MOLECULAR GENETIC STUDIES IN CORNEAL DYSTROPHIES

这项建议的总体目标是获得教育和 分子遗传学方法和人类基础实验室培训 遗传学，并将这些技能应用于眼科 精神错乱。目的是增进了解和了解 遗传性眼病的治疗，特别强调 角膜疾病。这些目标将通过参加研究生课程来实现 所述级别课程和会议(附录B-1)和 对Reis Buckler角膜病变家系进行基因连锁研究 营养不良(RBCD)。RBCD计划是我进行培训的平台 成为分子遗传学领域的独立内科医生兼科学家。 RBCD是一种以遗传性为主的角膜疾病，有婴儿发病， 完全的外显性和可变的表现力，主要影响 中心视力导致&lt；20/200视力，禁止畏光，以及 受严重影响的反复侵蚀引起的尖锐刺痛 个人。较轻病例的裂隙灯外观贴近 类似准分子激光术后角膜混浊的网状外观。 对RBCD的基本分子理解可能有助于揭示更常见的 角膜疤痕问题，角膜最常见的原因 美国老年人的移植。一旦RBCD基因被克隆并 具有特征性的，关于病理生理机制的知识 可能会导致对角膜伤口愈合、疤痕形成的更多了解 和复发性糜烂综合征。 简而言之，使用以下技术来进行基因连锁 分析。首先，确定血统，并从 DNA准备的重要家庭成员。如果需要，淋巴母细胞 建立了细胞系。然后，利用高信息量的微卫星 DNA标记，对家系进行检查。连锁分析在以下位置执行 为确定RBCD基因的染色体位置所做的努力。 一旦发现与染色体的连锁，精细结构连锁作图， 与物理映射结合使用，将用于缩小 包含RBCD基因座的区域。酵母人工染色体(YAC) 将构建(或识别)跨越该区域的重叠群。一个 外显子捕获等技术的数量(Duyk 1990，Buckler 1991)， 溶液杂交(Hoffman 1987)，以及Hpall微型体的检测 碎片(HTF)岛(Conneally 1984)将用于识别 来自RBCD区域的表达序列。 一旦RBCD基因被克隆和鉴定，理解 这种前基底膜疾病的病理生理过程将 对其他角膜相关的照明机制有帮助 条件。此外，这些知识将有助于合理规划。 诊断和咨询服务以及预防性治疗 以及对受影响患者的治疗。

The general objectives of this proposal are to gain an education and basic laboratory training in molecular genetic methods and human genetics, in general, and to apply these skills to ophthalmologic disorders. The purpose is to contribute to the understanding and treatment of inherited ocular disorders, with a special emphasis on corneal diseases. These objectives will be met by attending graduate level courses and conferences as described (Appendix B-1) and by performing gene linkage studies in families with Reis Bucklers Corneal Dystrophy (RBCD). The RBCD proposal is the platform for my training to be an independent physician-scientist in the field of molecular genetics. RBCD is a dominantly inherited corneal disorder, has an infantile onset, complete penetrance and variable expressivity, and affects primarily central vision causing <20/200 visual acuity, disabling photophobia, and sharp stabbing pain from recurrent erosions in severely affected individuals. The slit lamp appearance of the milder cases closely resembles the reticular appearance of post EXCIMER laser corneal haze. A basic molecular understanding of RBCD may shed light on the more common problem of corneal scarring, the most common cause for corneal transplantation in the American elderly. Once the RBCD gene is cloned and characterized, the knowledge gained about the pathophysiologic mechanisms may lead to a greater understanding of corneal wound healing, scarring and recurrent erosion syndrome. Briefly, the following techniques are employed to perform gene-linkage analysis. First, the pedigree is ascertained and blood is collected from significant family members for DNA preparation. If needed, lymphoblast cell lines are established. Then, using highly informative microsatellite DNA markers, the pedigree is examined. Linkage analysis is performed in an effort to establish the chromosomal location of the gene for RBCD. Once linkage to a chromosome is found, fine structure linkage mapping, in conjunction with physical mapping, will be used to narrow down the region containing the RBCD locus. Yeast artificial chromosome (YAC) contigs which span this region will be constructed (or identified). A number of techniques such as exon trapping (Duyk 1990, Bucklers 1991), solution hybridization (Hoffman 1987),and detection of Hpall tiny fragment (HTF) islands (Conneally 1984) will then be used to identify expressed sequences from the RBCD region. Once the RBCD gene is cloned and characterized, understanding the pathophysiologic process of this anterior basement membrane disorder will be helpful in illuminating mechanisms in other corneal related conditions. In addition, this knowledge will assist in rational planning of diagnostic and counseling services as well as preventative therapies and treatment for affected patients.