原发性视网膜色素变性与视网膜血管闭塞的相关性研究

发布时间：2018-10-20 17:21

【摘要】：研究目的视网膜色素变性(retinitis pigmentosa,RP)是视网膜变性中最常见的一组以进行性光感受器细胞及视网膜色素上皮(retinitis pigment epithelium,RPE)功能丧失为共同表现的遗传性疾病。而原发性视网膜色素变性合并视网膜血管进行性闭塞是近年来眼科医师逐步认识的一种疾病。国内曾以原发性血管闭塞性视神经视网膜病变做个案报道,强调不明原因,原发性血管闭塞为特点而国外未见报道。在进一步完善资料的补充搜集之后,本研究对此病的临床特点进行总结,并对部分患者进行既往未曾做过的全基因组外显子测序以试图阐明其发病机制并印证既往我们对此病的认识。研究方法回顾性分析23例(46只眼)原发性RP合并视网膜血管闭塞患者的临床资料。其中男性12例,女性11例;年龄从6～70岁平均49.96±15.80岁。患者均双眼受累。患者均进行了包括眼底检查、荧光素眼底血管造影、视网膜电图等检查。采集其中6个患者的血样,还采集了其中1个患者父母(无临床表现)血样,共8个样本。进行全基因组外显子测序。测序主要包括目标区域序列的富集、DNA测序、生物信息学统计三个主要步骤。测序结果中对出现在2个及以上case的位点进行了分析,重点关注没有注释到单核苷酸多态性数据库的位点,可能是潜在的与RP相关的新的位点;进一步关注筛选突变类型为错义突变等能直接引起氨基酸改变的位点,这样功能变化会相对比较明显。这样大概只有不到300个位点,81个基因;跟进位点depth≥10且quality≥20进行过滤。然后对这些潜在位点应用京都基因和基因组百科全书(Kyoto Encyclopedia of Genesand Genomes,KEGG)和基因本体论数据库(gene ontology,GO)进了功能富集分析。并结合临床背景、经验,进行综合筛选过滤,寻找潜在的位点和致病基因。研究结果原发性RP合并视网膜血管闭塞患者的临床表现为眼前节均无炎性反应,眼压正常,晶状体混浊20只眼,未见虹膜新生血管;患者眼底视乳头色淡,其中苍白如月者17只眼;视网膜血管变细,鼻侧血管更明显,血管旁可见白鞘。由于患者病程不一,其视网膜血管闭塞程度亦不同,严重者血管可闭塞成白线。视网膜色素紊乱和脱色素点,无典型骨细胞样色素沉着。未见视网膜新生血管,无增生性视网膜病变及玻璃体积血。20只眼眼底荧光血管造影示视乳头始终呈低荧光或晚期淡荧光充盈,局限于有光感眼或无光感眼。视乳头部位有一小段血管有荧光素充盈或完全无血管充盈者17只眼。整个眼底可见椒盐状荧光素充盈。吲哚青绿造影检查表现为脉络膜血管较细,荧光素充盈较弱,退行较快,视网膜血管闭塞表现同荧光素眼底血管造影所见。视网膜电图检查显示晚期明视、暗视ERG的a,b波幅为无波型或近无波型。患者多有夜盲史。8个样本全基因组外显子测序结果出现了已报道的RP相关致病基因的单核苷酸多态性位点,又在一些基因的编码区发现了新的突变位点,并导致了氨基酸改变。基因注释发现与组织凋亡,神经褪变,抑制细胞增殖等相关。应用KEGG和GO数据库寻找生物学相关通路,显示存在有凋亡通路,神经轴突导向通路,血管内皮生长因子信号通路,视黄醇代谢通路等。结论本组研究患者眼底均表现为视乳头色淡,视网膜血管变细,广泛视网膜色素上皮受累,可见脱色素斑点及有或无细小色素点;ERG检查a、b波均为无波型或近无波型,支持本组原发性RP合并视网膜血管闭塞患者属于原发性RP(毯层RP)范畴。同时也有其自身临床特点:1.晚期血管可完全闭塞成白线或近完全闭塞,视神经明显萎缩可以苍白如月。2.视网膜色素上皮以进行性萎缩为主、无典型骨细胞样色素增殖沉着,未见视网膜新生血管及玻璃体出血。3.此病病程进展速度似较原发性RP为快。全基因组外显子测序结果中出现了已报道的RP致病基因的单核苷酸多态性位点,是否这些位点在疾病中高发并与RP相关有待验证。在一些基因编码区发现了引起氨基酸改变的很多未见报道的新突变位点,基因注释发现与组织凋亡,神经褪变,抑制细胞增殖等相关。并有基因参与凋亡、神经轴突导向、血管内皮生长因子信号等生物学通路。若从理论上完全解释还有待进一步的工作来证实。
[Abstract]:Retinitis pigmentosa (RP) is one of the most common genetic diseases in the degeneration of retina, which is characterized by progressive photoreceptor cells and retinal pigment epithelium (RPE). Primary retinal pigment degeneration and progressive occlusion of retinal vessels are a disease which has been gradually recognized by the ophthalmologist in recent years. A case report of primary angiitis obliterans optic neuropathy was reported in China. It was emphasized that it was not reported in foreign countries due to unknown reasons and primary vascular occlusion. After further improvement of the data collection, this study summarized the clinical features of the disease and sequenced all genome exons that had not been previously done by some patients in an attempt to elucidate the pathogenesis of the disease and confirm our knowledge of the disease. Methods The clinical data of 23 cases (46 eyes) with primary RP combined with retinal vessel occlusion were analyzed retrospectively. Among them, 12 males and 11 females were females, and the average age ranged from 6 to 70 years, and the average age ranged from 49. 96 to 15. 80 years. Both eyes of the patient were involved. All patients underwent examinations including fundus examination, fluorescein angiography, electroretinogram, etc. Blood samples were collected from six of these patients, and a total of 8 samples were collected from one of the patients (no clinical presentation). All genome exons were sequenced. The sequencing mainly includes three main steps: enrichment of target region sequence, DNA sequencing and bioinformatics statistics. The results of sequencing showed that the sites of 2 and more cases were analyzed, and the sites that were not annotated to the single-point polymorphism database might be potential new sites related to RP. Further attention is paid to screening mutation types such as missense mutation and so on, which can directly cause amino acid changes, so that the functional changes can be relatively obvious. Thus, there are only less than 300 sites, 81 genes, a follow-up site depth column 10, and a quality table 20 for filtering. These potential