本文選題：低密度脂蛋白受體相關蛋白-5 + 基因突變　； 參考：《上海交通大學》2014年博士論文

【摘要】：目的 對家族性滲出性玻璃體視網(wǎng)膜病變(FEVR)的兩個家系行臨床表型分析和DNA測序，尋找致病基因的可能新突變位點。并進一步觀察低密度脂蛋白受體相關蛋白-5基因（LRP5）表達抑制后人視網(wǎng)膜血管內(nèi)皮細胞（hREC）轉(zhuǎn)錄組變化，初步探討其影響血管生成的可能機制。 方法 1.收集兩個FEVR家系的臨床資料并提取DNA，行FEVR四個已知致病基因（LRP5、FZD4、NDP和TSPAN12）的外顯子和相鄰內(nèi)含子區(qū)的Sanger測序，并構建相應LRP5突變質(zhì)粒行熒光素酶活性實驗，觀察其對Norrin/β-catenin信號途徑的影響，以判斷突變的致病性。 2. siRNA技術抑制hREC LRP5表達后，行轉(zhuǎn)錄組表達譜分析，篩選出差異顯著的基因，并行GO和KEGG Pathway分析。 結果 1.在兩個FEVR家系中各發(fā)現(xiàn)了LRP5基因上的一個未報道過的雜合突變：p.A422T和p.L540P，這兩個突變在各自的家系中與表型共分離，，熒光素酶活性實驗提示具有致病性。兩個先證者還另外各有一個LRP5基因的雜合突變，先證者一的p.Q816P（來自于無疾病的母親）和先證者二的新突變p.T852M（父母雙方均無），熒光素酶活性實驗提示p.Q816P可能無致病性，而p.T852M可能具有致病性。先證者的眼部表現(xiàn)均較患病的親代重，可能與聯(lián)合突變有關。所有患者均伴有骨密度下降。 2. hREC轉(zhuǎn)錄組表達譜比較結果顯示：LRP5基因表達抑制后，差異倍數(shù)超過2倍，p0.05的表達上調(diào)基因有87個，下調(diào)基因有52個；倍數(shù)超過2倍，p0.01的上調(diào)基因有28個，下調(diào)基因有22個。GO分析顯示有19個極顯著差異基因可投射至蛋白氨基酸磷酸化等6個GO term。KEGG Pathway分析顯示有31個差異基因與13條信號通路相關；局部粘附信號通路、細胞外基質(zhì)-受體相互作用、絲裂原活化蛋白激酶信號通路、JAK-STAT信號通路和ErbB信號通路等可能與LRP5調(diào)節(jié)視網(wǎng)膜血管發(fā)育相關。 結論 1.在中國人群中發(fā)現(xiàn)了LRP5基因上引起FEVR致病的兩個新突變位點，同時伴有骨密度異常；LRP5基因的聯(lián)合突變可能會加重臨床表型。 2. LRP5基因表達抑制前后hREC轉(zhuǎn)錄組的差異基因，蛋白氨基酸磷酸化及局部粘附信號通路等可能與LRP5調(diào)節(jié)視網(wǎng)膜血管發(fā)育相關。
[Abstract]:Objective to study the phenotypic analysis and DNA sequencing of two families with familial exudative vitreoretinopathy (FEVR) and to find out the possible new mutation sites of the pathogenic gene. The expression of low density lipoprotein receptor associated protein-5 (LRP5) in human retinal vascular endothelial cells (RECs) was observed and the possible mechanism of its influence on angiogenesis was investigated. Method 1. The clinical data of two FEVR families were collected and the DNA was extracted. The exons and adjacent intron regions of the four known pathogenic genes of FEVR, LRP5FZD4NDP and TSPAN12, were sequenced by Sanger sequencing, and the corresponding LRP5 mutant plasmids were constructed for luciferase activity experiments. After inhibiting the expression of hREC LRP5 by siRNA technique, the differentially expressed genes were screened out and analyzed by go and KEGG Pathway. Result 1. One unreported heterozygous mutation of LRP5 gene was found in two families of FEVR. The two mutations were separated from phenotypes in their respective families, and the luciferase activity test indicated pathogenicity. The two probands also had a heterozygous mutation of LRP5 gene, p. Q816P (from a disease-free mother) and p. T852Mof proband II (both parents had no LRP5 gene heterozyme). The results of luciferase activity test suggested that p. Q816P might not be pathogenicity. But P. T 852m may be pathogenicity. The ocular manifestations of the proband were heavier than those of the parent, and might be related to the joint mutation. In all patients, bone mineral density (BMD) decreased. 2. The results showed that after the expression of 1% LRP5 gene was inhibited, 87 genes were up-regulated and 52 genes were down-regulated. There were 28 up-regulated genes with multiple of 2 times p0.01, and 22 down-regulated genes with 22. Go analysis showed that 19 genes could project to protein amino acid phosphorylation and 6 go term.KEGG Pathway analysis showed that 31 differentially expressed genes were related to 13 signal pathways. Local adhesion signaling pathway, extracellular matrix receptor interaction, mitogen-activated protein kinase signaling pathway, JAK-STAT signal pathway and ErbB signal pathway may be related to the regulation of retinal vascular development by LRP5. Conclusion 1. Two new mutations in the LRP5 gene were found to cause FEVR in Chinese population, and the combined mutation of LRP5 gene with abnormal bone mineral density may aggravate the clinical phenotype. 2. The differentially expressed genes of hREC, protein amino acid phosphorylation and local adhesion signal pathway before and after LRP5 gene expression inhibition may be related to the regulation of retinal vascular development by LRP5.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R774.1

【參考文獻】

相關期刊論文 前1條

1 ;原發(fā)性骨質(zhì)疏松癥診治指南(2011年)[J];中華骨質(zhì)疏松和骨礦鹽疾病雜志;2011年01期

本文編號：2020482