Leber先天性黑矇致病基因研究及基因治疗进展

Leber先天性黑矇(LCA)是一种严重的遗传性视网膜病变,具有遗传异质性与表型多样性的特点。近年来的遗传学研究相继发现多个致病基因与LCA发病相关,并对这些致病基因的发病机制作了进一步研究。LCA基因治疗已经从临床前期动物研究阶段进入Ⅰ期临床试验阶段,尤其是在LCAⅡ型患者中进行的RPE65基因治疗方面取得了突破性进展,为其他遗传性视网膜疾病的基因治疗打下良好的基础。     

应用目标基因捕获结合二代测序技术检测无遗传性眼病家族史的遗传性视网膜疾病患者的致病基因

背景 遗传性视网膜疾病(HRDs)是以原发性视网膜病变为主要病理改变、视力和色觉等视敏度严重损害为主要临床表现的致盲眼病,其与遗传因素密切相关,是临床上难治性盲的首要原因. 目的 应用目标基因捕获结合二代测序技术检测HRDs的致病基因. 方法 选择2014年1月至2015年1月在宁夏眼科医院就诊的无遗传性眼病家族史的20例HRDs患者作为研究对象.收集所有患者及其家庭成员临床资料,完善眼科检查,抽取患者和家庭正常成员外周静脉血,提取DNA.针对232个HRDs已知致病基因的目标序列设计并定制目标序列捕获芯片,借助高通量二代测序技术检测患者的遗传变异,数据经分析滤过,候选突变进行Sanger测序验证和致病性评估,明确致病基因和突变,并对表型和基因型间的关系进行分析.结果 20例HRDs患者中有11例患者检测到致病性突变位点,共涉及9个基因,阳性率为55％.其中有8例患者检测到复合杂合突变,3例患者为纯合子突变,均为新发现的突变位点.通过对家系成员的基因筛查分析,6例HRDs患者为常染色体隐性遗传,5例遗传类型不确定.结合临床表型以及基因检测结果分析,11例HRDs患者的诊断分别为锥杆细胞营养不良5例,致病基因分别为ABCA4、RPE65、USH2A、RIMS1和RHO;Leber先天性黑曚3例,致病基因分别为CRB1(2例)和LCA5;先天性静止性夜盲l例,致病基因为PRPF3;Best卵黄样黄斑营养不良1例,致病基因为BEST1基因;Stargardt病1例,致病基因为ABCA4基因. 结论 目标基因捕获结合二代测序技术可以对视网膜疾病患者进行快速、有效的基因诊断,结合临床特征分析有助于提高隐性遗传及遗传方式不明的HRDs的临床诊断水平.     

重视开展我国遗传性视网膜疾病的基因治疗研究

遗传性视网膜疾病是临床上常见且危害严重的眼科遗传性致盲疾病.我国拥有丰富的遗传性视网膜疾病人群资源,人类基因组计划的完成及相关遗传学技术的广泛应用为遗传性视网膜疾病基因研究提供了良好的平台.经过近年来的不断探索,我国对遗传性视网膜疾病的基因研究水平明显提高,部分成果已经接近国际先进水平,如常染色体显性遗传视网膜色素变性新基因的发现及功能研究,有的遗传性视网膜疾病正在从"不治之症"逐渐过渡到"可治之症"等.但就整体水平而言,不论是在遗传性视网膜疾病的基础研究还是临床研究方面与国外同类研究水平相比均存在着明显的差距.因此,我们应当抓住目前基因研究不断深入发展的机遇,集中全国眼科优势力量,创造和提供条件让更多的遗传性视网膜疾病患者尽早得到基因诊断,为早日在我国尝试进行多种遗传性视网膜疾病的基因治疗提供新的契机.     

