本文選題：孤獨(dú)癥 + NLGN3　； 參考：《中南大學(xué)》2013年博士論文

【摘要】：孤獨(dú)癥是一種嚴(yán)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(huì)交往障礙、言語(yǔ)交流障礙、狹隘興趣和重復(fù)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(dú)癥患病率近年急劇上升,最新流行病學(xué)調(diào)查發(fā)現(xiàn)孤獨(dú)癥的發(fā)病率已高達(dá)1.14-2.6%。孤獨(dú)癥的病因?qū)W及發(fā)病機(jī)制尚未闡明,遺傳學(xué)研究發(fā)現(xiàn)一些比較確定的孤獨(dú)癥易感基因,這些基因主要參與神經(jīng)發(fā)育過(guò)程,如突觸發(fā)生和突觸可塑性等。 對(duì)孤獨(dú)癥發(fā)病機(jī)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(dú)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(dòng)物大腦突觸后細(xì)胞粘附因子,通過(guò)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(shí)別、突觸成熟及信息傳遞等過(guò)程中發(fā)揮重要作用。 方法：本研究前期工作中,通過(guò)直接測(cè)序?qū)?18名孤獨(dú)癥患者和453名正常對(duì)照的NLGN3和NLGN4X外顯子序列及外顯子側(cè)翼序列進(jìn)行分析,我們?cè)诨颊呓M中發(fā)現(xiàn)了NLGN3和NLGN4X的10個(gè)已知SNPs和4個(gè)未報(bào)道的錯(cuò)義變異(p.G426S-NLGN3、p.G84R-NLGN4X、 P.Q162K-NLGN4X和p.A283T-NLGN4X),在正常對(duì)照組中發(fā)現(xiàn)了13個(gè)SNPs(包括患者中發(fā)現(xiàn)的10個(gè)SNPs)。 本研究中針對(duì)這些變異,我們做了進(jìn)一步的研究與分析,希望能夠闡明他們?cè)诠陋?dú)癥發(fā)病中的作用和機(jī)制。我們主要從兩個(gè)方面進(jìn)行研究。1.針對(duì)患者組和對(duì)照組共有的已知SNPs,通過(guò)基于病例-對(duì)照(Case-Control)的關(guān)聯(lián)研究闡明NLGN3和NLGN4X與中國(guó)人群中孤獨(dú)癥的相關(guān)性。2.我們所發(fā)現(xiàn)的4個(gè)錯(cuò)義突變,建立穩(wěn)定表達(dá)的HEK293細(xì)胞模型,通過(guò)與內(nèi)質(zhì)網(wǎng)標(biāo)志蛋白免疫熒光染色,對(duì)NLGN3/4X野生型和相關(guān)突變體進(jìn)行亞細(xì)胞定位研究；通過(guò)蛋白酶體抑制劑(MG132)陽(yáng)溶酶體抑制劑(CQ)來(lái)阻斷neuroligin3和neuroligin4X可能的降解途徑確定相關(guān)突變體在細(xì)胞內(nèi)的降解途徑；通過(guò)CHX抑制蛋白合成,確定相關(guān)突變體的降解速率；通過(guò)neuroligin和IgG-△neurexin(-SS4)免疫共沉淀分析突變體是否影響neuroligin-neurexin復(fù)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(duì)突觸的影響。 結(jié)果：通過(guò)基于病例-對(duì)照(Case-Control)的關(guān)聯(lián)研究,我們發(fā)現(xiàn)NLGN4X的2個(gè)連鎖的常見(jiàn)SNP位點(diǎn)rs3747333和rs3747334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(OR=4.685,95%CI=2.073-10.592, p=5.09E-05)。 對(duì)所發(fā)現(xiàn)的4個(gè)錯(cuò)義突變進(jìn)行生物學(xué)功能分析,通過(guò)免疫熒光染色發(fā)現(xiàn)4個(gè)錯(cuò)義突變的相應(yīng)突變蛋白在非神經(jīng)元性的細(xì)胞HEK293細(xì)胞中呈現(xiàn)細(xì)胞膜分布,和neuroligin3或neuroligin4X野生型蛋白沒(méi)有差異性；通過(guò)MG132和CQ藥物作用于相應(yīng)的細(xì)胞系,4個(gè)被檢突變蛋白和野生型蛋白降解途徑主要通過(guò)泛素介導(dǎo)的蛋白酶體降解途徑進(jìn)行降解；通過(guò)CHX藥物作用于相應(yīng)的細(xì)胞系,4個(gè)被檢突變蛋白的降解速率和野生型蛋白沒(méi)有明顯差異；通過(guò)免疫共沉淀實(shí)驗(yàn)發(fā)現(xiàn)4個(gè)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(méi)有明顯的差異。 結(jié)論：本研究結(jié)果表明NLGN4X的2個(gè)連鎖的常見(jiàn)NP位點(diǎn)rs3747333和rs3747334與中國(guó)漢族人群孤獨(dú)癥顯著相關(guān),NLGN4X是中國(guó)人群孤獨(dú)癥的易感基因。但是,在孤獨(dú)癥患者中發(fā)現(xiàn)的4個(gè)錯(cuò)義突變的相關(guān)突變蛋白的表達(dá)、亞細(xì)胞定位、降解過(guò)程以及和neurexin的相互作用在非神經(jīng)元性的細(xì)胞HEK293細(xì)胞中沒(méi)有發(fā)生明顯的改變,可能通過(guò)其他尚未闡明的分子機(jī)制導(dǎo)致孤獨(dú)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(hào)：1745065