanned the whole glutamate decarboxylase gene 1, and these haplotypes contributed to the risk of neural disorders, especially schizophrenia. This meta-analysis may be the first to focus systematically on the genetic association of GAD1 in neural disorders. It suggests the potential roles of the whole GAD1 genome, yet not a single locus in the pathogenesis of schizophrenia and other psychic disorders.NRG1 is another most researched gene with schizophrenia. However, studies of the association of NRG1 with schizophrenia have so far produced inconclusive and even conflicting results. We performed a meta-analysis of 26 published case-control and family-based association studies up to September 2008. 8049 cases, 8869 controls and 1515 families were analyzed. We found that: ( 1 ) Kazeem’s or Lohmueller’s Method makes no difference to synthesize family and case-control studies; ( 2 ) the association analysis of case-control studies was statistically consistent with family studies;( 3 )Part of the markers showed significant association with schizophrenia and the population heterogeneity was evident; (4)The 22 risk genes in the network were unconnected to each other, underlining the independent multi-etiologies involved in schizophrenia.miRNA is reported to be involved in neurological and psychiatric disorders, especially schizophrenia. In addition, robust association studies of schizophrenia have been carried out to identify positive mutations of allele. As polymorphisms in miRNA-binding sites (PolymiRTS) might affect the binding of miRNA to its targets, whether polymorphisms in miRNA-binding sites of the positive genes in schizophrenia hold functions remain undetermined. In this study, 425 genes associated with schizophrenia or located in linkage regions were selected to predict the putative miRNA-binding sites by means of specialized algorithms (pictar, microinspector, rnahybrid, miRbase, miRanda). Then, 803 SNPs within the putative binding sites were identified and their ability to affect or impair the binding with the miRNA was weighted by assessing theΔG and the variation ofΔG (ΔΔG, Gibbs free energy), through comparing the wild-type and variant alleles. In addition, the association of such 803 SNPs with schizophrenia and other diseases was searched in Szdabase and Pubmed and their potential putative functions were analyzed. The findings are as follows: (1) Part of the putative PolymiRTS were associated with schizophrenia in more than 3 studies, including rs10759 of RGS4 , rs165599 of OMT, and rs372055 of PRODH, etc; (2) Putative polymiRTS associated with other disease might be potential positive SNPs in schizophrenia. (3) Putative polymiRTS not reported before might be potential positive SNPs in schizophrenia. All together, the identified potentially functional polymiRTS in this study hold great significance for the design of future association studies as well as experimental miRNA-function studies in schizophrenia.All together, great improvements have been achieved in the methods of meta-analysis of GAD1 and NRG1 with psychiatric disorders. And the results suggested strongly that a single SNP doesn’t contribute to the complex diseases significantly and the population heterogenetity are very important. Better hereditary methods are required urgently to manage such complex diseases. The PolymiRTS screening makes great significance for further case-control analysis for schizophreniagenetic research as well as for experimental research of SNPs functions in vitro or invivo.更多还原