中国人群儿童孤独症的NRXN1基因关联研究和两种疾病相关拷贝数变异的鉴定

发布时间：2018-05-09 20:41

本文选题：Neurexin-1 + 孤独症　；参考：《中南大学》2012年博士论文

【摘要】：第一部分中国人群儿童孤独症的NRXN1基因关联研究孤独症是一种严重影响儿童健康的神经发育性障碍疾病,以社交相互性受损、语言和沟通障碍以及重复刻板行为为主要特征。孤独症是广泛性发育障碍(PDD,Pervasive Developmental Disorder)的一个最重要的亚类。由于PDD以儿童孤独症为主,其他亚类也有类似的孤独症样表现,所以PDD又被称为孤独症谱群疾病(ASD,Autism Spectrum Disorder).孤独症的诊断缺乏客观指标,主要以行为障碍为依据。孤独症缺乏特效治疗,主要依赖行为学训练,其预后差,60-70%的患儿不能独立生活。流行病学研究显示,孤独症的发病率逐年上升,2012年3月美国疾病控制及预防中心(CDC)调查显示的最新孤独症发病率已上升至11.3‰(1/88),其中男孩被诊断为孤独症的比例是1/54,是女孩的5倍。据世界卫生组织(WHO)估计,中国有60-180万的孤独症患儿,给社会造成极大的负担。孤独症的病因复杂,家系和双生子研究结果表明遗传因素在孤独症致病因素中起重要作用。研究结果表明,已鉴定的孤独症相关基因多数与突触功能及神经元可塑性相关,如NRXN1,NLGN3和NLGN4, SHANK3,CNTNAP2,PCD9以及PCD10等。位于染色体2p16.3的NRXN1(neurexin-1)基因编码neurexin蛋白,neurexin家族是一类高度多态性细胞表面蛋白,特异地表达于哺乳动物神经元,参与细胞识别和细胞黏附,并可能介导细胞信号转导。α-neurexin或β-neurexin通过与neuroligin形成异四聚体,调控神经元的突触兴奋或突触抑制,而神经突触异常可能会导致孤独症的发生。白2006年Friedman等首次报道NRXN-1α的新发杂合缺失与神经发育疾病的相关性以来,在不同人群精神分裂症、孤独症以及智力低下等多种精神疾病患者中均检测到NRXN1基因外显子的缺失突变。2008年,Kim等在孤独症患者中对NRXN1进行重测序发现了罕见的错义突变,提示NRXN1与孤独症的发生和发展有关.NRXN-1α缺失小鼠和果蝇模型的神经行为学缺陷也为NRXN-1α参与孤独症发病机理提供了证据。这些研究结果显示NRXN1是孤独症重要的候选易感基因之一。中国的孤独症研究明显落后于国际前沿,中国汉族人群孤独症患者与NRXN1的相关性及突变筛查研究尚无报道,而不同种族的遗传易感因素存在着很大的差别,因此,对中国汉族孤独症患者及其家系进行NRXN1基因的突变筛查及相关性分析,有助于了解在中国汉族人群中,NRXN1基因的变异与孤独症的关系。本研究利用直接测序法,在中国汉族人群313个孤独症患者中对NRXN-1α的所有编码区外显子及外显子/内含子交界区序列,NRXN-1β的1号外显子及外显子/内含子交界区序列进行了突变检测。对于检测到的带有非同义突变的外显子,在500个正常对照样本中进一步检测,以验证被检突变是否为数据库中未报道过的多态。对于检测到所有非同义突变,尤其是仅在患者中出现而在对照中没有出现的非同义变异,进一步检测其父母亲DNA样本以证实该变异是父母遗传还是新发(de novo)。在鉴定罕见或新发变异的同时,再进行病例——对照(Case-Control)关联研究。我们的研究共发现22个位于neurexin-1外显子区域的变异,包括7个错义突变,3个缺失和12个同义突变。在这些变异中,有3个错义突变和3个同义突变是未报道过的新发突变且在500个正常对照中未检出。另有4个错义突变,3个缺失和3个同义突变在dbSNP数据库中末报道过,同时在500个正常对照中有检出。此外,我们的结果中还包含了2个编码区和4个内含子区的已知SNPs。对所有患者和对照中均检测到的变异的统计学分析结果表明,SNP (rs2303298)的等位基因频率和基因型频率在患者组和对照组之间的有显著统计学差异(26.2%vs.13.8%；χ2=22.487；p=3.45E-006；OR=2.152(1.559-2.970))。本研究结果显示NRXN1基因的一个常见SNP (rs2303298)与中国汉族人群孤独症的发生显著相关。在更多样本中发现孤独症相关的NRXN1基因变异,并开展其分子致病机制的研究,有望阐明NRXN1在孤独症发病机制中的作用。第二部分两种孤独症相关拷贝数变异的鉴定已有的研究显示,染色体异常及罕见基因变异等与孤独症的发生有关,随着基因组学研究的发展,拷贝数变异(Copy Number Variations, CNVs)也被认为与孤独症的发生有关,基于高密度芯片的拷贝数变异研究使孤独症易感位点的鉴定取得了很大的进展。孤独症是最早开展CNVs全基因组关联分析研究的疾病之一。2007年Sebat等最早报道了孤独症与新发CNVs显著相关,随后有研究小组在孤独症家系中发现16p11.2新发的约593kb的缺失,此研究结果得到了其他三个研究小组的验证。研究发现的孤独症相关CNVs区域包含了许多新的孤独症相关基因,例如SHANK2,SYNGAP1, DLGAP2以及X染色体连锁DDX53-PTCHD1位点等。