【作者】 余金丹；

【导师】 赵正言；

【作者基本信息】 浙江大学 ， 儿科学， 2011， 博士

【摘要】 目的：孤独症谱系障碍(Autism spectrum disorders,ASDs)是一种广泛发育障碍性疾病,以社会交往障碍、语言发育障碍、兴趣范围狭窄和刻板重复的行为方式为基本临床特征。近年来孤独症的发病率呈明显的上升趋势,已影响到越来越多儿童的健康发育,给社会和家庭带来巨大的经济和精神负担,成为需要紧急关注的公共健康问题。因此积极探索孤独症的病因,阐明孤独症的致病机制,为其有效预防和早期干预提供依据也已成为当务之急。迄今孤独症确切的发病机制尚不清楚,但越来越多的研究显示遗传因素起了主要作用。目前普遍认为孤独症是一种复杂的多基因遗传疾病,由许多微效基因相互作用,并由环境致病因子诱发,共同导致孤独症的发生。基于全基因的连锁分析发现2q、7q、22q、Xq和Xp等多个部位与孤独症高度连锁。而通过对孤独症病理生理的初步研究,提出神经生物学假说参与孤独症的病理发病机制,并认为突触发育障碍是其中一个重要组成部分,推测突触结构异常或功能失衡会造成脑功能紊乱,导致孤独症发生。因此近十年来突触相关基因,特别是NRXNs-NLGNs-SHANK3通路和孤独症相关性的研究成为新的热点。因此本研究选取该通路中5个功能相关且位于上述连锁区的的突触基因NRXN1、NLGN3、NLGN4X、CNTNAP2、SHANK3作为候选基因,以标签SNPs作为标记,运用病例对照方法分析上述基因的单个SNP位点及单体型和孤独症的关联,探讨NRXN1、NLGN3、NLGN4X、CNTNAP2和SHANK3基因是否为中国汉族儿童孤独症的易感基因。作为复杂的多基因疾病,孤独症基因-基因间交互作用被认为可能在决定个体对于疾病的易感性方面具有极为重要的作用,因此很有必要将不同基因的各自的微效作用联合起来,进行基因交互作用分析。因此,我们采用多因子降维分析方法研究上述突触相关基因NRXN1、CNTNAP2、NLGN3、NLGN4X、SHANK3的交互作用对中国汉族儿童孤独症致病易感性的影响。随着孤独症遗传学研究的深入和基因检测技术的发展,在孤独症病人中检测出越来越多的罕见基因突变,研究者认为虽然这些基因突变发生频率极低,但可能对某些孤独症病人起强效作用,因此有助于我们更好理解孤独症的发病机制及潜在的病理基础。为此,本研究筛检了上述5个突触基因中21个已知的孤独症相关罕见突变,初步了解其是否存在于中国汉族儿童孤独症中并参与疾病发生。第一部分突触相关基因NRXN1、NLGN3、NLGN4X、CNTNAP2、SHANK3单核苷酸多态性与孤独症相关性分析方法：1.样本采集和基因组DNA抽提：本研究共包含229例孤独症儿童患者和191例无精神神经疾病的正常对照儿童。孤独症诊断按照国际疾病分类(ICD-10)和精神疾病诊断和统计手册(DSM-Ⅵ)。采集病例组和对照组外周血,并采用AxyPrep血基因组DNA小量试剂盒抽提基因组DNA,并于-80℃保存备用。2. SNPs位点的选择和分型：使用SNPbrowser软件,用SNP标签法(SNP-tagging)来选择标签SNP,再从中选取最优的31个SNPs位点作为分子标记,运用基质辅助激光解吸电离飞行时间质谱(Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry,MALDI-TOF MS)方法进行基因分型。3.NRXN1、NLGN3、NLGN4X、CNTNAP2、SHANK3基因和孤独症相关性的统计学分析：包括Hardy-Weinberg遗传平衡检验；单个SNP与孤独症的相关性分析；不同SNPs标记之间的连锁不平衡分析；构建单倍型,估算单倍型频率以及不同单倍型和孤独症的相关性分析,并进行性别分层分组病例对照相关分析。统计分析的显著性差异的P值水平为<0.05。结果：1. NRXN1单核苷酸多态性与孤独症的病例-对照相关分析结果：(1)单个SNP和孤独症的相关分析显示,NRXN1基因中的内含子部位的SNPrs1421589与女性孤独症患者存在显著性相关(P=0.038),其T等位基因可以增加女性患孤独症的危险(OR=1.955,95%CI：1.035-3.69),并且在加性遗传模式下,其基因型分布也与女性孤独症存在显著性相关(P=0.023)。单倍型相关分析显示包含该SNP位点的单倍型T-T(rs1421589-rs1563018)可以增加女性患孤独症的危险(P=0.039,OR=1.925,95%CI：1.029-3.604)。(2)NRXN1基因中外显子SNP rs1045874与孤独症中存在显著性相关(P=0.016,OR：1.85,95%CI：1.11-3.07),遗传模式是超显性模式。2.NLGN3基因单核苷酸多态性与孤独症的病例-对照相关分析结果：(1)单个SNP和孤独症的相关分析显示,各有3个SNP分别与男性孤独症和女性孤独症患者存在显著性相关,男性为rs11795613,rs4844285,rs5981079；女性为：rs11795613,rs4844285,rs7051529,且在加性遗传模式下,此3个SNP基因型分布与女性孤独症患者存在显著性相关(rs11795613,P=0.036；rs4844285,P=0.036；rs7051529,P=0.038)。同时显示rs4844286也与孤独症存在相关性,但其相关性不存在性别差异,rs4844286-T为危险性等位基因(P=0.020,OR=1.595,95%CI：1.076-2.365)。(2)连锁不平衡和单倍型相关分析显示,NLGN3基因的单倍型域Block1构建4-SNP单倍型A-G-T-T、3-SNP单倍型A-G-T、2-SNP单倍型A-G、G-A和T-T,其单倍型频率在男性病例组与对照组之间存在显著性差异,单倍型A-G-T-T、A-G-T、A-G和T-T可以增加男性患孤独症的危险(A-G-T-T:P=0.031,OR:1.633,95%CI：1.046-2.549；A-G-T：P=0.015,OR：1.758,95%CI：1.112-2.778：A-G：P=0.0113,OR：1.803,95%CI：1.14-2.854：T-T：P=0.047,OR：1.571,95%CI：1.005-2.457),而单倍型G-A则可以降低男性患孤独症的危险(P=0.0184,OR：0.575,95%CI: 0.361-0.913)。NLGN3基因的单倍型域Block1构建3-SNP单倍型A-G-T、2-SNP单倍型A-G、G-A和单倍型域Block2构建G-T单倍型,其单倍型频率在女性病例组与对照组存在显著性差异,其单倍型A-G-T和A-G可以增加女性患孤独症的危险(A-G-T: P=0.03,OR: 2.16, 95%CI: 1.071-4.357; A.G: P=0.035,OR：2.147,95%CI：1.048-4.400),而单倍型G-A和G-T则可以降低女性患孤独症的危险(G-A：P=0.035,OR：0.466,95%CI：0.227-0.954；G-T：P=0.044,OR：0.383,95%CI：0.147-0.999)。