中国人群儿童孤独症的CORO2B基因关联研究及PAK2基因突变筛查

发布时间：2018-10-19 17:14

【摘要】：孤独症谱系障碍性疾病(Autism Spectrum Disorders, ASD)是一种严重影响儿童健康的神经发育性疾病,一般为3岁前发病,男女发病比例为4：1,最新流行病学调查显示ASD发病率已高达1/50。ASD具有高度的临床异质性,其三个典型的临床症状为语言障碍、社交障碍以及重复刻板的行为。近年来,通过染色体核型分析,全基因组连锁分析,全基因组拷贝数变异研究以及全基因组关联研究,已经鉴定了多个ASD相关的易感位点和基因。这些基因大多与神经发育、神经元可塑性、突触连接功能以及神经环路相关。第一章：中国人群儿童孤独症的CORO2B基因关联研究研究背景：我们前期的研究发现一例孤独症患者携带一个新发单拷贝数缺失,这个拷贝数变异包含CLN6、CORO2B、SENP8等31个基因。通过功能候选,我们认为CORO2B很可能与孤独症发病相关。另外我们之前的全基因组关联研究(GWAS)结果也表明CORO2B上的多个单核苷酸多态(SNP)与孤独症相关。研究目的：本研究将在我们新收集的一批孤独症样本中对我们前期的关联结果进行进一步验证,以探索CORO2B与中国人群孤独症发病的相关性。实验方法：利用Haploview挑选前期研究中与孤独症具有相关性的连锁不平衡区域的标签SNP (rs1465997),用primer3(http://frodo.wi.mit.edu/primer3/)对该位点两侧的碱基序列设计引物。PCR扩增该片段并用Sanger测序法进行直接测序。用DNAStar中的SeqMan进行分析,检测该位点的基因型。用plink (http://pngu.mgh.harvard.edu/～purcell/plink/)做哈代温伯格平衡(HWE)检验和传递不平衡关联分析(TDT)。实验结果：分型的270个核心家系在该SNP位点均符合孟德尔遗传定律。最小等位基因频率为0.24,基因型分布符合HWE定律(p=0.25)。传递不平衡关联分析结果无显著统计学意义(p=0.0522)且优势比与前期GWAS结果不一致(OR=0.76)。结论：本实验结果未能验证之前GWAS的结果。CORO2B该位点在本研究样本中与中国人群孤独症不相关。第二章：中国人群儿童孤独症的PAK2基因突变筛查研究背景：PAK2(P21蛋白激酶2)基因位于染色体3q29,含14个外显子,编码524个氨基酸。已有研究表明PAK1和PAK3突变与认知障碍疾病相关。PAK2位于3q29微缺失综合症(OMIM#609425)和3q29微重复综合症(OMIM#611936)拷贝数变异区间内。这两个综合征均存在智力低下和／或学习障碍,语音延迟和孤独症表型。这些证据表明PAK蛋白对神经发育至关重要,PAK家族基因的变异很可能与神经发育性疾病相关,如孤独症等。研究目的：本研究在中国人群孤独症患者中筛查PAK2基因突变,探索PAK2基因突变是否与中国人群孤独症相关。实验方法：在UCSC (http://genome.ucsc.edu/)中找到PAK2基因编码区和5'UTR区碱基序列。以PAK2基因编码区和5'UTR区碱基序列为目的片段用primer3设计引物(http://frodo.wi.mit.edu/primer3/)。对设计出的引物片段进行PCR扩增并用S anger测序法进行直接测序。用DNAStar中的SeqMan进行分析。以UCSU中查到的序列(NM_002577)为标准,分析PAK2基因的编码区和5'UTR区变异位点。实验结果：本研究在312个孤独症患者中共鉴定了10个变异。其中4个位于PAK2基因编码区域,均为同义突变。2个位于5'UTR区,为c.-191CT和c.-167GT。变异c.-191CT在dbSNP数据库中未见报道,变异c.-167GT为已知多态。另外4个变异位于外显子剪接位点附近的内含子区(c.289-3ta, c.576+14ga,c.1154-21_1154-20instgttt,c.1350+8gc)。变异c.576+14ga在dbSNP数据库中未见报道。变异c.289-3ta, c.1154-21_1154-20instgttt和c.1350+8gc均为已知多态。结论：本研究未在中国人群儿童孤独症患者中发现PAK2基因改变氨基酸编码的突变。不支持PAK2基因编码区变异与中国人群儿童孤独症相关。
[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
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.94;Q987

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