RET基因3’UTR和SOX10基因多态性与中国汉族人群先天性巨结肠症的相关性研究

发布时间：2018-04-19 10:28

本文选题：先天性巨结肠症 + 中国汉族人群　；参考：《浙江大学》2012年博士论文

【摘要】：先天性巨结肠症(Hirschsprung's disease, HSCR),又称肠无神经节细胞症(aganglionosis),是小儿外科常见的消化道畸形病症,是典型的肠神经系统先天发育异常疾病。HSCR的病变特点是病变肠段肌间神经丛和粘膜下神经丛神经纤维增生伴神经节细胞缺如,肠道神经调节紊乱,以致受累肠段异常收缩,近端结肠代偿性扩张与增厚,形成巨结肠。先天性巨结肠的平均发病率为1/5000,以亚洲人群发病最高,为2.8/10000,高加索人群为1.5/10000,西班牙人群最低为1/10000。男女发生之比为4：1,在短段型HSCR中这种性别差异尤其显著。现已明确HSCR是一种神经嵴源疾病,发病与胚胎时期神经嵴细胞迁移过程异常有关。因此,目前鉴定的HSCR相关易感基因,主要来自于肠神经系统发育中起主要作用的2个信号通路相关基因,RET信号通路和EDNRB信号通路。迄今已发现11个HSCR相关易感基因,分别是RET原癌基因(RET)及其配体胶质神经源性神经生长因子基因(GDNF)和GDNF辅助受体家族基因神经营养蛋白基因(NTN),内皮素受体B基因(EDNRB)及其配体内皮素3(END3)和内皮素转化酶基因1(EC E1),此外还有3个信号通路相关的转录因子基因：SOX10. ZFHX1B、PHOX2B,以及在神经系统发育中起重要作用的KIAA1279基因、L1CAM基因和TITF1基因。本课题应用病例-对照分析方法对RET基因3'UTR和SOX10基因外显子上的相关多态性位点及单体型与HSCR之间进行相关分析研究,探讨RET3'UTR和SOX10基因在中国汉族儿童先天性巨结肠发病中的作用机制。以上研究结果,为进一步阐明中国汉族儿童先天性巨结肠发病的分子机制提供实验依据。第一部分RET基因3'UTR多态性与中国汉族人群先天性巨结肠症的相关性研究材料与方法：1.标本采集和基因组DNA抽提：根据2005年第四届国际巨结肠与相关神经嵴源性疾病会议制定的巨结肠诊断标准,确定研究病例。选择HSCR患者107例,同时收集无消化道和神经嵴相关疾病的正常血液标本89例为对照组。本研究经浙江大学医学院伦理委员会批准,全部患者和对照组均获得家长知情同意。基因组DNA抽提采用试剂盒提供的方法抽提,保存备用。 2.SNP的确定和分型：利用生物信息数据库查找目的基因的多态性位点(http://www.ncbi.nlm.nih.gov/snp/),共选取RET基因3'UTR的多态性位点7个采用PCR扩增目的基因、直接测序法对所选SNPs位点进行分型分析。 3.多态性位点和HSCR相关性的统计学分析软件包括：所测得基因型频率的Hardy-Weinberg在对照组中的遗传平衡检验；每个SNP与HSCR之间在不同遗传模式下的相关性分析；不同SNPs标记之间的连锁不平衡分析；构建单体型,估算单体型频率以及不同单体型与HSCR的相关性分析。所以以上统计分析的显著性差异的P值水平为0.5。 4. RNAstructure4.5和RNA二级结构在线分析软件(http://www.introni.it/rnafold.html)分析多态性位点对RNA二级结构的影响。结果： 1.单个SNP与HSCR的相关性分析结果显示：rs17028、rs3026782、rs2742240、rs2435355、rs2742241在中国人群为多态性,rs76759170、rs3026785在中国汉族人群不具有多态性；SNP1-5等位基因型频率在健康人群和HSCR病例之间的分布存在极显著性差异,P0.001,且与HSCR发病呈负相关,OR值从0.2834-0.5117之间,为HSCR的保护性位点；SNP1-5多态性位点等位基因型频率低于对照人群,OR值位于2.52-7.64之间。 2.根据最小AIC值,我们认为：rs17028,rs2435355为显性遗传模式；rs2742240和rs2742241的最佳遗传模式为加性；rs3026782的最佳遗传模式为超显性。 3.根据连锁不平衡分析结果,构建了由相邻位点存在显著相关(D'0.75)的SNPs组成的单体型(SNP1/SNP2/SNP3/SNP4/SNP5). 4.不同单体型和HSCR的相关性分析：单体型SNP1/SNP2/SNP3/SNP4/SNP5/SNP6/SNP7与HSCR存在负相关。默认的遗传模式为加性模式。 