3.NLGN4X基因单核苷酸多态性与孤独症的病例-对照相关分析结果：(1)单个SNP和孤独症的相关分析显示,NLGN4X基因内含子SNP rs5961397与女性孤独症患者存在显著相关性,其A等位基因可以增加女性患孤独症的危险(P=0.047,OR=2.133,95%CI：1.001-4.545),其基因型分布在加性遗传模式下也与女性孤独症患者存在显著性相关(P=0.039)。(2)单倍型相关分析显示单倍型域Block1构建的单倍型G-G和Block3构建的单倍型A-C分别与女性和男性孤独症存在显著性相关,单倍型G-G可以降低女性患孤独症的危险(P=0.024,OR：0.404,95%CI：0.180-0.904),单倍型A-C则可以增加男性患孤独症的危险(P=0.022,OR：2.551,95%CI：1.121-5.804)4.CNTNAP2和SHANK3基因单核苷酸多态性和孤独症的相关性分析结果：(1)单个SNP和孤独症的相关性分析结果未发现两个基因中单个SNP位点和孤独症存在显著相关性。(2)连锁不平衡及单倍型相关分析显示,也未发现CNTNAP2和SHANK3基因的的单倍型与孤独症存在显著性相关。结论：本研究基于候选基因SNP的病例-对照关联分析,对5个突触相关基因NRXN1、NLGN3、NLGN4X、CNTNAP2、SHANK3和中国汉族儿童孤独症的相关性进行了初步研究,结果显示NRXN1、NLGN3和NLGN4X基因可能是中国汉族儿童孤独症的易感基因,而CNTNAP2和SHANK3基因可能不是中国汉族儿童孤独症的易感基因。第二部分突触相关基因的交互作用与汉族儿童孤独症关联分析方法：1.数据筛选：来源于本研究第一部分通过MALDI-TOF MS技术得到的5个突触相关基因的单核甘酸多态性信息。剔除不符合Hardy-Weinberg平衡的SNP位点和有缺失数据的研究个体,共计29个SNP位点和348个研究对象进入多因子降维分析。2.统计分析：基因-基因交互作用的多因子降维分析方法采用MDR version 2.0软件包。选择具有最大的交叉验证一致性、最大检验准确度且排列检验结果具有显著性的组合作为所有组合的最佳模型。结果：两位点最佳模型(rs4844286-rs5916352)具有最高的交叉验证一致性(9/10)和检验准确度(0.617),故选择该两位点基因型组合(NLGN3-NLGN4X)作为突触相关基因间交互作用的最佳模型。结论：本研究应用MDR分析方法研究突触通路上5个功能相关基因NRXN1、NLGN3、NLGN4X、CNTNAP2、SHANK3的交互作用,第一次报道了一个两位点基因型组合(NLGN3-NLGN4X)的基因-基因交互作用与中国汉族儿童孤独症的易感性相关。第三部分突触相关基因NRXN1、NLGN3、NLGN4X、CNTNAP2、SHANK3孤独症相关罕见突变的研究方法：1.样本采集和基因组DNA抽提同第一部分。2.罕见突变的选择和检测方法：查找国内外文献中已报道的与孤独症相关的罕见突变,并选择其中21个突触相关基因的罕见突变,运用基质辅助激光解吸电离飞行时间质谱(MALDI-TOFMS)方法进行突变筛检。结果：采用MALDI-TOF MS方法成功检测了上述5个突触相关基因中的21个孤独症相关罕见突变的位点,但仅检测到3种罕见变异,分别是NRXN1基因的c.83G>C、CNTNAP2基因的c.3385G>C和CNTNAP2基因的c.3385G>C。在13例孤独症患者和4例正常对照组中均检测的NRXN1的c.83G>C错义突变,且均为杂合突变。在1例正常对照组中检测到了CNTNAP2基因的c.3385G>C错义突变,也为杂合突变。在1例有严重语言发育缺陷的女性孤独症患者中,检测到SHANK3基因的c.203T>C错义突变,突变率为0.43%。结论：本研究在中国孤独症儿童中筛检NRXN1、NLGN3、NLGN4X、CNTNAP2和SHANK3基因中21个已知的孤独症相关罕见突变,首次在不同的独立样本中重复验证SHANK3基因的c.203T>C错义突变,推测其可能是孤独症致病相关罕见变异。NRXN1、NLGN3、NLGN4X和CNTNAP2基因中的已知孤独症罕见突变可能不是中国汉族儿童孤独症的致病突变。更多还原

【Abstract】 Objective:Autism spectrum disorders (ASDs) are severe neurodevelopmental syndrome with a complex genetic etiology, which characterized by impaired reciprocal social interactions, deficient communication, restricted interests and stereotyped activity patterns. ASDs are acknowledged to be among the most heritable neuropsychiatric disorders and etiologically heterogeneous, probably associated with a combination of the effects of multiple genes and environmental factors, and the role of genetic factors in the pathogenesis of ASDs has been definite established. Many independent genome-wide scans for ASDs susceptibility loci have been carried out. The region on chromosome 2q、7q、22q、Xq and Xp stands out as the region of suggestive linkage to atiology of ASDs in many independent genome-wide scans. The synaptic hypothesis was also proposed that alteration of synaptic homeostasis and/or impairments in synapse development is