本文選題：Neurexin-1 + 孤獨(dú)癥��； 參考：《中南大學(xué)》2012年博士論文

【摘要】：第一部分中國(guó)人群兒童孤獨(dú)癥的NRXN1基因關(guān)聯(lián)研究 孤獨(dú)癥是一種嚴(yán)重影響兒童健康的神經(jīng)發(fā)育性障礙疾病,以社交相互性受損、語(yǔ)言和溝通障礙以及重復(fù)刻板行為為主要特征。孤獨(dú)癥是廣泛性發(fā)育障礙(PDD,Pervasive Developmental Disorder)的一個(gè)最重要的亞類(lèi)。由于PDD以?xún)和陋?dú)癥為主,其他亞類(lèi)也有類(lèi)似的孤獨(dú)癥樣表現(xiàn),所以PDD又被稱(chēng)為孤獨(dú)癥譜群疾病(ASD,Autism Spectrum Disorder).孤獨(dú)癥的診斷缺乏客觀(guān)指標(biāo),主要以行為障礙為依據(jù)。孤獨(dú)癥缺乏特效治療,主要依賴(lài)行為學(xué)訓(xùn)練,其預(yù)后差,60-70%的患兒不能獨(dú)立生活。流行病學(xué)研究顯示,孤獨(dú)癥的發(fā)病率逐年上升,2012年3月美國(guó)疾病控制及預(yù)防中心(CDC)調(diào)查顯示的最新孤獨(dú)癥發(fā)病率已上升至11.3‰(1/88),其中男孩被診斷為孤獨(dú)癥的比例是1/54,是女孩的5倍。據(jù)世界衛(wèi)生組織(WHO)估計(jì),中國(guó)有60-180萬(wàn)的孤獨(dú)癥患兒,給社會(huì)造成極大的負(fù)擔(dān)。 孤獨(dú)癥的病因復(fù)雜,家系和雙生子研究結(jié)果表明遺傳因素在孤獨(dú)癥致病因素中起重要作用。研究結(jié)果表明,已鑒定的孤獨(dú)癥相關(guān)基因多數(shù)與突觸功能及神經(jīng)元可塑性相關(guān),如NRXN1,NLGN3和NLGN4, SHANK3,CNTNAP2,PCD9以及PCD10等。位于染色體2p16.3的NRXN1(neurexin-1)基因編碼neurexin蛋白,neurexin家族是一類(lèi)高度多態(tài)性細(xì)胞表面蛋白,特異地表達(dá)于哺乳動(dòng)物神經(jīng)元,參與細(xì)胞識(shí)別和細(xì)胞黏附,并可能介導(dǎo)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)。α-neurexin或β-neurexin通過(guò)與neuroligin形成異四聚體,調(diào)控神經(jīng)元的突觸興奮或突觸抑制,而神經(jīng)突觸異常可能會(huì)導(dǎo)致孤獨(dú)癥的發(fā)生。白2006年Friedman等首次報(bào)道NRXN-1α的新發(fā)雜合缺失與神經(jīng)發(fā)育疾病的相關(guān)性以來(lái),在不同人群精神分裂癥、孤獨(dú)癥以及智力低下等多種精神疾病患者中均檢測(cè)到NRXN1基因外顯子的缺失突變。2008年,Kim等在孤獨(dú)癥患者中對(duì)NRXN1進(jìn)行重測(cè)序發(fā)現(xiàn)了罕見(jiàn)的錯(cuò)義突變,提示NRXN1與孤獨(dú)癥的發(fā)生和發(fā)展有關(guān).NRXN-1α缺失小鼠和果蠅模型的神經(jīng)行為學(xué)缺陷也為NRXN-1α參與孤獨(dú)癥發(fā)病機(jī)理提供了證據(jù)。這些研究結(jié)果顯示NRXN1是孤獨(dú)癥重要的候選易感基因之一。中國(guó)的孤獨(dú)癥研究明顯落后于國(guó)際前沿,中國(guó)漢族人群孤獨(dú)癥患者與NRXN1的相關(guān)性及突變篩查研究尚無(wú)報(bào)道,而不同種族的遺傳易感因素存在著很大的差別,因此,對(duì)中國(guó)漢族孤獨(dú)癥患者及其家系進(jìn)行NRXN1基因的突變篩查及相關(guān)性分析,有助于了解在中國(guó)漢族人群中,NRXN1基因的變異與孤獨(dú)癥的關(guān)系。 本研究利用直接測(cè)序法,在中國(guó)漢族人群313個(gè)孤獨(dú)癥患者中對(duì)NRXN-1α的所有編碼區(qū)外顯子及外顯子/內(nèi)含子交界區(qū)序列,NRXN-1β的1號(hào)外顯子及外顯子/內(nèi)含子交界區(qū)序列進(jìn)行了突變檢測(cè)。