本文選題：先天性巨結腸癥 + 中國漢族人群��； 參考：《浙江大學》2012年博士論文

【摘要】：先天性巨結腸癥(Hirschsprung's disease, HSCR),又稱腸無神經(jīng)節(jié)細胞癥(aganglionosis),是小兒外科常見的消化道畸形病癥,是典型的腸神經(jīng)系統(tǒng)先天發(fā)育異常疾病。HSCR的病變特點是病變腸段肌間神經(jīng)叢和粘膜下神經(jīng)叢神經(jīng)纖維增生伴神經(jīng)節(jié)細胞缺如,腸道神經(jīng)調節(jié)紊亂,以致受累腸段異常收縮,近端結腸代償性擴張與增厚,形成巨結腸。先天性巨結腸的平均發(fā)病率為1/5000,以亞洲人群發(fā)病最高,為2.8/10000,高加索人群為1.5/10000,西班牙人群最低為1/10000。男女發(fā)生之比為4：1,在短段型HSCR中這種性別差異尤其顯著。 現(xiàn)已明確HSCR是一種神經(jīng)嵴源疾病,發(fā)病與胚胎時期神經(jīng)嵴細胞遷移過程異常有關。因此,目前鑒定的HSCR相關易感基因,主要來自于腸神經(jīng)系統(tǒng)發(fā)育中起主要作用的2個信號通路相關基因,RET信號通路和EDNRB信號通路。迄今已發(fā)現(xiàn)11個HSCR相關易感基因,分別是RET原癌基因(RET)及其配體膠質神經(jīng)源性神經(jīng)生長因子基因(GDNF)和GDNF輔助受體家族基因神經(jīng)營養(yǎng)蛋白基因(NTN),內皮素受體B基因(EDNRB)及其配體內皮素3(END3)和內皮素轉化酶基因1(EC E1),此外還有3個信號通路相關的轉錄因子基因：SOX10. ZFHX1B、PHOX2B,以及在神經(jīng)系統(tǒng)發(fā)育中起重要作用的KIAA1279基因、L1CAM基因和TITF1基因。 本課題應用病例-對照分析方法對RET基因3'UTR和SOX10基因外顯子上的相關多態(tài)性位點及單體型與HSCR之間進行相關分析研究,探討RET3'UTR和SOX10基因在中國漢族兒童先天性巨結腸發(fā)病中的作用機制。以上研究結果,為進一步闡明中國漢族兒童先天性巨結腸發(fā)病的分子機制提供實驗依據(jù)。 第一部分RET基因3'UTR多態(tài)性與中國漢族人群先天性巨結腸癥的相關性研究 材料與方法：1.標本采集和基因組DNA抽提：根據(jù)2005年第四屆國際巨結腸與相關神經(jīng)嵴源性疾病會議制定的巨結腸診斷標準,確定研究病例。選擇HSCR患者107例,同時收集無消化道和神經(jīng)嵴相關疾病的正常血液標本89例為對照組。本研究經(jīng)浙江大學醫(yī)學院倫理委員會批準,全部患者和對照組均獲得家長知情同意�；蚪MDNA抽提采用試劑盒提供的方法抽提,保存?zhèn)溆谩?2.SNP的確定和分型：利用生物信息數(shù)據(jù)庫查找目的基因的多態(tài)性位點(http://www.ncbi.nlm.nih.gov/snp/),共選取RET基因3'UTR的多態(tài)性位點7個采用PCR擴增目的基因、直接測序法對所選SNPs位點進行分型分析。 3.多態(tài)性位點和HSCR相關性的統(tǒng)計學分析軟件包括：所測得基因型頻率的Hardy-Weinberg在對照組中的遺傳平衡檢驗；每個SNP與HSCR之間在不同遺傳模式下的相關性分析；不同SNPs標記之間的連鎖不平衡分析；構建單體型,估算單體型頻率以及不同單體型與HSCR的相關性分析。所以以上統(tǒng)計分析的顯著性差異的P值水平為0.5。 4. RNAstructure4.5和RNA二級結構在線分析軟件(http://www.introni.it/rnafold.html)分析多態(tài)性位點對RNA二級結構的影響。 結果： 1.單個SNP與HSCR的相關性分析結果顯示：rs17028、rs3026782、rs2742240、rs2435355、rs2742241在中國人群為多態(tài)性,rs76759170、rs3026785在中國漢族人群不具有多態(tài)性；SNP1-5等位基因型頻率在健康人群和HSCR病例之間的分布存在極顯著性差異,P0.001,且與HSCR發(fā)病呈負相關,OR值從0.2834-0.5117之間,為HSCR的保護性位點；SNP1-5多態(tài)性位點等位基因型頻率低于對照人群,OR值位于2.52-7.64之間。 