thought to be a major cause of brain disorders such as autism and mental retardation. A genetics topic in ASDs has emerged focusing on identification of the synaptic genes contributing to the formation and function of the synapse. Of particular interests are synaptic genes of the NRXNs-NLGNs-SHANK3 pathway in ASDs pathogenesis. Five synaptic associated genes NRXN1、NLGN3、NLGN4X、CNTNAP2 and SHANK3 selected in this study, are located within these regions. By using the case-control association analysis with 31 tagging SNPs of these five syanptic genes, we will investigate the relationship between ASDs and the sigle SNP and the haplotypes, also analysis with gender stratification. As a result, we will try to find out if these five genes are susceptibility genes of the ASDs.Moreover, ASDs are hereditary heterogeneous with a combination of the effects of multiple genes. So it is importance to analysis the gene-gene interaction of multiple genes for etiology of ASDs.It has also been considered that genes contributing to ASDs are likely a combination of rare variants in fewer individuals with a dramatic effect and common variants in majority individuals with small increments of risk. So we attempted to replicate rare mutations in genes of this pathway, which were previously described to be associated with ASDs, to validate the initial findings and evaluate if they also play a potentially role for ASDs in Chinese Han population.PartⅠAssociation analysis of SNPs of synaptic gene NRXN1、NLGN3、NLGN4X, CNTNAP2、SHANK3 with autism spectrum disorders in a Chinese Han cohortMethods:1. We studied a Chinese sample of Han origin consisting of 229 unrelated patients with ASDs and 191 ethnically and geographically matched controls in this study. All affected subjects were diagnosed according to the diagnostic and statistical manual of mental disorders (DSM-IV) criteria or International Classification of Diseases-10 (ICD-10) from outpatients of Children’s Hospital, Zhejiang University School of Medicine and from several special autism-training schools. All controls were randomly drawn from out-patients of our hospital with no personal history of psychiatric disorders.2. SNPs selection and genotyping:Based on the genotype data from the SNP databases and International HapMap Project,31 tagging SNPs within these five candidate genes with a minor allele frequency (MAF) greater than 5% in the Han_Chinese or in the HCB_Asian population were selected to capture the majority of the common variations. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS) was used for SNP genotyping.3. Statistic Analysis:The Hardy-Weinberg equilibrium (HWE) was assessed with chi-square test. SNPs were used in case-control study for allelic, genotypic and haplotypic association and were also performed with genders stratification.Results:1. Association analysis of SNPs of NRXN1 with autism spectrum disorders(1) A significant genetic association between an intronic SNP rs1421589 and female ASDs was observed, with T-allele having an increased risk to develop to autism(P=0.038, OR=1.955,95%CI:1.035-3.69), and the log-additive model was accepted as the best inhericance model fitting this data(P=0.023, OR=2.28,95%CI: 1.09-4.74). Haplotype-specific association analysis revealed that the haplotype T-T (rs1421589-rs1563018) showed significant association with female ASDs and has increased risk to develop to ASDs (P=0.039, OR=1.925,95%CI:1.029-3.604).(2) One exonic SNP rs1045874 showed significant differences in genotypic frequencies between the individuals with ASDs and controls under overdominant model (P=0.016, OR:1.85,95%CI:1.11-3.07) by logistic regression analysis, although no significant difference in allelic frequencies was observed.2. Association analysis of SNPs of NLGN3 with autism spectrum disorders (1) Significant differences of allele frequencies were detected for 3 SNPs in NLGN3 gene contrasted between cases and controls for male and female samples separately (male:rs11795613, rs4844285 and rs5981079, female:rs11795613, rs4844285 and rs7051529). In addition, significant differences in genotype distributions of these three SNPs were also detected in female samples under a log-additive model: rs11795613, p=0.036; rs4844285, p=0.036; rs7051529, p=0.038.(2) Haplotype-specific association analysis revealed that 4-SNPs haplotype A-G-T-T,3-SNPs haplotype A-G-T,2-SNPs haplotype A-G and T-T play a role as a susceptibility factor for male ASDs (A-G-T-T:P=0.031, OR:1.633,95%CI: 1.046-2.549; A-G-T:P=0.015, OR:1.758,95%CI:1.112-2.778; A-G:P=0.0113, OR:1.803,95%CI:1.14-2.854; T-T:P=0.047, OR:1.571,95%CI:1.005-2.457), 2-SNPs haplotype G-A play a role as a protective factor for male ASDs(P=0.0184, OR: 0.575,95%CI:0.361-0.913). Moreover, the haplotype A-G-T and A-G play a role as a susceptibility factor for female ASDs (A-G-T:P=0.03, OR:2.16,95%CI: 1.071-4.357; A-G:P=0.035, OR:2.147,95%CI:1.048-4.400), the haplotype G-A and G-T play a role as a protective factor for female ASDs (G-A:P=0.035, OR:0.466, 95%CI:0.227-0.954; G-T:P=0.044, OR:0.383,95%CI:0.147-0.999)3. Association analysis of SNPs of NLGN4X with autism spectrum disorders(1) Significant difference in allelic and genotypic frequencies of SNP rs5961397 were detected between the female individuals with autism and controls (allelic:p=0.047, genotypic under log-additive model:p=0.039). The rs5961397-A allele was overrepresented in female ASDs (OR=2.133,95% CI=1.001-4.545), and the rs5961397-G was a protective allele. Haplotype G-G (rs6529901-sr5961397) showed significant association with female ASDs and has a protective haplotype (P=0.024, OR: 0.404,95%CI:0.180-0.904).