关注先天性白内障的疾病相关基因研究

先天性白内障是儿童期视力损害的首要病因之一.随着分子生物学技术的发展,对遗传性先天性白内障相关基因的研究从致病基因定位及突变的筛查,逐渐发展到致病基因突变的机制探索.围绕先天性白内障相关基因研究的主题,本文着重对晶状体蛋白、缝隙连接蛋白、主要内源性蛋白基因等常见候选基因对白内障影响的机制进行归纳,并对先天性白内障相关基因在眼球发育过程、年龄相关性白内障、表观遗传学等其他领域的拓展研究进行阐述与展望.     

先天性视网膜变性及功能不良性疾病的分子遗传缺陷研究

1　 L eber先天性黑目蒙 (L eber congenital amourosis,L CA)遗传病因新发现本病是一组发病最早、最严重的遗传性视网膜变性疾病。常因视网膜发育缺陷 ,早期严重变性或功能不良 ,患者在生后 1岁内即有盲目。本病在遗传性视网膜变性中约占 5 % ,主要是AR型遗传。自从 1995年 L CA的第 1个致病基因定位于 17号染色体短臂后 ,至今已发现并定位了 5个 L CA致病基因 ,包括最近 Stockton等 (1998)定位到 14q2 4的 L CA3的突变基因。其中 3个 L CA致病基因已被克隆出 ,并在 L CA患者中检测出突变。这是最近几年本病的分子遗传病因学研究…     

应用外显子结合目标区域捕获测序芯片对一回族先天性白内障家系致病基因的检测

背景 先天性白内障是造成儿童盲和弱视的重要原因,其中约50％的先天性白内障具有遗传性.目的 应用眼遗传病外显子结合目标区域捕获测序芯片检测一常染色体显性遗传先天性白内障家系的致病基因.方法 于2011年在宁夏眼科医院收集一回族常染色体显性遗传先天性白内障家系,采集家系中患者及表型正常成员的临床资料.对家系成员进行眼科检查,抽取患者、表型正常家系成员及300名正常对照者的外周静脉血各5 ml,提取DNA,利用眼遗传病外显子结合目标区域捕获测序芯片筛查和检测候选致病突变位点.采用PCR和直接测序法对家系成员和正常对照者进行突变位点验证,最终确定致病突变位点.结果 该家系共6代61名成员,均为回族,先天性白内障患者18例,为5代遗传,符合常染色体显性遗传特征.患者中合并眼球震颤和斜视者7例,合并高度近视者4例,来诊前均已实施白内障摘出术.利用眼遗传病外显子结合目标区域捕获测序芯片检测结合生物信息学方法筛查后共得到8个候选致病突变位点,其中5个在非编码区,3个在编码区,通过PCR和直接测序法验证确定CRYGD基因上的P24T突变是该家系的致病突变位点.该突变与家系内所有患者表型共分离,在家系表型正常者及300名正常对照者均未发现此突变.结论 外显子结合目标区域捕获测序技术快速检测CRYGD基因P24T突变为该先天性白内障家系致病突变,该技术为临床表型多样、致病基因众多的先天性白内障的致病基因检测提供新的手段.     

Leber先天性黑朦致病基因及相关临床表型研究进展

Leber先天性黑嚎（LCA）是导致先天性盲的主要遗传性视网膜疾病,具有遗传异质性与临床表型多样性的特点。近年来其分子遗传学研究成为国内外热点,相继明确了20个与LCA相关的致病基因。多项研究表明LCA的基因型和临床表型之间存在关联,了解不同致病基因对应的临床表型特点有助于致病基因的筛查。就当前发现的LCA致病基因、可能的发病机制以及特定基因型与临床表型的关系进行综述,以期有助于临床诊断和咨询。     

原发性先天性青光眼分子遗传学研究进展

原发性先天性青光眼（primarycongenital aucoma,PCG）是一种发生于婴幼儿的遗传性致盲性眼病,也是先天性青光眼中最常见的类型,遗传因素为该病的主要致病原因,目前报道的与PCG相关的候选基因为CYPIBl、LTBP2和MYOC,并对这些基因的遗传方式、突变筛查及突变后的基因结构、功能改变等方面做了大量研究。然而,上述基因突变并不能解释所有PCG患者的发病机制,因此,发现新的致病基因和突变位点将成为研究工作者关注的焦点。f国际眼科纵览,2012,36：293—297）     