到目前为止,已有研究显示约7%-20%的孤独症患者存在疾病相关的CNVs,涉及染色体主要包括1、3、5、7、15、16和22等。这些研究为拷贝数变异参与孤独症等遗传异质性很强的复杂疾病的发病机理提供了重要证据。而所有这些已发现的CNVs在不同的患者身上可能影响不同的基因,这意味着尽管不同的孤独症患者拥有相似症状,但引起症状的原因却可能是不同的遗传缺陷。因此,鉴定新的CNVs位点将为定位CNVs累及的可能易感基因提供研究基础。为了阐明中国汉族人群的孤独症遗传基础,本研究采用HumanCNV370-Duo芯片对455例孤独症患者样本进行全基因组基因分型,并用软件cnv Partition v2.4.4对患者和对照样本的分型数据进行全基因组CNVs分析。对得到的CNVs结果与正常对照、Database of Genomic Variants(DGV)数据库等比较,分类已报道的CNVs和我们新发现的罕见CNVs.对新发罕见CNVs利用实时荧光定量PCR(Real-time PCR)技术进行验证。进一步地利用生物信息学分析方法对这些罕见CNVs区域内的基因进行筛选,评价该位点与孤独症的相关性以及对发病风险的贡献。孤独症是一种与神经发育有关的疾病,遗传学因素导致神经发育有关的蛋白质、酶、受体、神经递质等的异常表达,引起神经元增殖和分化异常,提供了孤独症易感性的生物学基础。因此,在进行生物信息学分析时重点关注相关CNVs中与神经发生和发育有关的基因。本研究在455例孤独症患者中发现了大量CNVs,通过与正常对照CNVs进行病例——对照(Case-Control)分析以及与DGV数据库进行对比,我们在两个孤独症患者中分别发现了两个新发的罕见CNVs。其一是位于5号染色体5p15.33-p15.2区域的一个8MB的杂合缺失,属于拷贝数减少改变,为新发CNV,该区域内的SLC6A3基因被报道参与多种与神经发育异常有关的精神和神经系统疾病的发生；其二是位于10号染色体10p12.33区域的一个1MB的杂合重复,属于拷贝数增加改变,该区域内的CACNB2基因属于钙离子通道基因,已有研究表明异常的钙离子信号通道可能导致孤独症相关表型。此外,该区域内的PTPLA和STAM基因参与信号转导,被认为与神经退行性疾病相关。这些基因可能影响神经发生或发育有关蛋白质的表达,提示这两个新发CNVs可能与孤独症发生相关。本研究在455例孤独症患者中鉴定了两种新的孤独症相关CNVs位点,这两个CNVs所包含的基因将成为我们接下来的重点研究的对象。本研究为孤独症易感基因的定位和克隆研究提供了一定分子研究基础。
[Abstract]:Part one NRXN1 gene association in Chinese children with autism
Autism is a neurodevelopmental disorder that seriously affects children's health. It is characterized by impaired social interaction, language and communication disorders and repeated stereotypes. Autism is the most important subclass of PDD (Pervasive Developmental Disorder). Because PDD is the main child of autism, the other is the other. Subclasses also have similar autism - like manifestations, so PDD is also known as ASD (Autism Spectrum Disorder). The diagnosis of autism lacks objective indicators, mainly based on behavioral disorders. Autism lacks special treatment, mainly relies on behavioral training, its prognosis is poor, and 60-70% children can't live independently. Epidemiology research The incidence of autism increased year by year. In March 2012, the US Center for Disease Control and Prevention (CDC) survey showed that the incidence of the latest autism increased to 11.3 per thousand (1/88), of which boys were diagnosed as autism by 1/54 and 5 times that of girls. According to the WHO (WHO), there were 60-180 autistic children in China. Society creates a great burden.