對(duì)于檢測(cè)到的帶有非同義突變的外顯子,在500個(gè)正常對(duì)照樣本中進(jìn)一步檢測(cè),以驗(yàn)證被檢突變是否為數(shù)據(jù)庫(kù)中未報(bào)道過(guò)的多態(tài)。對(duì)于檢測(cè)到所有非同義突變,尤其是僅在患者中出現(xiàn)而在對(duì)照中沒(méi)有出現(xiàn)的非同義變異,進(jìn)一步檢測(cè)其父母親DNA樣本以證實(shí)該變異是父母遺傳還是新發(fā)(de novo)。在鑒定罕見(jiàn)或新發(fā)變異的同時(shí),再進(jìn)行病例——對(duì)照(Case-Control)關(guān)聯(lián)研究。 我們的研究共發(fā)現(xiàn)22個(gè)位于neurexin-1外顯子區(qū)域的變異,包括7個(gè)錯(cuò)義突變,3個(gè)缺失和12個(gè)同義突變。在這些變異中,有3個(gè)錯(cuò)義突變和3個(gè)同義突變是未報(bào)道過(guò)的新發(fā)突變且在500個(gè)正常對(duì)照中未檢出。另有4個(gè)錯(cuò)義突變,3個(gè)缺失和3個(gè)同義突變?cè)赿bSNP數(shù)據(jù)庫(kù)中末報(bào)道過(guò),同時(shí)在500個(gè)正常對(duì)照中有檢出。此外,我們的結(jié)果中還包含了2個(gè)編碼區(qū)和4個(gè)內(nèi)含子區(qū)的已知SNPs。對(duì)所有患者和對(duì)照中均檢測(cè)到的變異的統(tǒng)計(jì)學(xué)分析結(jié)果表明,SNP (rs2303298)的等位基因頻率和基因型頻率在患者組和對(duì)照組之間的有顯著統(tǒng)計(jì)學(xué)差異(26.2%vs.13.8%；χ2=22.487；p=3.45E-006；OR=2.152(1.559-2.970))。 本研究結(jié)果顯示NRXN1基因的一個(gè)常見(jiàn)SNP (rs2303298)與中國(guó)漢族人群孤獨(dú)癥的發(fā)生顯著相關(guān)。在更多樣本中發(fā)現(xiàn)孤獨(dú)癥相關(guān)的NRXN1基因變異,并開(kāi)展其分子致病機(jī)制的研究,有望闡明NRXN1在孤獨(dú)癥發(fā)病機(jī)制中的作用。 第二部分兩種孤獨(dú)癥相關(guān)拷貝數(shù)變異的鑒定 已有的研究顯示,染色體異常及罕見(jiàn)基因變異等與孤獨(dú)癥的發(fā)生有關(guān),隨著基因組學(xué)研究的發(fā)展,拷貝數(shù)變異(Copy Number Variations, CNVs)也被認(rèn)為與孤獨(dú)癥的發(fā)生有關(guān),基于高密度芯片的拷貝數(shù)變異研究使孤獨(dú)癥易感位點(diǎn)的鑒定取得了很大的進(jìn)展。 孤獨(dú)癥是最早開(kāi)展CNVs全基因組關(guān)聯(lián)分析研究的疾病之一。2007年Sebat等最早報(bào)道了孤獨(dú)癥與新發(fā)CNVs顯著相關(guān),隨后有研究小組在孤獨(dú)癥家系中發(fā)現(xiàn)16p11.2新發(fā)的約593kb的缺失,此研究結(jié)果得到了其他三個(gè)研究小組的驗(yàn)證。研究發(fā)現(xiàn)的孤獨(dú)癥相關(guān)CNVs區(qū)域包含了許多新的孤獨(dú)癥相關(guān)基因,例如SHANK2,SYNGAP1, DLGAP2以及X染色體連鎖DDX53-PTCHD1位點(diǎn)等。到目前為止,已有研究顯示約7%-20%的孤獨(dú)癥患者存在疾病相關(guān)的CNVs,涉及染色體主要包括1、3、5、7、15、16和22等。這些研究為拷貝數(shù)變異參與孤獨(dú)癥等遺傳異質(zhì)性很強(qiáng)的復(fù)雜疾病的發(fā)病機(jī)理提供了重要證據(jù)。而所有這些已發(fā)現(xiàn)的CNVs在不同的患者身上可能影響不同的基因,這意味著盡管不同的孤獨(dú)癥患者擁有相似癥狀,但引起癥狀的原因卻可能是不同的遺傳缺陷。