先天性小耳畸形候选致病基因筛选和畸形耳组织中microRNA表达谱的研究

发布时间：2018-04-28 14:03

本文选题：先天性小耳畸形 + 芯片　；参考：《复旦大学》2011年博士论文

【摘要】：第一部分先天性小耳畸形候选致病基因的筛选和验证目的：(1)应用Affymetrix SNP6.0芯片和直接测序验证相结合的方法,筛选先天性小耳畸形的候选致病基因。 (2)检测中国Treacher Collins综合征患者TCOF1基因的突变情况。方法：(1)收集先天性小耳畸形患者112例,对其中的3例患者血液基因组进行Affymetrix SNP6.0芯片分析获得候选致病基因,然后在112例患者中对候选基因的外显子直接测序验证。 (2)采用直接测序的方法对3例Treacher Collins综合征患者血液基因组中TCOF1所有外显子,外显子内含子交界区和5’端上游1200bp进行突变分析。结果：(1)经过生物信息学的分析筛选出5个候选致病基因MSX1, MSX2,GSC, HOXA2和PACT。 (2)在112例患者中检测了GSC, HOXA2和PACT的外显子序列,检测出3个多态性改变和位于HOXA2的5’非翻译区的新发改变90GGA和114AAC,未发现致病突变。 (3)在3个Treacher Collins综合征患者鉴定出12个TCOF1基因序列改变,包含4个新发的多态性改变-26TA,17693GA,21761-21765delCTCTC和21968GT。结论：(1) GSC, HOXA2和PACT不是先天性小耳畸形的热点突变基因。位于HOXA2的5’非翻译区的新发改变的功能有待进一步研究。 (2)未在3例TCS患者血液基因组中发现TCOF1基因的致病突变,Treacher Collins综合征患者可能存在其他致病基因或致病因素。第二部分畸形患耳组织中microRNA表达谱的研究目的：通过研究先天性小耳畸形患耳组织中microRNA异常表达情况,探讨其在先天性小耳畸形发生过程中的作用,为今后基因功能研究奠定基础。方法：收集先天性小耳畸形患耳组织19例和正常耳廓组织5例,选取4例患耳组织和2例正常组织,抽取总RNA,采用北京博奥公司的Affymetrix miRNA芯片检测microRNA表达情况,利用实时定量PCR技术对芯片结果验证,挑选出表达有显著差异的microRNA预测靶基因。结果：(1)芯片筛查出表达上调的microRNA有7个,分别是hsa-miR-16, hsa-miR-140-3p, hsa-miR-126, hsa-miR-185, hsa-miR-378, hsa-miR-451和hsa-miR-486-5p,表达下调的microRNA有5个,分别是hsa-miR-203, hsa-miR-205, hsa-miR-200c, hsa-miR-708和hsa-miR-1308. (2)实时定量PCR验证结果与芯片结果基本相符,差异有显著性的表达上调microRNA有hsa-miR-126和hsa-miR-451,差异有显著性的表达下调microRNA有hsa-miR-203, hsa-miR-205和hsa-miR-200c。 (3)靶基因预测：hsa-miR-126的靶基因筛选后得到26个,hsa-miR-451的靶基因筛选后得到15个,其中有很多基因的功能是未知的。结论：建立了先天性小耳畸形microRNA表达谱,初步筛查出7个表达上调和5个表达下调的microRNA。采用实时定量PCR验证得到了5个表达差异有显著性的microRNA,证明芯片结果准确可靠。靶基因中有些基因的功能尚未有报道,本研究将挑选其中的基因继续进行动物模型实验,以研究未知基因的功能,探索与先天性小耳畸形发生的关系。
[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.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R764.7

【参考文献】