5.RNA二级结构在线分析软件和RNA structure4.5分析显示：SNP1和SNP7的野生型和突变型结构最小自由能分别从-73.0kcal/mol到-75.6kcal/mol-34.7kcal/mol到-37.1kcal/mol,表明突变型等位基因对RNA二级结构的稳定作用；其余位点的最小自由能均出现增加,显示了这些位点的等位基因可能影响RNA二级结构的不稳定性。结论： 1)首次对RET基因3’UTR的SNPs与中国汉族人群HSCR的相关性进行研究,结果显示这一区域的SNPs与HSCR的发病呈负相关。 2)这一区域的SNP位点可能是HSCR的保护性位点。 3)这些SNP位点可能通过影响RNA的二级结构从而影响RET基因的表达水平。第二部分SOX10基因多态性与中国汉族人群先天性巨结肠症的相关性研究材料与方法：1.标本采集和基因组DNA抽提：根据2005年第四届国际巨结肠与相关神经嵴源性疾病会议制定的巨结肠诊断标准,确定研究病例。选择HSCR患者104例,同时收集无消化道和神经嵴相关疾病的正常血液标本94例为对照组。本研究经浙江大学医学院伦理委员会批准,全部患者和对照组均获得家长知情同意。基因组DNA抽提采用试剂盒提供的方法抽提,保存备用。 2.基因突变检测和SNPs分型：采用PCR-直接测序法对SOX10基因外显子和部分内含子序列进行PCR扩增,直接测序法进行相关突变和多态性扫描,并对结果进行分型。 3.多态性位点和HSCR相关性的统计学分析软件包括：所测得基因型频率的Hardy-Weinberg在对照组中的遗传平衡检验；每个SNP与HSCR之间在不同遗传模式下的相关性分析；不同SNPs标记之间的连锁不平衡分析；构建单体型,估算单体型频率以及不同单体型与HSCR的相关性分析。所以以上统计分析的显著性差异的P值水平为0.05。结果： 1.SOX10基因突变检测：在SOX10基因上发现4个SNPs。第一个SNP在第2外显子编码区,序列改变为c.18CT(GAC→GAT),为SNP1,编码氨基酸为D6D；第二个SNP位于第2外显子编码区,序列改变为c.122GT(GGC→GTC),导致甘氨酸到缬氨酸的改变,本研究命名为SNP2；第三个SNP位于第三内含子区域,序列改变为IVS3+10C→G,本研究命名为SNP3；第四个SNP位于第4外显子编码区,序列改变为c.927TC(CAT→CAC),组氨酸同义突变,本研究命名为SNP4。 2.相关性分析：4个SNP基因型分布和等位基因频率在病例组和对照组中无显著性差异。运用逻辑回归分析法,在五种不同的遗传模式下分析单个SNP和先天性巨结肠症的相关性,未发现SNP位点与HSCR有相关性。结论： 1)首次在HSCR病例中进行SOX10基因突变和多态性检测分析,发现一个错义突变和两个同义突变位点,这些位点经分析与HSCR发病无相关性。 2) SOX10可能不是中国汉族人群散发性HSCR的易感基因。
[Abstract]:Congenital megacolon (Hirschsprung's disease, HSCR), also called intestinal aganglionosis (aganglionosis), is a disease of digestive tract malformation in pediatric surgery is common, enteric nervous system congenital dysplasia disease pathological characteristics of.HSCR lesions is a typical intestinal myenteric plexus and submucosal plexus nerve fiber hyperplasia with the absence of intestinal ganglion cells, neural regulation disorder that affected intestinal segment abnormal contraction, proximal colon compensatory enlargement and thickening of the formation of megacolon. The average incidence of Hirschsprung's disease rate is 1/5000, the Asian mass is 2.8/10000, the highest disease, Caucasian 1.5/10000, Spanish population minimum 1/10000. sex ratio for 4:1, especially the gender difference in short type HSCR.
