LRP5基因突变与FEVR发病关系及其对hREC转录组影响

发布时间：2018-06-15 03:46

本文选题：低密度脂蛋白受体相关蛋白-5 + 基因突变　；参考：《上海交通大学》2014年博士论文

【摘要】：目的对家族性渗出性玻璃体视网膜病变(FEVR)的两个家系行临床表型分析和DNA测序，寻找致病基因的可能新突变位点。并进一步观察低密度脂蛋白受体相关蛋白-5基因（LRP5）表达抑制后人视网膜血管内皮细胞（hREC）转录组变化，初步探讨其影响血管生成的可能机制。方法 1.收集两个FEVR家系的临床资料并提取DNA，行FEVR四个已知致病基因（LRP5、FZD4、NDP和TSPAN12）的外显子和相邻内含子区的Sanger测序，并构建相应LRP5突变质粒行荧光素酶活性实验，观察其对Norrin/β-catenin信号途径的影响，以判断突变的致病性。 2. siRNA技术抑制hREC LRP5表达后，行转录组表达谱分析，筛选出差异显著的基因，并行GO和KEGG Pathway分析。结果 1.在两个FEVR家系中各发现了LRP5基因上的一个未报道过的杂合突变：p.A422T和p.L540P，这两个突变在各自的家系中与表型共分离，，荧光素酶活性实验提示具有致病性。两个先证者还另外各有一个LRP5基因的杂合突变，先证者一的p.Q816P（来自于无疾病的母亲）和先证者二的新突变p.T852M（父母双方均无），荧光素酶活性实验提示p.Q816P可能无致病性，而p.T852M可能具有致病性。先证者的眼部表现均较患病的亲代重，可能与联合突变有关。所有患者均伴有骨密度下降。 2. hREC转录组表达谱比较结果显示：LRP5基因表达抑制后，差异倍数超过2倍，p0.05的表达上调基因有87个，下调基因有52个；倍数超过2倍，p0.01的上调基因有28个，下调基因有22个。GO分析显示有19个极显著差异基因可投射至蛋白氨基酸磷酸化等6个GO term。KEGG Pathway分析显示有31个差异基因与13条信号通路相关；局部粘附信号通路、细胞外基质-受体相互作用、丝裂原活化蛋白激酶信号通路、JAK-STAT信号通路和ErbB信号通路等可能与LRP5调节视网膜血管发育相关。结论 1.在中国人群中发现了LRP5基因上引起FEVR致病的两个新突变位点，同时伴有骨密度异常；LRP5基因的联合突变可能会加重临床表型。 2. LRP5基因表达抑制前后hREC转录组的差异基因，蛋白氨基酸磷酸化及局部粘附信号通路等可能与LRP5调节视网膜血管发育相关。
[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.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R774.1

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