The causes of autism are complex, and the results of family and twin studies have shown that genetic factors play an important role in the pathogenesis of autism. The results show that most of the identified autism related genes are associated with synaptic function and neuronal plasticity, such as NRXN1, NLGN3 and NLGN4, SHANK3, CNTNAP2, PCD9, and PCD10, etc. located in chromosome 2p16.3. The NRXN1 (neurexin-1) gene encodes the neurexin protein, and the neurexin family is a highly polymorphic cell surface protein, which is specifically expressed in mammalian neurons, participates in cell recognition and cell adhesion, and may mediate cell signal transduction. Alpha -neurexin or beta -neurexin forms an isooligomer with neuroligin to regulate the synapse of neurons. Exp, synaptic inhibition, and synapse abnormalities may lead to autism. In white 2006 Friedman, the first report of the new heterozygosity of NRXN-1 A and the correlation of neurodevelopmental disorders, the NRXN1 exons were detected in a variety of people with schizophrenia, autism, and mental retardation in different populations. The deletion mutation.2008 years, Kim and other NRXN1 re sequencing in autistic patients found a rare missense mutation, suggesting that NRXN1 and the development of autism related to the neurobehavioral defects of the.NRXN-1 alpha deficient mice and the Drosophila melanogaster models also provide evidence for the mechanism of NRXN-1 alpha participation in the pathogenesis of autism. These findings suggest that NRXN1 is the same. One of the important candidate susceptibility genes of autism is one of the candidate susceptibility genes. The study of autism in China is obviously lagging behind the international frontier. There is no report on the correlation between autistic patients and NRXN1 in Chinese Han population and the mutation screening study, while the genetic susceptibility factors of different races are very different. Therefore, the N of Chinese autistic patients and their families are carried out in China. The mutation screening and correlation analysis of RXN1 gene can help to understand the relationship between NRXN1 gene mutation and autism in Chinese Han population.
In this study, the direct sequencing method was used to detect all the exons and exons / intron sequences of NRXN-1 alpha in 313 autistic patients in Chinese Han population, and the sequence of NRXN-1 beta exon 1 exon and exon / intron junction sequence. The exons with unsynonymous mutations were detected in 500 positive cases. Further tests were carried out in the control samples to verify whether the detected mutation was not reported in the database. For all non synonymous mutations, especially in the patients, there was no unsynonymous variation in the control, and the parent DNA samples were further tested to confirm whether the mutation was inherited or new (de Nov). O). A case - control (Case-Control) association study was conducted to identify rare or new mutations.