靶向治疗视网膜疾病的研究进展

作为一种非传统但十分新颖而精准的疗法,基因手段在多种遗传性视网膜疾病的诊治中代表了一种全新而可靠的希望,尤其是先天性黑曚与Stargardt病.近年来,随着临床应用技术和诊断水平的更新及提高,利用基因手段来诊治遗传性视网膜疾病成效颇丰.本文旨在概述测序技术和基因治疗在遗传性视网膜疾病中的作用和最新的研究进展,并对目前使...     

Leber先天性黑蒙基因研究进展

Leber先天性黑蒙(LCA)是导致婴幼儿先天性盲的严重遗传性视网膜疾病。近年发现数种与LCA相关的致病基因,主要包括GUCY2D、RPE6 5、CRX、AIPL1、RPGRIP1和CRB1,其功能涉及视网膜光电信号的传导、维生素A在视网膜的代谢、光感受器细胞的分化和形态发育、蛋白的转运和分布等。针对RPE6 5的基因治疗在动物实验中取得了一定的成果,将是未来LCA临床治疗的主要研究方向。本文就当前LCA的致病基因及其可能的发病机制、基因治疗等方面的研究进展作一综述。     

Molecular genetics of macular degeneration

Macular degeneration is a leading cause of blindness that affects the aged population. The complexity of the molecular basis of macular disease is now beginning to be elucidated with the identification of disease-causing genes. For example, mutations in the ABCR gene, (recently identified in cones as well) which codes for retinal rod-specific ABCR protein is responsible for Stargardt macular dystrophy/fundus flavimaculatus, an autosomal recessive macular dystrophy with juvenile onset, which accounts for 7% of human retinal degenerative diseases. The gene mutant in X-linked juvenile retinoschisis, XLRS1, is the first macular dystrophy gene to be isolated by positional cloning. Mutations in the peripherin/RDS gene have been shown to be associated with a variety of distinct forms of macular degenerations. The tissue inhibitor of metalloproteinase 3 (TIMP3) is implicated in autosomal dominant Sorsby fundus dystrophy. Best vitelliform macular dystrophy was mapped to 11q12–q13. The cloned gene product is the protein bestrophin, which is a retinal specific gene expressed in the RPE and possibly involved in the metabolism and transport of polyunsaturated fatty acids. The cloning of genes for rare heritable forms of macular degeneration will increase our understanding of the basic pathogenesis of the disease process. In the future this should also allow us to test the hypothesis that the coincidence of subclinical mutations in a number of genes involved in the formation and function of the macula can be responsible for cases of age-related macula-degeneration which is by far the most common form of these macular disorders.     

Gene therapy in inherited retinal degenerative diseases,a review

Hereditary diseases of the retina represent a group of diseases with several heterogeneous mutations that have the common end result of progressive photoreceptor death leading to blindness. Retinal degenerations encompass multifactorial diseases such as age-related macular degeneration, Leber congenital amaurosis, Stargardt disease, and retinitis pigmentosa. Although there is currently no cure for degenerative retinal diseases, ophthalmology has been at the forefront of the development of gene therapy, which offers hope for the treatment of these conditions. This article will explore an overview of the clinical trials of gene supplementation therapy for retinal diseases that are underway or planned for the near future.     

RDH5 gene mutations and electroretinogram in fundus albipunctatus with or without macular dystrophy: RDH5 mutations and ERG in fundus albipunctatus