It is clear that HSCR is a kind of neural crest derived diseases, and the incidence of embryonic neural crest cell migration process abnormalities. Therefore, the identification of HSCR related susceptibility genes, 2 signal pathway related genes mainly from the enteric nervous system plays a major role in the development of the RET and EDNRB signal pathways have been found so far. 11 HSCR susceptible genes are RET oncogene (RET) and its ligand glial derived neurotrophic factor gene (GDNF) gene and neural GDNF coreceptor protein gene family nutrition (NTN), endothelin receptor B gene (EDNRB) and its ligand endothelin 3 and endothelin (END3) invertase gene 1 (EC E1), in addition to the 3 signal pathway related transcription factor gene: SOX10. ZFHX1B, PHOX2B, as well as in the nervous system play an important role in the development of KIAA1279 gene, L1CAM gene and TITF1 gene.
The control method for RET 3'UTR gene and SOX10 gene exon between loci associated polymorphisms and the haplotypes with HSCR were investigated by applying the case, to explore the mechanism of RET3'UTR and SOX10 gene in the pathogenesis of China Han children in Hirschsprung's disease. The research results provide an experimental basis for further to elucidate the molecular mechanism of Chinese Han children in Hirschsprung's disease.
The correlation between the first part of the RET gene 3'UTR polymorphism and Hirschsprung's disease in Chinese Han population
Materials and methods: 1. specimens were collected and genomic DNA extraction: according to the diagnostic criteria of Hirschsprung's disease in 2005 fourth session of the international giant colon and related neural crest derived disease made at the meeting, determine the research cases. 107 cases of HSCR were collected at the same time, digestive tract and neural crest related diseases of normal blood samples of 89 patients in the control group. This study was approved by the ethics committee of the Zhejiang University School of medicine, all the patients and the control group received parental consent. Extraction method of genomic DNA extraction kit provided by preservation reserve.
Identify and type 2.SNP: the use of biological information database to find polymorphic loci gene (http://www.ncbi.nlm.nih.gov/snp/), selected RET gene 3'UTR 7 polymorphic loci amplified by PCR gene sequencing typing analysis of selected SNPs sites.
Statistics of 3. polymorphic loci and HSCR correlation analysis software includes genetic equilibrium test measured the genotype frequency of Hardy-Weinberg in the control group; SNP and HSCR correlation analysis between each under different genetic model between different SNPs markers; linkage disequilibrium; haplotype, haplotype analysis and frequency estimation correlation of different haplotypes with HSCR. So the significant differences between the above statistical analysis of the P level for 0.5.
4. RNAstructure4.5 and RNA two level structure online analysis software (http://www.introni.it/rnafold.html) was used to analyze the effect of polymorphic loci on the structure of RNA two.
Result:
Correlation analysis of 1. single SNP and HSCR results showed that rs17028, rs3026782, rs2742240, rs2435355, rs2742241 in Chinese population polymorphism, rs76759170, rs3026785 in Chinese Han population had no polymorphism; there are significant differences, the distribution of SNP1-5 allele frequencies between healthy people and HSCR cases of P0.001, and a negative correlation with the incidence of HSCR, OR from 0.2834-0.5117, for the protection of the site of HSCR; SNP1-5 polymorphism allele frequency is lower than the control group, the OR value in 2.52-7.64.