2.根據(jù)最小AIC值,我們認為：rs17028,rs2435355為顯性遺傳模式；rs2742240和rs2742241的最佳遺傳模式為加性；rs3026782的最佳遺傳模式為超顯性。 3.根據(jù)連鎖不平衡分析結果,構建了由相鄰位點存在顯著相關(D'0.75)的SNPs組成的單體型(SNP1/SNP2/SNP3/SNP4/SNP5). 4.不同單體型和HSCR的相關性分析：單體型SNP1/SNP2/SNP3/SNP4/SNP5/SNP6/SNP7與HSCR存在負相關。默認的遺傳模式為加性模式。 5.RNA二級結構在線分析軟件和RNA structure4.5分析顯示：SNP1和SNP7的野生型和突變型結構最小自由能分別從-73.0kcal/mol到-75.6kcal/mol-34.7kcal/mol到-37.1kcal/mol,表明突變型等位基因對RNA二級結構的穩(wěn)定作用；其余位點的最小自由能均出現(xiàn)增加,顯示了這些位點的等位基因可能影響RNA二級結構的不穩(wěn)定性。 結論： 1)首次對RET基因3’UTR的SNPs與中國漢族人群HSCR的相關性進行研究,結果顯示這一區(qū)域的SNPs與HSCR的發(fā)病呈負相關。 2)這一區(qū)域的SNP位點可能是HSCR的保護性位點。 3)這些SNP位點可能通過影響RNA的二級結構從而影響RET基因的表達水平。 第二部分SOX10基因多態(tài)性與中國漢族人群先天性巨結腸癥的相關性研究 材料與方法：1.標本采集和基因組DNA抽提：根據(jù)2005年第四屆國際巨結腸與相關神經(jīng)嵴源性疾病會議制定的巨結腸診斷標準,確定研究病例。選擇HSCR患者104例,同時收集無消化道和神經(jīng)嵴相關疾病的正常血液標本94例為對照組。本研究經(jīng)浙江大學醫(yī)學院倫理委員會批準,全部患者和對照組均獲得家長知情同意。基因組DNA抽提采用試劑盒提供的方法抽提,保存?zhèn)溆谩?2.基因突變檢測和SNPs分型：采用PCR-直接測序法對SOX10基因外顯子和部分內含子序列進行PCR擴增,直接測序法進行相關突變和多態(tài)性掃描,并對結果進行分型。 3.多態(tài)性位點和HSCR相關性的統(tǒng)計學分析軟件包括：所測得基因型頻率的Hardy-Weinberg在對照組中的遺傳平衡檢驗；每個SNP與HSCR之間在不同遺傳模式下的相關性分析；不同SNPs標記之間的連鎖不平衡分析；構建單體型,估算單體型頻率以及不同單體型與HSCR的相關性分析。所以以上統(tǒng)計分析的顯著性差異的P值水平為0.05。 結果： 1.SOX10基因突變檢測：在SOX10基因上發(fā)現(xiàn)4個SNPs。第一個SNP在第2外顯子編碼區(qū),序列改變?yōu)閏.18CT(GAC→GAT),為SNP1,編碼氨基酸為D6D；第二個SNP位于第2外顯子編碼區(qū),序列改變?yōu)閏.122GT(GGC→GTC),導致甘氨酸到纈氨酸的改變,本研究命名為SNP2；第三個SNP位于第三內含子區(qū)域,序列改變?yōu)镮VS3+10C→G,本研究命名為SNP3；第四個SNP位于第4外顯子編碼區(qū),序列改變?yōu)閏.927TC(CAT→CAC),組氨酸同義突變,本研究命名為SNP4。 2.相關性分析：4個SNP基因型分布和等位基因頻率在病例組和對照組中無顯著性差異。運用邏輯回歸分析法,在五種不同的遺傳模式下分析單個SNP和先天性巨結腸癥的相關性,未發(fā)現(xiàn)SNP位點與HSCR有相關性。 結論： 1)首次在HSCR病例中進行SOX10基因突變和多態(tài)性檢測分析,發(fā)現(xiàn)一個錯義突變和兩個同義突變位點,這些位點經(jīng)分析與HSCR發(fā)病無相關性。 2) SOX10可能不是中國漢族人群散發(fā)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.

【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R726.5

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