全外显子组测序和目标序列靶向捕获测序在遗传性视网膜变性基因诊断中的差异

目的:对比外显子组测序(whole exome sequencing,WES)和目标序列靶向捕获测序检测中国遗传性视网膜变性(inherited retinal dystrophies,IRDs)患者致病基因变异的差异。方法:收集182例IRDs家系,所有先证者均接受系统的眼科检查和必要的全身检查,采集患者及家属血样并提取基因组DNA。按照就诊的时间顺序将患者平均分为两组,一组91例接受WES,另一组91例应用本课题组设计并定制的“遗传性眼病基因诊断芯片” (hereditary eye disease enrichment panel,HEDEP)进行IRDs致病基因外显子区域靶向捕获测序。对候选致病基因用Sanger测序进行验证,并对家系成员进行共分离分析,使用多重连接依赖的探针扩增技术对拷贝数变异进行验证,针对二代测序捕获效率低的区域如RPGR ORF15区,应用Sanger 测序补充检测。根据美国医学遗传学与基因组学学会和分子病理学协会(American College of Medical Genetics and Genomics and the Association for Molecular Pathology,ACMG/AMP)制定的《ACMG/AMP基因变异分类标准与指南》将检测到的所有基因变异进行分类,本文只包含“致病的”、“可能致病的”的基因变异,不包含“意义不明确的”、“可能良性的”和“良性的”基因变异。结果:应用HEDEP确诊的家系共51例,阳性率为56.04%(51/91);应用WES确诊的家系共30例,阳性率为33.00%(30/91);总阳性率44.51%(81/182)。平均测序深度以及测序覆盖度方面,HEDEP优于WES,此外HEDEP具有检测拷贝数变异潜力。本研究共检测到29个IRDs基因的致病突变,最常见的致病基因为USH2A、ABCA4和RPGR,基因突变频率分别为11.54%(21/182)、6.59%(12/182)、3.85%(7/182);共发现43个新的致病突变,并检测到6例家系携带RPGR ORF15区的突变。结论:针对临床确诊的IRDs病例,HEDEP较WES能获得更高的基因诊断阳性率和更精确的诊断结果,可作为IRDs基因诊断的首选方法,WES可作为其他基因诊断方法的补充手段。同时,本研究丰富了IRDs致病基因的突变频谱,为将来IRDs基因诊断、遗传咨询和基因治疗奠定了基础。     

孔源性视网膜脱离患者视网膜下液维A酸含量的分析

采用高效液相色谱法研究 5 6例孔源性视网膜脱离患者的视网膜下液 (SRF)及血清维A酸的含量 ,并观察部分患者服用维生素A对其的影响。结果示 ,患者SRF中维A酸的含量与增殖性玻璃体视网膜病变 (PVR)程度、视网膜脱离范围和术前服用维生素A的天数呈正相关 (r1=0 .85 1,P <0 .0 0 ;r2 =0 .4 5 2 ,P <0 .0 1;r3=0 .4 3 1,P <0 .0 5 ) ;血清维A酸的含量与眼部病变无关 ;SRF中维A酸在口服维生素服药第 4d时显著增高。结果表明视网膜脱离后 ,色素上皮和神经上皮之间代谢关系发生紊乱 ,造成维A酸循环利用障碍。服用维生素A有利于阻止PVR的发生和发展。     

血栓通联合等容血液稀释对视网膜静脉阻塞患者视网膜微循环的影响

目的 :探讨血栓通 (PNS)、等容血液稀释 (IHD)及二者结合治疗视网膜静脉阻塞 (RVO)对视网膜微循环的影响。方法 :采用PNS ,IHD及PNS +IHD随机对照治疗RVO患者 73例 ,观察治疗前后视网膜微循环时间 (RCT)和视网膜出血、水肿、毛细血管渗漏及黄斑囊样水肿 (CME)、无灌注区面积等的改变。结果 :与另两种方法比较 ,PNS +IHD使RCT ,尤其非缺血型和分支静脉阻塞患者RCT缩短更明显 ,视网膜出血、水肿、毛细血管渗漏及CME消退更快 ,并使病程小于两周的早期患者无灌注区发生率减低 ,缺血型者无灌注区边缘毛细血管充盈增加 ,但无灌注区面积无显著缩小。结论 :PNS结合IHD能显著缩短RVO患者RCT ,促进视网膜出血、水肿和毛细血管渗漏及CME的消退 ,可能还有减少早期患者无灌注区形成的作用。     

黄斑裂孔性视网膜脱离的玻璃体手术

目的:研究玻璃体切除术对黄斑裂孔性视网膜脱离的治疗效果.方法:对黄斑裂孔性视网膜脱离患者16例(16只眼),施行玻璃体切除及空气、C3F8或硅油充填术,术后随访6个月以上.结果:16例患者,术后11例裂孔封闭、视网膜解剖复位,5例未愈,术后最佳视力0.05～0.2,近期治愈率达68%.结论:玻璃体手术中充分剥离黄斑前膜是手术成功的关键,膨胀气体及促进黄斑裂孔愈合的辅助药物的应用对提高手术效果起着重要作用.     