sites were then subjected to functional enrichment analysis using Kyoto Encyclopedia of Genesand Genes (KEGG) and Gene Ontology Database (GO). Combined with the clinical background and experience, the comprehensive screening and filtration were carried out to find the potential sites and pathogenic genes. Results The clinical manifestations of primary RP-combined retinal vascular occlusion were no inflammatory reaction in anterior segment, normal intraocular pressure, 20 eyes with posterior lens opacity, and no iris neovascularization. The blood vessels at the nose side are more obvious, and white blood vessels can be seen near the blood vessels. Because the course of the patient is not one, the degree of retinal blood vessel occlusion is different, and the blood vessel of the serious person can be blocked into white line. Retinal pigment disorder and depigmentation point, no typical osteoclast-like pigmentation. No retinal neovascularization, no proliferative retinopathy and vitreous hemorrhage were seen. 20 eyes with fundus fluorescein angiography showed that the nipple was always low or late fluorescent filling, limited to light-sensitive eyes or no light-sensitive eyes. There is a small segment of blood vessel at the papillary site with fluorescein filling or no blood vessel filling 17 eyes. The whole fundus of the fundus can be filled with salt-like fluorescein. Angiographic examination showed that the choroid vessels were thinner, the fluorescein filling was weak, the regression line was faster, and the retinal vessel occlusion was seen with fluorescein angiography. The examination of electroretinogram showed that the amplitude of a and b of dark ERG was wave-free or nearly wave-free. The results showed that there was a single polymorphic site of the reported RP-related pathogenic gene, and a new mutation site was found in the coding region of some genes, which led to amino acid change. Gene annotation has been found to be related to tissue apoptosis, neurodegeneration, inhibition of cell proliferation, and the like. The KEGG and GO databases were used to find biological correlation pathways, and there were apoptotic pathways, neurite-directed pathways, vascular endothelial growth factor signaling pathways, retinal metabolism pathways, and the like. Conclusion The fundus of the patients in this group is characterized by pale nipple color, thinning retinal vein, extensive retinal pigment epithelium involvement, visible depigmentation spot and or without fine pigment spot, ERG examination a and b wave are wave-free or near-wave type, The patients with primary RP combined with retinal vascular occlusion belong to the category of primary RP (blanket layer RP). At the same time, it has its own clinical features: 1. Late blood vessels may be completely occluded into white lines or near complete occlusion, and the optic atrophy may pale as pale as month. Retinal pigment epithelium is mainly composed of progressive atrophy, no typical osteoclast-like pigment proliferation, no retinal neovascularization and vitreous hemorrhage. The progress of the disease progression seems to be faster than that of the primary RP. The results of all-genome exon sequencing showed the reported single-site polymorphic sites of RP-pathogenic genes, and whether these sites were high in the disease and associated with RP-related needs to be verified. In some gene coding regions, many novel mutation sites have been found to cause amino acid changes, and gene annotation has been found to be related to tissue apoptosis, neurodegeneration, inhibition of cell proliferation, and the like. and has genes involved in biological pathways such as apoptosis, neurite outgrowth, vascular endothelial growth factor signaling, and the like. If the theory is fully explained and the work to be further worked out, it is proved.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R774.1

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