本文選題：孤獨癥 + NLGN3�。� 參考：《中南大學》2013年博士論文

【摘要】：孤獨癥是一種嚴重影響兒童健康的神經(jīng)發(fā)育性疾病,其主要的臨床特征包括社會交往障礙、言語交流障礙、狹隘興趣和重復刻板行為等,通常3歲以內(nèi)發(fā)病,男女比例約為4：1-10：1。孤獨癥患病率近年急劇上升,最新流行病學調(diào)查發(fā)現(xiàn)孤獨癥的發(fā)病率已高達1.14-2.6%。孤獨癥的病因?qū)W及發(fā)病機制尚未闡明,遺傳學研究發(fā)現(xiàn)一些比較確定的孤獨癥易感基因,這些基因主要參與神經(jīng)發(fā)育過程,如突觸發(fā)生和突觸可塑性等。 對孤獨癥發(fā)病機制的研究中最為廣泛的是neurexin家族蛋白和neuroligin家族蛋白。而孤獨癥患者的男女差異性使得位于X染色體上的NLGN3和NLGN4X的研究尤為引人注目。NLGN3和NLGN4X編碼的蛋白屬哺乳動物大腦突觸后細胞粘附因子,通過和neurexin相互作用在突觸結(jié)構(gòu)形成、神經(jīng)遞質(zhì)釋放、突觸的識別、突觸成熟及信息傳遞等過程中發(fā)揮重要作用。 方法：本研究前期工作中,通過直接測序?qū)?18名孤獨癥患者和453名正常對照的NLGN3和NLGN4X外顯子序列及外顯子側(cè)翼序列進行分析,我們在患者組中發(fā)現(xiàn)了NLGN3和NLGN4X的10個已知SNPs和4個未報道的錯義變異(p.G426S-NLGN3、p.G84R-NLGN4X、 P.Q162K-NLGN4X和p.A283T-NLGN4X),在正常對照組中發(fā)現(xiàn)了13個SNPs(包括患者中發(fā)現(xiàn)的10個SNPs)。 本研究中針對這些變異,我們做了進一步的研究與分析,希望能夠闡明他們在孤獨癥發(fā)病中的作用和機制。我們主要從兩個方面進行研究。1.針對患者組和對照組共有的已知SNPs,通過基于病例-對照(Case-Control)的關(guān)聯(lián)研究闡明NLGN3和NLGN4X與中國人群中孤獨癥的相關(guān)性。2.我們所發(fā)現(xiàn)的4個錯義突變,建立穩(wěn)定表達的HEK293細胞模型,通過與內(nèi)質(zhì)網(wǎng)標志蛋白免疫熒光染色,對NLGN3/4X野生型和相關(guān)突變體進行亞細胞定位研究；通過蛋白酶體抑制劑(MG132)陽溶酶體抑制劑(CQ)來阻斷neuroligin3和neuroligin4X可能的降解途徑確定相關(guān)突變體在細胞內(nèi)的降解途徑；通過CHX抑制蛋白合成,確定相關(guān)突變體的降解速率；通過neuroligin和IgG-△neurexin(-SS4)免疫共沉淀分析突變體是否影響neuroligin-neurexin復合體的形成。從而初步闡明NLGN突變蛋白能否正常行使其生理功能以及對突觸的影響。 結(jié)果：通過基于病例-對照(Case-Control)的關(guān)聯(lián)研究,我們發(fā)現(xiàn)NLGN4X的2個連鎖的常見SNP位點rs3747333和rs3747334等位基因頻率和基因型頻率在患者組和對照組之間的有顯著統(tǒng)計學差異(OR=4.685,95%CI=2.073-10.592, p=5.09E-05)。 對所發(fā)現(xiàn)的4個錯義突變進行生物學功能分析,通過免疫熒光染色發(fā)現(xiàn)4個錯義突變的相應突變蛋白在非神經(jīng)元性的細胞HEK293細胞中呈現(xiàn)細胞膜分布,和neuroligin3或neuroligin4X野生型蛋白沒有差異性；通過MG132和CQ藥物作用于相應的細胞系,4個被檢突變蛋白和野生型蛋白降解途徑主要通過泛素介導的蛋白酶體降解途徑進行降解；通過CHX藥物作用于相應的細胞系,4個被檢突變蛋白的降解速率和野生型蛋白沒有明顯差異；通過免疫共沉淀實驗發(fā)現(xiàn)4個被檢突變蛋白及野生型蛋白都能夠與IgG-Aneurexin1β融合蛋白相結(jié)合而被沉淀,沒有明顯的差異。 結(jié)論：本研究結(jié)果表明NLGN4X的2個連鎖的常見NP位點rs3747333和rs3747334與中國漢族人群孤獨癥顯著相關(guān),NLGN4X是中國人群孤獨癥的易感基因。但是,在孤獨癥患者中發(fā)現(xiàn)的4個錯義突變的相關(guān)突變蛋白的表達、亞細胞定位、降解過程以及和neurexin的相互作用在非神經(jīng)元性的細胞HEK293細胞中沒有發(fā)生明顯的改變,可能通過其他尚未闡明的分子機制導致孤獨癥的發(fā)生。
[Abstract]:Autism is a neurodevelopmental disorder that affects children ' s health . The main clinical features include social interaction disorders , verbal communication disorders , narrow interests and repetitive stereotypes . The prevalence of autism is about 4 : 1 - 10 : 1 . The prevalence of autism has risen sharply in recent years . Recent epidemiological studies have found that the incidence of autism is up to 1.14 - 2.6 % . The etiology and pathogenesis of autism have not yet been elucidated . Genetic studies have found that some of the more specific susceptibility genes of autism are involved in neurodevelopmental processes , such as synaptic transmission and synaptic plasticity .

The most extensive research on the pathogenesis of autism is neurexin family protein and neuroligin family protein . The difference of male and female in autism makes the study of NLGN3 and NLGN4X located on X chromosome particularly notable . The proteins encoded by NLGN3 and NLGN4X belong to the postsynaptic cell adhesion factor of mammalian brain . The interaction of NLGN3 and NLGN4X plays an important role in synaptic structure formation , neurotransmitter release , synaptic identification , synaptic maturation and information transmission .

Methods : The exon sequences of NLGN3 and NLGN4X exon sequences and exon flanking sequences were analyzed by direct sequencing in 318 autism patients and 453 normal controls , and we found 10 known SNPs in NLGN3 and NLGN4X and four unreported ( p . G426S - NLGN3 , p . G84R - NLGN4X , P . Q162K - NLGN4X and p . A283T - NLGN4X ) in the patient group , and 13 SNPs were found in the normal control group ( including 10 SNPs found in patients ) .

In order to clarify their roles and mechanisms in the pathogenesis of autism , we hope to clarify the role and mechanism of NLGN3 and NLGN4X in the pathogenesis of autism .
The possible degradation pathways of neurotrophin - 3 and neurotrophin 4X were blocked by proteasome inhibitor ( MG132 ) .
determining the degradation rate of the relevant mutant by CHX inhibiting protein synthesis ;
The formation of neuroligin - neurexin complex was influenced by neuroligin and IgG - 鈻，

本文編號：1745065