Our study found 22 mutations in the exons of neurexin-1, including 7 missense mutations, 3 deletions and 12 synonymous mutations. Among these, 3 missense and 3 synonymous mutations were unreported new mutations and were not detected in 500 normal controls. There were 4 missense mutations, 3 missing and 3 synonymous processes. It was reported at the end of the dbSNP database and detected in 500 normal controls. In addition, our results also included the statistical analysis of the variations detected by the known SNPs. in all patients and controls in 2 coding regions and 4 introns. The results showed that the allele frequency and genotype frequency of SNP (rs2303298) were affected. There was a significant difference between the group and the control group (26.2%vs.13.8%; Chi 2=22.487; p=3.45E-006; OR=2.152 (1.559-2.970)).
The results of this study show that a common SNP (rs2303298) of the NRXN1 gene is significantly related to the occurrence of autism in Chinese Han population. The discovery of autism related NRXN1 gene mutations in more samples and the study of its molecular pathogenicity mechanism are expected to elucidate the role of NRXN1 in the pathogenesis of autism.
The second part is the identification of two types of autism related copy number variation.
Previous studies have shown that chromosomal abnormalities and rare genetic variations are associated with autism. With the development of genomic research, the Copy Number Variations (CNVs) is also considered to be associated with autism. The identification of the susceptibility loci of autism based on the study of the copy number variation of high density chips has made the identification of the susceptible loci of autism Great progress.
Autism is one of the earliest diseases of the CNVs genome association analysis, one of the earliest reports of Sebat,.2007, which was the first to report on autism and new CNVs, and then the research team found the missing of the new 593kb of 16p11.2 in the autism family. The results were verified by three other research groups. The disease related CNVs region contains a number of new autism related genes, such as SHANK2, SYNGAP1, DLGAP2, and X chromosome linked DDX53-PTCHD1 loci. Up to now, there have been studies showing the existence of disease related CNVs in approximately 7%-20% autistic patients, involving chromosomes mainly including 1,3,5,7,15,16 and 22. These studies are copies of the number. All these found CNVs may affect different genes in different patients, which means that although different autistic patients have similar symptoms, the causes of symptoms may be different genetic defects. Therefore, identification of new CNVs sites will provide a basis for locating susceptible genes involved in CNVs.
In order to elucidate the genetic basis of autism in Chinese Han population, the whole genome genotyping was carried out in 455 autistic patients with HumanCNV370-Duo chip, and the whole genome CNVs analysis was carried out with the software CNV Partition v2.4.4 for the patients and the control samples. The results of the obtained CNVs were compared with the normal control, Database Compared with the of Genomic Variants (DGV) database, the reported CNVs and our newly discovered rare CNVs. were used to verify the new rare CNVs using real time fluorescent quantitative PCR (Real-time PCR) technology. Further, the bioinformatics analysis method was used to screen the genes in these rare CNVs region regions to evaluate the loci and autism. The correlation and contribution to the risk of disease. Autism is a disease associated with neurodevelopment. Genetic factors lead to abnormal expression of proteins, enzymes, receptors, neurotransmitters and other abnormalities of neurodevelopment, causing neuronal proliferation and differentiation, providing a biological basis for the susceptibility of autism. Therefore, biological information is carried out. In the analysis, we focus on genes related to neurogenesis and development in CNVs.
In this study, a large number of CNVs were found in 455 autistic patients. Through the case control (Case-Control) analysis with the normal control CNVs and the DGV database, we found two newly rare CNVs. in two autistic patients, one of which was a 8MB in the 5p15.33-p15.2 region of chromosome 5. The SLC6A3 gene in the region is reported to be involved in a variety of mental and nervous system diseases associated with neurodevelopmental abnormalities, and the other is a heterozygous repetition of a 1MB located in the 10p12.33 region of chromosome 10, belonging to the increase of the copy number, and the CACNB2 gene in the region. In calcium channel genes, some studies have shown that abnormal calcium signaling pathways may lead to autism related phenotypes. In addition, PTPLA and STAM genes involved in signal transduction in this region are considered to be associated with neurodegenerative diseases. These genes may affect neurogenesis or the expression of proteins related to neurogenesis, suggesting these two new CN Vs may be associated with autism.
In this study, two new autism related CNVs loci were identified in 455 autistic patients. These two CNVs genes will be the focus of our next focus. This study provides a basis for molecular research for the location and cloning of autistic susceptibility genes.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R749.94

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