关思友治疗疑难病的临床辨证思路撷菁

关思友先生治疗疑难病时,在辨证论治的基础上,借鉴前医经验,注重逆向思维,取得良好的临床疗效。临证时,不囿表面所见,把细究病因放在重要地位,并提倡“治病不可因循守旧,固守古训,要圆机活法,因人、因地、因时治宜,有是证用是药。”     

复杂原发性视网膜脱离的综合疗法

目的：探讨综合疗法对原发性视网膜脱离的手术效果及并发症。方法：采用环扎巩膜外势压及眼内注气等综合疗法。结果：治疗成功率由８５％提高到９０％。结论：综合疗法治疗原发性视网膜脱离是一种行之有效的方法。     

Immunohistochemistry of anterior proliferative vitreoretinopathy

Anterior proliferative vitreoretinopathy is characterized by epiretinal proliferation that extends anteriorly over the vitreous base, and may, in addition to the cells usually contributing to proliferative vitreoretinopathy, also contain cells of ocular structures located in that area. We examined 11 complete globes with aPVR that were enucleated after previous severe trauma or perforating injuries (n=8) and complicated retinal detachment (n=3) by a panel of immunohistochemical markers. We found presence of RPE, glial cells, macrophages and fibrocytes, as consistently reported in PVR membranes. In addition, T-cell lymphocytes were present in 6 of the cases, and cells expressing the common leucocyte antigen on 8 cases. Cells staining positive for the intracytoplasmic contractile filament-smooth muscle actin were present in 5 cases and cells staining for desmin in one case. Collagen type IV was part of most of the membranes, and vessels with leakage of plasma factors were present in more than half of the cases.This paper was presented in part at the First Annual Meeting of the European Community Ophthalmic Research Association (ECORA), Bonn, Germany, 6.10.1993.     

Steps to optimize transscleral photocoagulation

Background: In transscleral photocoagulation, the desired effect is coagulation of parts of the ciliary body or of the peripheral retina. However, the application is often limited by the unwanted effect of coagulation of the sclera. to reduce this effect, the ratio of incident radiation flux to radiation flux transported through the sclera (and able to coagulate the target tissue) should be minimized by the incident beam characteristics.Methods: Monte Carlo simulations for the radiation transport problem of multiple scattering in the sclera were used to calculate the ratio of transported to incident radiation for different parameter settings of beam diameters, optical thicknesses of the sclera and beam angles. To verify the theoretical calculations, an simple optical device utilizing a bulb instead of a laser source was constructed and applied to enucleated porcine eyes.Results: The theoretical calculations showed that the ratio of incident to transported radiation flux can typically be decreased by a factor of three by increasing the beam radius from 0.35 mm (as used in state-of-the-art laser devices) to 2 mm. This was confirmed by the experiments. Coagulations of the ciliary body or of the peripheral retina were possible with power densities an order of magnitude below the values normally applied with laser sources.Conclusion: To improve transscleral photocoagulation, beam diameters should be increased.     

Shiva-1: in vitro and in vivo tests of the effects of a novel,synthetic, lytic peptide on ocular cells

An investigation was undertaken to determine the toxicity of an intravitreal injection of a novel peptide drug, Shiva-1, in rabbits. The drug, a synthetic peptide modeled after lytic peptides secreted by certain insects, has antiproliferative and antibacterial properties. Initial in vitro experiments showed that the drug, at a concentration of 100 M, was toxic to both Y-79 retinoblastoma cells and human retinal pigment epithelial cells. A wide range of doses (6–1200 g) was injected into the rabbit vitreous in an attempt to determine the maximum tolerated dose. Retinal toxicity was evaluated clinically, by electroretinography, and by light microscopy. Some localized toxicity was evident at 200 g; all doses of 240 g and above were toxic. While the drug appears to exhibit a narrow range between effective and toxic doses, the results suggest that this and other peptides of similar design merit further investigation for the treatment of proliferative and infectious diseases of the eye.Supported in part by U.S. Public Health Service grants EY07541 and EY02377 from the National Eye Institute, the National Institutes of Health Services, Bethesda, MD, USA