因此,鑒定新的CNVs位點(diǎn)將為定位CNVs累及的可能易感基因提供研究基礎(chǔ)。 為了闡明中國(guó)漢族人群的孤獨(dú)癥遺傳基礎(chǔ),本研究采用HumanCNV370-Duo芯片對(duì)455例孤獨(dú)癥患者樣本進(jìn)行全基因組基因分型,并用軟件cnv Partition v2.4.4對(duì)患者和對(duì)照樣本的分型數(shù)據(jù)進(jìn)行全基因組CNVs分析。對(duì)得到的CNVs結(jié)果與正常對(duì)照、Database of Genomic Variants(DGV)數(shù)據(jù)庫(kù)等比較,分類(lèi)已報(bào)道的CNVs和我們新發(fā)現(xiàn)的罕見(jiàn)CNVs.對(duì)新發(fā)罕見(jiàn)CNVs利用實(shí)時(shí)熒光定量PCR(Real-time PCR)技術(shù)進(jìn)行驗(yàn)證。進(jìn)一步地利用生物信息學(xué)分析方法對(duì)這些罕見(jiàn)CNVs區(qū)域內(nèi)的基因進(jìn)行篩選,評(píng)價(jià)該位點(diǎn)與孤獨(dú)癥的相關(guān)性以及對(duì)發(fā)病風(fēng)險(xiǎn)的貢獻(xiàn)。孤獨(dú)癥是一種與神經(jīng)發(fā)育有關(guān)的疾病,遺傳學(xué)因素導(dǎo)致神經(jīng)發(fā)育有關(guān)的蛋白質(zhì)、酶、受體、神經(jīng)遞質(zhì)等的異常表達(dá),引起神經(jīng)元增殖和分化異常,提供了孤獨(dú)癥易感性的生物學(xué)基礎(chǔ)。因此,在進(jìn)行生物信息學(xué)分析時(shí)重點(diǎn)關(guān)注相關(guān)CNVs中與神經(jīng)發(fā)生和發(fā)育有關(guān)的基因。 本研究在455例孤獨(dú)癥患者中發(fā)現(xiàn)了大量CNVs,通過(guò)與正常對(duì)照CNVs進(jìn)行病例——對(duì)照(Case-Control)分析以及與DGV數(shù)據(jù)庫(kù)進(jìn)行對(duì)比,我們?cè)趦蓚�(gè)孤獨(dú)癥患者中分別發(fā)現(xiàn)了兩個(gè)新發(fā)的罕見(jiàn)CNVs。其一是位于5號(hào)染色體5p15.33-p15.2區(qū)域的一個(gè)8MB的雜合缺失,屬于拷貝數(shù)減少改變,為新發(fā)CNV,該區(qū)域內(nèi)的SLC6A3基因被報(bào)道參與多種與神經(jīng)發(fā)育異常有關(guān)的精神和神經(jīng)系統(tǒng)疾病的發(fā)生；其二是位于10號(hào)染色體10p12.33區(qū)域的一個(gè)1MB的雜合重復(fù),屬于拷貝數(shù)增加改變,該區(qū)域內(nèi)的CACNB2基因?qū)儆阝}離子通道基因,已有研究表明異常的鈣離子信號(hào)通道可能導(dǎo)致孤獨(dú)癥相關(guān)表型。此外,該區(qū)域內(nèi)的PTPLA和STAM基因參與信號(hào)轉(zhuǎn)導(dǎo),被認(rèn)為與神經(jīng)退行性疾病相關(guān)。這些基因可能影響神經(jīng)發(fā)生或發(fā)育有關(guān)蛋白質(zhì)的表達(dá),提示這兩個(gè)新發(fā)CNVs可能與孤獨(dú)癥發(fā)生相關(guān)。 本研究在455例孤獨(dú)癥患者中鑒定了兩種新的孤獨(dú)癥相關(guān)CNVs位點(diǎn),這兩個(gè)CNVs所包含的基因?qū)⒊蔀槲覀兘酉聛?lái)的重點(diǎn)研究的對(duì)象。本研究為孤獨(dú)癥易感基因的定位和克隆研究提供了一定分子研究基礎(chǔ)。
[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.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R749.94

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