(2) Haplotype-specific association analysis revealed that one haplotype block defined by two SNPs (rs1882409-rs5916352) showed a significant association with male ASDs and the haplotype A-C play a risk role for male ASDs (P=0.022, OR:2.551, 95%CI:1.121-5.804).4. Association analysis of SNPs of CNTNAP2 and SHANK3 with autisms spectrum disordersNeither single SNP nor haplotype in CNTNAP2 and SHANK3 was detected to show significant association with ASDs, even after gender stratification analysis.Conclusions:Our study suggest the possible involvement of NRXN1、NLGN3、NLGN4X genes in the suscepitibility to ASDs. Future replications are warrented before definitive conclusion can be drawn.PartⅡGene-gene interaction of synaptic gene NRXN1、NLGN3、NLGN4X、CNTNAP2、SHANK3 with autism spectrum disorders in a Chinese Han cohortMethods:Twenty-nine SNPs of synaptic genes were involved in the analysis of gene-gene interaction using multifactor dimensionality reduction (MDR). Model with the highest CV consistency, the highest testing accuracy as well as the significant permutation test results was considered as the best interaction-model.Results:MDR analysis revealed that the two-loci genotypic combination (rs4844286-rs5916352) was the best gene-gene interaction model of synaptic genes in the etiology of autism(CV consistency=9/10, testing accuracy=0.617).Conclusions:Gene-gene interaction of NLGN3-NLGN4X may be involved in the etiology of autism in Chinese Han cohort. PartⅢStudy of rare mutations of synaptic gene NRXN1、CNTNAP2、NLGN3、NLGN4X、SHANK3 with autism spectrum disoreders in a Chinese Han cohortMethods:Twenty-one rare variants in these synaptic genes (NRXN1, NLGN3, NLGN4X, CNTANP2 and SHANK3) previously reported to be associated with ASDs were selected to perform for mutations screening, using MALDI-TOF mass spectrometry and analyzed with the SpectroTYPER RT 2.0 software.Results:We successfully detected 21 rare variants previously described to be associated with ASDs in these five genes and three missense mutations were identified. One missense mutations, c.83G>C polymorphism in exonl in NRXN1 gene, were identified in both affected patients and control samples and the rare allele C was present in 13 cases (2.97%) and 4 controls (1.09%). The difference is not significant either for allelic frequencies (χ2=3.269, p=0.118) or for genotypic (χ2=3.339, p=0.114). Another missense mutation, c.3385G>C polymorphism in CNTNAP2 gene was only detected in a single control sample and absent from patients with ASDs. One missense mutation in SHANK3 gene, c.203T>C, was identified only in a female autism with serious language impairment, and was absent in normal control samples.Conclusion:It is for the first time, a specific mutation of SHANK3 gene (c.203T>C) is confirmed by a molecular screening in another ethinic population, suggesting this missense mutation may be a cause of ASDs. However, functional studies will be required to confirm that this mutation is indeed pathogenic.更多还原