【摘要】：孤獨(dú)癥譜系障礙性疾病(Autism Spectrum Disorders, ASD)是一種嚴(yán)重影響兒童健康的神經(jīng)發(fā)育性疾病,一般為3歲前發(fā)病,男女發(fā)病比例為4：1,最新流行病學(xué)調(diào)查顯示ASD發(fā)病率已高達(dá)1/50。ASD具有高度的臨床異質(zhì)性,其三個(gè)典型的臨床癥狀為語(yǔ)言障礙、社交障礙以及重復(fù)刻板的行為。近年來(lái),通過(guò)染色體核型分析,全基因組連鎖分析,全基因組拷貝數(shù)變異研究以及全基因組關(guān)聯(lián)研究,已經(jīng)鑒定了多個(gè)ASD相關(guān)的易感位點(diǎn)和基因。這些基因大多與神經(jīng)發(fā)育、神經(jīng)元可塑性、突觸連接功能以及神經(jīng)環(huán)路相關(guān)。 第一章：中國(guó)人群兒童孤獨(dú)癥的CORO2B基因關(guān)聯(lián)研究 研究背景：我們前期的研究發(fā)現(xiàn)一例孤獨(dú)癥患者攜帶一個(gè)新發(fā)單拷貝數(shù)缺失,這個(gè)拷貝數(shù)變異包含CLN6、CORO2B、SENP8等31個(gè)基因。通過(guò)功能候選,我們認(rèn)為CORO2B很可能與孤獨(dú)癥發(fā)病相關(guān)。另外我們之前的全基因組關(guān)聯(lián)研究(GWAS)結(jié)果也表明CORO2B上的多個(gè)單核苷酸多態(tài)(SNP)與孤獨(dú)癥相關(guān)。 研究目的：本研究將在我們新收集的一批孤獨(dú)癥樣本中對(duì)我們前期的關(guān)聯(lián)結(jié)果進(jìn)行進(jìn)一步驗(yàn)證,以探索CORO2B與中國(guó)人群孤獨(dú)癥發(fā)病的相關(guān)性。 實(shí)驗(yàn)方法：利用Haploview挑選前期研究中與孤獨(dú)癥具有相關(guān)性的連鎖不平衡區(qū)域的標(biāo)簽SNP (rs1465997),用primer3(http://frodo.wi.mit.edu/primer3/)對(duì)該位點(diǎn)兩側(cè)的堿基序列設(shè)計(jì)引物。PCR擴(kuò)增該片段并用Sanger測(cè)序法進(jìn)行直接測(cè)序。用DNAStar中的SeqMan進(jìn)行分析,檢測(cè)該位點(diǎn)的基因型。用plink (http://pngu.mgh.harvard.edu/～purcell/plink/)做哈代溫伯格平衡(HWE)檢驗(yàn)和傳遞不平衡關(guān)聯(lián)分析(TDT)。 實(shí)驗(yàn)結(jié)果：分型的270個(gè)核心家系在該SNP位點(diǎn)均符合孟德爾遺傳定律。最小等位基因頻率為0.24,基因型分布符合HWE定律(p=0.25)。傳遞不平衡關(guān)聯(lián)分析結(jié)果無(wú)顯著統(tǒng)計(jì)學(xué)意義(p=0.0522)且優(yōu)勢(shì)比與前期GWAS結(jié)果不一致(OR=0.76)。 結(jié)論：本實(shí)驗(yàn)結(jié)果未能驗(yàn)證之前GWAS的結(jié)果。CORO2B該位點(diǎn)在本研究樣本中與中國(guó)人群孤獨(dú)癥不相關(guān)。 第二章：中國(guó)人群兒童孤獨(dú)癥的PAK2基因突變篩查 研究背景：PAK2(P21蛋白激酶2)基因位于染色體3q29,含14個(gè)外顯子,編碼524個(gè)氨基酸。已有研究表明PAK1和PAK3突變與認(rèn)知障礙疾病相關(guān)。PAK2位于3q29微缺失綜合癥(OMIM#609425)和3q29微重復(fù)綜合癥(OMIM#611936)拷貝數(shù)變異區(qū)間內(nèi)。這兩個(gè)綜合征均存在智力低下和／或?qū)W習(xí)障礙,語(yǔ)音延遲和孤獨(dú)癥表型。這些證據(jù)表明PAK蛋白對(duì)神經(jīng)發(fā)育至關(guān)重要,PAK家族基因的變異很可能與神經(jīng)發(fā)育性疾病相關(guān),如孤獨(dú)癥等。 研究目的：本研究在中國(guó)人群孤獨(dú)癥患者中篩查PAK2基因突變,探索PAK2基因突變是否與中國(guó)人群孤獨(dú)癥相關(guān)。 實(shí)驗(yàn)方法：在UCSC (http://genome.ucsc.edu/)中找到PAK2基因編碼區(qū)和5'UTR區(qū)堿基序列。以PAK2基因編碼區(qū)和5'UTR區(qū)堿基序列為目的片段用primer3設(shè)計(jì)引物(http://frodo.wi.mit.edu/primer3/)。對(duì)設(shè)計(jì)出的引物片段進(jìn)行PCR擴(kuò)增并用S anger測(cè)序法進(jìn)行直接測(cè)序。用DNAStar中的SeqMan進(jìn)行分析。以UCSU中查到的序列(NM_002577)為標(biāo)準(zhǔn),分析PAK2基因的編碼區(qū)和5'UTR區(qū)變異位點(diǎn)。 實(shí)驗(yàn)結(jié)果：本研究在312個(gè)孤獨(dú)癥患者中共鑒定了10個(gè)變異。其中4個(gè)位于PAK2基因編碼區(qū)域,均為同義突變。2個(gè)位于5'UTR區(qū),為c.-191CT和c.-167GT。變異c.-191CT在dbSNP數(shù)據(jù)庫(kù)中未見報(bào)道,變異c.-167GT為已知多態(tài)。另外4個(gè)變異位于外顯子剪接位點(diǎn)附近的內(nèi)含子區(qū)(c.289-3ta, c.576+14ga,c.1154-21_1154-20instgttt,c.1350+8gc)。變異c.576+14ga在dbSNP數(shù)據(jù)庫(kù)中未見報(bào)道。變異c.289-3ta, c.1154-21_1154-20instgttt和c.1350+8gc均為已知多態(tài)。 結(jié)論：本研究未在中國(guó)人群兒童孤獨(dú)癥患者中發(fā)現(xiàn)PAK2基因改變氨基酸編碼的突變。不支持PAK2基因編碼區(qū)變異與中國(guó)人群兒童孤獨(dú)癥相關(guān)。
[Abstract]:Autism Spectrum Disorders (ASD) is a neurodevelopmental disorder that severely affects children's health, generally 3 years of age, with a prevalence of 4: 1. The latest epidemiological survey shows that the incidence of ASD has been up to 1/ 50. ASD has a high clinical heterogeneity, Three typical clinical symptoms are language barriers, social disorders, and repetitive stereotypes. In recent years, multiple ASD-related susceptibility sites and genes have been identified by chromosome karyotype analysis, genome-wide linkage analysis, genome-wide copy number variation studies, and genome-wide association studies. Most of these genes are associated with neural development, neuronal plasticity, synaptic connectivity, and nerve loops. Chapter One: CORO2B Gene Off of Children with Autism in Chinese Population Background of the study: our previous study found that a patient with autism carries a new single copy number deletion, which contains CLIA, CORO2B, SENP8, etc. 31 genes. Through functional candidates, we think that CORO2B is very likely to be isolated from loneliness The