本文選題：先天性小耳畸形 + 芯片　； 參考：《復旦大學》2011年博士論文

【摘要】：第一部分先天性小耳畸形候選致病基因的篩選和驗證 目的：(1)應用Affymetrix SNP6.0芯片和直接測序驗證相結合的方法,篩選先天性小耳畸形的候選致病基因。 (2)檢測中國Treacher Collins綜合征患者TCOF1基因的突變情況。 方法：(1)收集先天性小耳畸形患者112例,對其中的3例患者血液基因組進行Affymetrix SNP6.0芯片分析獲得候選致病基因,然后在112例患者中對候選基因的外顯子直接測序驗證。 (2)采用直接測序的方法對3例Treacher Collins綜合征患者血液基因組中TCOF1所有外顯子,外顯子內(nèi)含子交界區(qū)和5’端上游1200bp進行突變分析。 結果：(1)經(jīng)過生物信息學的分析篩選出5個候選致病基因MSX1, MSX2,GSC, HOXA2和PACT。 (2)在112例患者中檢測了GSC, HOXA2和PACT的外顯子序列,檢測出3個多態(tài)性改變和位于HOXA2的5’非翻譯區(qū)的新發(fā)改變90GGA和114AAC,未發(fā)現(xiàn)致病突變。 (3)在3個Treacher Collins綜合征患者鑒定出12個TCOF1基因序列改變,包含4個新發(fā)的多態(tài)性改變-26TA,17693GA,21761-21765delCTCTC和21968GT。 結論：(1) GSC, HOXA2和PACT不是先天性小耳畸形的熱點突變基因。位于HOXA2的5’非翻譯區(qū)的新發(fā)改變的功能有待進一步研究。 (2)未在3例TCS患者血液基因組中發(fā)現(xiàn)TCOF1基因的致病突變,Treacher Collins綜合征患者可能存在其他致病基因或致病因素。 第二部分畸形患耳組織中microRNA表達譜的研究 目的：通過研究先天性小耳畸形患耳組織中microRNA異常表達情況,探討其在先天性小耳畸形發(fā)生過程中的作用,為今后基因功能研究奠定基礎。 方法：收集先天性小耳畸形患耳組織19例和正常耳廓組織5例,選取4例患耳組織和2例正常組織,抽取總RNA,采用北京博奧公司的Affymetrix miRNA芯片檢測microRNA表達情況,利用實時定量PCR技術對芯片結果驗證,挑選出表達有顯著差異的microRNA預測靶基因。 結果：(1)芯片篩查出表達上調(diào)的microRNA有7個,分別是hsa-miR-16, hsa-miR-140-3p, hsa-miR-126, hsa-miR-185, hsa-miR-378, hsa-miR-451和hsa-miR-486-5p,表達下調(diào)的microRNA有5個,分別是hsa-miR-203, hsa-miR-205, hsa-miR-200c, hsa-miR-708和hsa-miR-1308. (2)實時定量PCR驗證結果與芯片結果基本相符,差異有顯著性的表達上調(diào)microRNA有hsa-miR-126和hsa-miR-451,差異有顯著性的表達下調(diào)microRNA有hsa-miR-203, hsa-miR-205和hsa-miR-200c。 (3)靶基因預測：hsa-miR-126的靶基因篩選后得到26個,hsa-miR-451的靶基因篩選后得到15個,其中有很多基因的功能是未知的。 結論：建立了先天性小耳畸形microRNA表達譜,初步篩查出7個表達上調(diào)和5個表達下調(diào)的microRNA。采用實時定量PCR驗證得到了5個表達差異有顯著性的microRNA,證明芯片結果準確可靠。靶基因中有些基因的功能尚未有報道,本研究將挑選其中的基因繼續(xù)進行動物模型實驗,以研究未知基因的功能,探索與先天性小耳畸形發(fā)生的關系。
[Abstract]:Part 1 screening and validation of candidate genes for congenital microtia
Objective: (1) to screen candidate pathogenic genes of congenital microtia by using Affymetrix SNP6.0 chip and direct sequencing verification.
(2) detect the mutation of TCOF1 gene in Chinese patients with Treacher Collins syndrome.
Methods: (1) 112 cases of congenital microtia were collected. The blood genome of 3 of the patients was analyzed by Affymetrix SNP6.0 chip analysis to obtain the candidate genes. Then the exons of the candidate genes were directly sequenced in 112 patients.
(2) a direct sequencing method was used to analyze all the exons of TCOF1 in the blood genome of 3 patients with Treacher Collins syndrome, the intron junctional region of exons and the upstream 1200bp of the 5 'end.
Results: (1) 5 candidate genes MSX1, MSX2, GSC, HOXA2 and PACT. were screened out by bioinformatics analysis.
(2) the exons of GSC, HOXA2 and PACT were detected in 112 patients, and 3 polymorphic changes and new changes in the 5 'untranslated region of HOXA2 were detected, 90GGA and 114AAC, and no pathogenic mutation was found.
(3) 12 TCOF1 gene sequences were identified in 3 Treacher Collins syndrome patients, including 4 new polymorphic changes in -26TA, 17693GA, 21761-21765delCTCTC and 21968GT.
Conclusion: (1) GSC, HOXA2 and PACT are not the hot Mutation Genes of congenital microtia. The function of the new changes in the 5 'non translation region of HOXA2 needs further study.
(2) no pathogenic mutation of TCOF1 gene was found in the blood genome of 3 patients with TCS, and other pathogenic genes or pathogenic factors may exist in patients with Treacher Collins syndrome.
MicroRNA expression profiles in second parts of deformed ear tissue
Objective: To study the abnormal expression of microRNA in the ear tissue of congenital microtia and explore its role in the development of congenital microtia, and lay a foundation for the study of gene function in the future.
Methods: 19 cases of congenital microtia and 5 cases of normal auricle tissue were collected. 4 cases of ear tissue and 2 normal tissues were selected and total RNA was selected. The expression of microRNA was detected by Affymetrix miRNA chip of BOO company in Beijing. The results were verified by real-time quantitative PCR technique, and the significant difference of microRN was selected. A predicts the target gene.
Results: (1) 7 microRNA, hsa-miR-16, hsa-miR-140-3p, hsa-miR-126, hsa-miR-185, hsa-miR-378, hsa-miR-451 and hsa-miR-486-5p, and 5 down regulated microRNA, respectively, are hsa-miR-203, hsa-miR-205, hsa-miR-200c, etc.
(2) the results of real-time quantitative PCR verification are basically consistent with the results of the chip. There is a significant difference in the expression of microRNA with hsa-miR-126 and hsa-miR-451. There is a significant difference in the expression of microRNA with hsa-miR-203, hsa-miR-205 and hsa-miR-200c..
(3) target gene prediction: the target gene of hsa-miR-126 is screened 26, and the target gene of hsa-miR-451 is screened 15, of which many genes are unknown.
Conclusion: the microRNA expression profile of congenital microtia was established. A preliminary screening of 7 up-regulated and 5 down-regulated expressions of microRNA. by real-time quantitative PCR was used to obtain 5 microRNA with significant difference in expression. The function of some genes in the target gene has not been reported. This study will be selected in this study. The gene continues to carry out animal model experiments to study the function of unknown genes and explore the relationship with the occurrence of congenital microtia.

【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R764.7

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