The aim of this study was to analyze the RDH5 gene in patients with fundus albipunctatus with and without macular dystrophy, and correlate the identified mutations with the electrophysiological results. Twenty-one patients from 19 unrelated Japanese families with fundus albipunctatus were examined. Ten unrelated patients had macular dystrophy. In 18 patients, either a homozygous or a compound heterozygous mutation in the RDH5 gene was identified. The bright-flash, mixed rod-cone ERG had a negative configuration with reduced a-wave amplitudes after a short period of dark-adaptation (20 or 30 min). After a prolonged dark-adaptation period (2 or 3 h), the waveform attained normal amplitudes in patients without macular dystrophy but the a-waves were still subnormal in patients with macular dystrophy. The photopic ERG responses were significantly reduced in patients with macular dystrophy, indicating that they also had cone dystrophy. The photopic ERGs were reduced in only some of the patients without macular dystrophy. In patients without macular dystrophy, the scotopic b-wave amplitudes were nonrecordable or significantly reduced after a short dark-adaptation period but then improved to normal levels. However, they did not fully recover in some patients with macular dystrophy. Three patients with macular dystrophy in whom a RDH5 gene mutation could not be detected by our routine method had atypical ERG responses. We conclude that RDH5 gene mutations cause a progressive cone dystrophy or macular dystrophy as well as night blindness. The clinical phenotype including electrophysiological responses varied among patients with the RDH5 gene mutations.     

Hereditary macular dystrophies

Hereditary macular dystrophies are degenerative diseases of the central area of the retina associating primary anomalies of the retinal pigment epithelium and sensory retina. These conditions, whose hallmark is a loss of visual acuity, are a major cause of blindness and affect patients at all ages. Macular dystrophies group diseases that are heterogenous at the genetic level, as well as at the clinical, histological and physiopathological levels. Monogenic macular dystrophies are rare autosomal dominant conditions, with the exception of Stargardt disease in its typical form, which is not only relatively frequent but is also inherited as an autosomal recessive trait. During the last few years, the molecular bases of these conditions have begun to be elucidated with the identification of several responsible genes. For some macular dystrophies, this new information has confirmed pre-existing hypotheses on their pathophysiology, but for others, the discovery of the disease gene has added further complexity to the disease process. Two contradictory concepts were particularly highlighted by these genetic studies. Several phenotypes previously described as different clinical entities were brought together by the identification of mutations in the same gene, and converselyome conditions that were clinically assigned the same name, often heterogeneous at the clinical level, appeared genetically and physiopathologically heterogeneous. In addition, it is worth noting that the monogenic macular dystrophy genes were often regarded as potential factors for susceptibility to age-related macular degenerations. However, to date, only ABCA4 mutations have been associated with a minority of this frequent multifactorial condition. The aim of this article is to give a progress report on the monogenic macular dystrophy genes and to review current knowledge concerning the pathophysiology of these conditions.     

Corneal dystrophies among patients undergoing keratoplasty in Saudi Arabia

Diseases leading to corneal opacities are inflammations, infections, metabolic causes, trauma, or genetically determined diseases. Corneal dystrophies may lead to corneal blindness. A clinical and pathological review of 2,108 corneal specimens received from December 1983 to January 1988 revealed 86 (4%) corneal specimens with corneal dystrophies. Fifty-three (62%) patients had macular dystrophy, 11 (13%) had congenital hereditary endothelial dystrophy (CHED), 12 (14%) had Fuchs' corneal dystrophy, 5 (6%) had lattice corneal dystrophy, and 3 (4%) had granular dystrophy. Macular dystrophy appears to be the most common cause of corneal dystrophy in Saudi Arabia. Twenty-two (42%) patients with macular dystrophy and 10 (91%) patients with CHED were the result of consanguineous marriages. Genetic counseling is advisable among families who carry the trait of CHED or macular corneal dystrophy.     

Molecular genetics and emerging therapies for retinitis pigmentosa: Basic research and clinical perspectives

