视网膜色素变性的遗传背景及分子机制的研究

发布时间：2019-01-06 15:18

【摘要】：视网膜色素变性(retinitis pigmentosa,RP)是其中常见的一种遗传性视网膜疾病,全球发病率为1/3000-1/5000,主要特征是一组以进行性光感受Photoreceptors,PRs)细胞及视网膜色素上皮细胞(Retinal Pigment Epithelium,RPE)功能丧失为共同表现的遗传性视网膜变性疾病。目前已知与视网膜色素变性相关的致病基因及位点共有63个,其中22个是与常染色体显性遗传相关。目前仅有50%-60%的RP找到了明确的致病基因,仍有大于40%的RP患者的致病基因尚未找到。传统的连锁分析定位克隆的方法具有局限性。随着二代测序技术的进一步发展,外显子组测序技术(whole exome sequencing),全基因组(Whole-genome sequencing,WGS)和靶基因芯片捕获技术被认为是RP的相关致病基因筛查的福音。如何选择高效,合理的分子诊断技术并尽量控制成本是目前对于遗传性疾病基因诊断亟待解决的共同问题。本研究在二代测序联合目标靶基因捕获技术,全外显子组测序以及连锁分析等方法分别在RP的遗传家系中找到新的致病突变PRPF4和SPP2。其中PRPF4和我们前期已经发现的SNRNP200是前体m RNA剪切子,在m RNA合成过程中发挥着重要的生理功能。SPP2作为cystatins超家族的成员,但其具体功能目前尚不清楚。在本研究中,我们通过构建细胞和斑马鱼模式动物,分别观察了这些突变对于细胞正常生理功能,以及组织形态结构的改变。并深入探讨了其组织特异性可能的机制。在PRPF4中,我们发现两个突变c.-114_-97del和hPrp4~(Pro315Leu)。其中c.-114_-97del可以妨碍两个转录因子结合位点,我们用荧光素酶报告基因的方法观察到PRPF4的启动活性明显降低。从患者身上提取的皮肤成纤维细胞可以观察到h Prp4~(Pro315Leu)可以明显的上调多种剪切复合体中剪切因子水平(包括prpf4),改变剪切复合体SC35聚集状态。以上的结果说明剪切复合体其他剪切因子的升高可能是一种代偿效应。在动物模型中,我们发现过表达h Prp4~(Pro315Leu)后可以导致斑马鱼致死致畸率的升高,并且可以引起视网膜原发性的损害。当h Prp4~(Pro315Leu)与MO(沉默prpf4基因)共同作用时,其致死致畸率明显升高。以上结果显示PRPF4是RP新的致病突变并且分别是通过单倍体不足或者显性负性抑制作用。SNRNP200实验中,我们观察到SNRNP200在斑马鱼中是广泛表达的。并在斑马鱼模型中沉默掉SNRNP200后,发现其剪切功能紊乱(cbln1),并且可以升高剪切复合体中其他剪切子的水平,对于某些视网膜特异表达的基因明显下调。并且当沉默掉SNRNP200后,其大体出现致死致畸率的增加,并且视杆细胞早期受累,这与RP临床病程是一致的。SPP2实验中,我们发现了SPP2的p.Gly97Arg突变(位于SPP24两个高度保守二硫键环的第一个上面)。过表达p.Gly97Arg和p.Gly29Asp(本实验拟定的阳性参照)后会导致SPP2在细胞中潴留并最终引起内质网应激的发生。在斑马鱼模式动物中,我们观察到过表达SPP2 mutation后会特异性的影响视杆细胞的活性。对于这些RP突变的筛查和研究能够更好的理解RP疾病发生的病理病生过程,从而为临床治疗提供帮助。
[Abstract]:Retina pigmentosa (RP) is one of the most common hereditary retinal diseases, with a global incidence of 1/ 3000-1/ 5000. The main features are a group of photoreceptors (PRs) and retinal pigment epithelial cells (RPE). the function of the RPE) is lost to a common manifestation of a hereditary retinal degenerative disease. There are 63 pathogenic genes and sites associated with retinitis pigmentosa, 22 of which are associated with autosomal dominant inheritance. At present, only 50% to 60% of the RP has identified a clear pathogenic gene, and still more than 40% of the pathogenic genes of the RP patients have not yet been found. The traditional method of chain analysis and location cloning has the limitation. With the further development of the second generation sequencing technology, the exon group sequencing technology, the whole-genome sequencing, the WGS and the target gene chip capture technology are considered to be the gospel of the relevant pathogenic gene screening of the RP. How to select efficient and reasonable molecular diagnosis technology and to control the cost as much as possible is a common problem to be solved urgently for genetic disease gene diagnosis. In this study, new pathogenic mutations, PRPF4 and SPP2, were found in the genetic family of RP by the methods of second generation sequencing combined target gene capture, full-exon sequencing and linkage analysis, respectively. PRPF4 and the SNRNP200, which we have found in the earlier stage, are the front body m RNA shear, and play an important physiological function in the synthesis of m-RNA. SPP2 is a member of the caystins superfamily, but its specific function is not yet clear. in this study, we respectively observe that normal physiological function of the cell and the change of the structure of the tissue by construct the cell and the zebrafish model animal. The possible mechanism of its tissue specificity was discussed in detail. In PRPF4, we found two mutations c. -114 _-97del and hPrp4 ~ (Pro315Leu). c. -114 _-97del may interfere with the two transcription factor binding sites, and we have observed a significant decrease in the activation activity of the PRPF4 using the luciferase reporter gene. The skin fibroblasts extracted from the patient can observe that h Prp4 ~ (Pro315Leu) can increase the shear factor level (including prpf4) in a variety of shear complex, and change the aggregation state of the shear complex SC35. The above results indicate that the increase in other shear factors of the shear complex may be a compensatory effect. In animal model, we have found that after the expression of h Prp4 ~ (Pro315Leu) can lead to a rise in the teratogenic rate of zebrafish, and can cause primary damage of the retina. When h Prp4 ~ (Pro315Leu) and MO (silent prpf4 gene) co-act, the lethal teratogenic rate of h Prp4 ~ (Pro315Leu) increased significantly. The above results show that PRPF4 is a new pathogenic mutation of RP and is induced by either a haploid or a dominant negative. In the SNRNP200 experiment, we observed that SNRNP200 is widely expressed in zebrafish. After the SNRNP200 was silent in the zebrafish model, it was found that its shear function was disturbed (cbln1), and the level of other shear sublevels in the shear complex could be increased, and the genes for certain retinal-specific expression were significantly down-regulated. and when the SNRNP200 is silent, it generally has an increase in the teratogenic rate, and the stem cell is involved in the early stage, which is consistent with the clinical course of the RP. In the SPP2 experiment, we found the p. Gly97Arg mutation of SPP2 (above the first of the two highly conserved disulfide bonds of the SPP24). Overexpression of p. Gly97Arg and p. Gly29Asp (a positive reference prepared in this experiment) results in the retention of SPP2 in the cells and ultimately the occurrence of endoplasmic reticulum stress. In a zebrafish-mode animal, we observed that the activity of the rod-like cells will be specific after the expression of SPP2 muation. The screening and study of these RP mutations can provide a better understanding of the pathogenesis of the pathology of the RP disease, thereby providing assistance for clinical treatment.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R774.1

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