results of the whole genome association study (GWAS) before us also indicate that multiple monogenic polymorphisms (SNPs) on CORO2B and Autism-related. Research purposes: This study will further validate the results of our previous association in a new collection of autism samples to explore CORO2B and Chinese population. The correlation between the pathogenesis of Sudoku was studied: the label SNP (rs1465997) of the linkage disequilibrium region with the correlation with autism was selected by Haploview, and the site was treated with primer3 (http:// frodo. wi. mit. edu/ primer3/). Base sequence design primers on both sides. PCR amplification of the fragment and using Sang Direct sequencing was carried out using the ser-sequencing method. Using the SeqMan in DNAStar, The genotype of the site was detected. Hardenberg equilibrium (HWE) was tested and delivered with plink (http:// pngu. mgh. harvard. edu/ ~ purcell/ plink/). Equilibrium Association Analysis (TDT). Experimental results: 270 core families of the classification are in the S The NP sites were consistent with Mendelian inheritance law. The minimum allele frequency was 0. 24, genotype distribution. HWE's law (p = 0. 25). There was no significant difference in the results of transfer disequilibrium (p = 0.0522) and the odds ratio was higher than that of early GWAS. Results were not consistent (OR = 0. 76). Conclusion: Ben The results of the previous GWAS were not verified by the experimental results. CORO2B was located at There is no correlation with autism in the Chinese population in this study sample. Chapter II Background: PAK2 (P21 protein kinase 2) gene is located in staining in Chinese population of children with autism. Body 3q29 contains 14 exons and encodes 524 amino acids. The study showed that PAK1 and PAK3 mutations were associated with cognitive disorders. PAK2 was located in 3q29 microdeletion syndrome (OMIM # 609425) and 3q29 microrepeats Mitosis (OMIM # 611936) copy number variation interval. Both syndromes are stored In mental retardation and/ or learning disorders, speech delays, and autism phenotypes, these evidence suggests that tau protein is essential to neurodevelopment, including family groups. The mutation is likely to be related to neurodevelopmental disorders, such as autism. The purpose of this study is to screen PAK2 in patients with autism in China. To explore whether PAK2 mutations are associated with autism in Chinese populations due to mutations. Test methods: UCSC (http:// genome. ucsc The PAK2 gene encoding region and the 5 'UTR region base sequence are found in. edu/). Primer3 primer (http) is used as the target fragment of the PAK2 gene encoding region and the 5' UTR region base sequence.:// frodo. wi. mit. edu/ primer3/). pairs are designed The primer fragment was amplified by PCR and tested with S-actin. Direct sequencing by sequence method. Analysis using SeqMan in DNAStar. Sequence found in UCSU (NM _ 00257 7) For standard, analyze the coding region of PAK2 gene and the mutation site of 5 'UTR region The results showed that 10 mutations were identified in 312 patients with autism. Four of them were located in the coding region of PAK2 gene. Mutation. 2 in 5' UTR region, c.-191CT and c. -167GT. Varic. -191 The CT is not reported in the dbSNP database, and the variation c.-167GT is known polymorphism. The other 4 variants are located in intron regions (c.289-3ta, c.576 + 14ga, c.115) near the exon splicing site. 4-21_1154-20instgttt,c.1350 + 8gc). Variation c. 576 + 14ga is not reported in dbSNP database. Variation c. 289-3ta, c. 1154-21 _ 1154-20instgttt and c.1350 + 8gc are known polymorphism. Conclusion: In this study, PAK2 gene changes were not found in children with autism in China
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R749.94;Q987

【共引文獻(xiàn)】

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