Retinitis Pigmentosa (RP) is a hereditary retinopathy that affects about 2.5 million people worldwide. It is characterized with progressive loss of rods and cones and causes severe visual dysfunction and eventual blindness in bilateral eyes. In addition to more than 3000 genetic mutations from about 70 genes, a wide genetic overlap with other types of retinal dystrophies has been reported with RP. This diversity of genetic pathophysiology makes treatment extremely challenging. Although therapeutic attempts have been made using various pharmacologic agents (neurotrophic factors, antioxidants, and anti-apoptotic agents), most are not targeted to the fundamental cause of RP, and their clinical efficacy has not been clearly proven. Current therapies for RP in ongoing or completed clinical trials include gene therapy, cell therapy, and retinal prostheses. Gene therapy, a strategy to correct the genetic defects using viral or non-viral vectors, has the potential to achieve definitive treatment by replacing or silencing a causative gene. Among many clinical trials of gene therapy for hereditary retinal diseases, a phase 3 clinical trial of voretigene neparvovec (AAV2-hRPE65v2, Luxturna) recently showed significant efficacy for RPE65-mediated inherited retinal dystrophy including Leber congenital amaurosis and RP. It is about to be approved as the first ocular gene therapy biologic product. Despite current limitations such as limited target genes and indicated patients, modest efficacy, and the invasive administration method, development in gene editing technology and novel gene delivery carriers make gene therapy a promising therapeutic modality for RP and other hereditary retinal dystrophies in the future.     

Introduction to genetics in ophthalmology, value of family studies

This paper reviews the author's personal experience with genetic eye diseases and discusses the significance of family studies in providing key information for the advancement of molecular research. Choroideremia: This disease has long been known as an X-linked progressive tapetoretinal degeneration, but it was first described in Japan in 1974 after finding asymptomatic fundus changes in heterozygous female carriers that are compatible with X chromosomal inactivation. Mutations in the disease-causing gene (REP-1) provide a clue to the diagnosis and pathophysiology of the disease.Leber's Hereditary Optic Neuropathy: The clinical expression is so variable among affected individuals and families that mild optic nerve disease of insidious onset should be differentiated from autosomal dominant optic atrophy. Molecular assessment of mitochondrial DNA leads to a definite diagnosis of the disease, but mitochondrial DNA mutations do not fully account for the clinical manifestation and phenotypic variability of the disease.Norrie Disease: This rare X-linked vitreoretinal dysplasia, characterized by congenital bilateral blindness, was documented in Japan some twenty years ago and the disease has been identified in four unrelated Japanese families. The disease, once diagnosed on the basis of elaborate clinical and familial studies, can now be defined by molecular assessment of the Norrie disease gene.Congenital Nystagmus: A four-generation family was described which presented with autosomal dominantly inherited congenital nystagmus, peripheral corneal opacity, and foveal hypoplasia without any iris tissue malformation. The diagnosis of this family was established by detection of a missense mutation in the paired domain of the PAX 6 gene, hence conforming to a forme fruste of congenital aniridia.Sorsby's Fundus Dystrophy: Two Japanese families with Sorsby's fundus dystrophy showed late-onset retinal dystrophy characterized by submacular hemorrhage and atrophy. Our patients presented with visual loss as late as 50 years of age or older due to macula-confined degenerative changes that were similar in all respects to exudative age-related macular degeneration and showed a novel mutation in the tissue inhibitor of the metalloproteinases-3 gene.Age-Related Macular Degeneration (ARMD): We have studied whether there is any association of candidate polymorphic genes involving xenobiotic or antioxidant metabolism with susceptibility to ARMD. Preliminary results suggest that the genetic polymorphism of microsomal epoxide hydrolase is related to potential risk of ARMD.     

Animal models in research on retinal degenerations: past progress and future hope

The retinal degenerations (RDs) are a family of inherited retinal degenerative diseases (dystrophies) that lead to vision loss. Although phenotypically very different, the RDs have several characteristics in common. They all are caused by gene mutations or at least have a genetic component in the etiology. They all lead to photoreceptor dysfunction, many leading to the death of both rod and cone photoreceptors. The mechanism of cell death in most of the RDs seems to be through the process of apoptosis. It is estimated that more than fifteen million people around the world have vision loss due to an inherited RD. Many of these are patients with the dry form of age-related macular degeneration (AMD) who retain partial functional vision. However, some have other degenerative conditions such as retinitis pigmentosa, Leber congenital amaurosis or wet AMD and can suffer from severe vision loss or total blindness.