MOLECULAR GENETIC STUDIES IN CORNEAL DYSTROPHIES

角膜营养不良的分子遗传学研究

• 批准号: 2882859
• 负责人: Richard W Yee
• 金额: $ 15万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-03-01 至 2001-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2882859
• 关键词: artificial chromosomes; autosomal dominant trait; blood chemistry; clinical research; complementary DNA; cornea disorder; family genetics; gene mutation; genetic mapping; genetic markers; genetic polymorphism; genome; human genetic material tag; human subject; linkage mapping; lymphoblast; molecular cloning; molecular genetics; nucleic acid hybridization; polymerase chain reaction

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.

该提案的总体目标是获得教育和 分子遗传学方法和人类的基础实验室培训 一般而言，遗传学，并将这些技能应用于眼科 失调。目的是促进理解和 治疗遗传性眼部疾病，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 级别课程和会议（附录 B-1）和 在 Reis Bucklers 角膜家族中进行基因连锁研究 营养不良（RBCD）。 RBCD 提案是我训练的平台 成为分子遗传学领域的独立医师科学家。 RBCD 是一种显性遗传性角膜疾病，在婴儿期发病， 完全外显率和可变表达率，并且主要影响 中央视力导致视力<20/200，导致畏光，以及 严重受影响的人会因反复侵蚀而出现剧烈刺痛 个人。较轻病例的裂隙灯表现密切相关 类似于准分子激光角膜混浊后的网状外观。 对 RBCD 的基本分子理解可能有助于揭示更常见的现象 角膜疤痕问题，这是角膜的最常见原因 美国老年人的移植。一旦 RBCD 基因被克隆并 表征，获得有关病理生理机制的知识 可能会加深对角膜伤口愈合、疤痕的了解 和复发性糜烂综合征。 简而言之，采用以下技术进行基因连锁 分析。首先，确定血统并采集血液 DNA 制备的重要家庭成员。如果需要，淋巴母细胞 细胞系已建立。然后，利用信息丰富的微卫星 DNA标记，检查谱系。连锁分析在 努力确定 RBCD 基因的染色体位置。 一旦发现与染色体的连锁，精细结构连锁图谱， 与物理测绘相结合，将用于缩小范围 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 多种技术，例如外显子捕获（Duyk 1990，Bucklers 1991）， 溶液杂交（Hoffman 1987），以及 Hpall 微小蛋白的检测 然后将使用片段（HTF）岛屿（Conneally 1984）来识别 RBCD 区域的表达序列。 一旦 RBCD 基因被克隆和表征，了解 这种前基底膜疾病的病理生理过程将 有助于阐明其他角膜相关的机制 状况。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 以及受影响患者的治疗。