2. according to the minimum AIC value, we think that rs17028 and rs2435355 are dominant genetic models. The best genetic pattern of rs2742240 and rs2742241 is additive; the best genetic mode of rs3026782 is overdominance.
3. based on the results of linkage disequilibrium analysis, a haplotype (SNP1/SNP2/SNP3/SNP4/SNP5) composed of SNPs with significant correlation (D'0.75) is constructed.
4. correlation analysis between different haplotypes and HSCR: there was a negative correlation between SNP1/SNP2/SNP3/SNP4/SNP5/SNP6/SNP7 and HSCR in haplotype. The default genetic model was additive model.
Online 5.RNA two level structure analysis software and RNA structure4.5 analysis showed that wild-type SNP1 and SNP7 mutant type and structure of minimum free energy respectively from -73.0kcal/mol to -75.6kcal/mol-34.7kcal/mol to -37.1kcal/mol, showed that the stabilizing effect of the mutant allele of RNA two level structure; the rest of the minimum free sites can increased, shows these sites the allele may affect the RNA two level structure instability.
Conclusion:
1) the first study of the correlation between the SNPs of the RET gene 3 'UTR and the HSCR of the Chinese Han population showed that the SNPs in this region was negatively correlated with the incidence of HSCR.
2) the SNP locus in this region may be a protective site for HSCR.
3) these SNP loci may affect the expression level of the RET gene by affecting the two grade structure of the RET.
The relationship between the second part of SOX10 gene polymorphism and Hirschsprung's disease in Chinese Han population
Materials and methods: 1. specimens were collected and genomic DNA extraction: according to the diagnostic criteria of Hirschsprung's disease in 2005 fourth session of the international giant colon and related neural crest derived disease made at the meeting, determine the research cases. 104 cases of HSCR were collected at the same time, digestive tract and neural crest related diseases of normal blood samples of 94 patients in the control group. This study was approved by the ethics committee of the Zhejiang University School of medicine, all the patients and the control group received parental consent. Extraction method of genomic DNA extraction kit provided by preservation reserve.
2. gene mutation detection and SNPs typing: PCR- direct sequencing method was used to amplify the exon and partial intron sequences of SOX10 gene by PCR amplification. Direct sequencing method was used to carry out related mutation and polymorphism scanning, and the results were classified.
Statistics of 3. polymorphic loci and HSCR correlation analysis software includes genetic equilibrium test measured the genotype frequency of Hardy-Weinberg in the control group; SNP and HSCR correlation analysis between each under different genetic model between different SNPs markers; linkage disequilibrium; haplotype, haplotype analysis and frequency estimation correlation of different haplotypes with HSCR. So the significant differences between the above statistical analysis of the P level for 0.05.
Result:
Detection of 1.SOX10 gene mutation was found: 4 SNPs. SNP in the first exon encoding region in second in the SOX10 gene, sequence change to c.18CT (GAC to GAT), SNP1, encoding amino acid D6D; second SNP located in the second exon encoding region, sequence change to c.122GT (GGC, GTC), cause to change the glycine valine, this study named SNP2; third SNP located in the third intron region, sequence change of IVS3+10C to G, this study named SNP3; fourth SNP located in the fourth exon encoding region, sequence change to c.927TC (CAT, CAC), group of amino acids synonymous mutation, this study named SNP4.
2. correlation analysis: 4 SNP genotype distribution and no significant difference in allele frequency between cases and controls. Using logistic regression analysis, correlation analysis of single SNP and Hirschsprung's disease in five different genetic models, found no correlation between SNP and HSCR sites.
Conclusion:
1) for the first time, SOX10 gene mutation and polymorphism were detected in HSCR cases, and a missense mutation and two synonymous mutation sites were found. These loci were not correlated with the onset of HSCR.
2) SOX10 may not be a susceptible gene for sporadic HSCR in Chinese Han population.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R726.5

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