GYRATE ATROPHY-MODEL FOR MOLECULAR STUDY OF EYE DISEASES

用于眼部疾病分子研究的旋转萎缩模型

• 批准号: 3266278
• 负责人: GEORGE INANA
• 金额: $ 16.4万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-30 至 1992-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3266278
• 关键词: complementary DNA; disease /disorder model; eye disorder; gene deletion mutation; gene expression; gene mutation; gene rearrangement; genetic disorder diagnosis; laboratory rat; mitochondria; molecular genetics; molecular pathology; mutant; nucleic acid probes; nucleic acid sequence; ornithinemia; point mutation; polymerase chain reaction; retina degeneration; retinitis pigmentosa; tissue /cell culture

DESCRIPTION: (Applicant's abstract). The broad, long-term goal of this work is to understand the molecular basis of hereditary retinal degenerative diseases, which involves identification of the disease genes, demonstration of the specific nature of the gene defects, and elucidation of the pathophysiologlogical mechanisms involved, all of which may enable gene replacement therapy in the future. Among the various inherited retinal degenerative diseases that lead to blindness in man, gyrate atrophy (GA) serves as a unique model because knowledge of the underlying biochemical defect, i.e. a generalized deficiency of the mitochondrial enzyme ornithine animotransferase (OAT), has enabled us to take a relatively straightforward approach to tackling this disease at the gene level. Specific Aims 1 and 2 Analysis of the OAT gene and its expression in GA cases: correlation of the gene defects to disease phenotypes. GA is a disease that exhibits considerable heterogeneity at the clinical level. Molecular genetic analysis of GA cases by us and others are revealing a remarkably complex picture in which not one, but a variety of different mechanisms of OAT gene inactivation results in disease. Almost every case analyzed so far has provided valuable new information not only about the disease but also on fundamental mechanisms of gene expression and regulation. For this reason, analysis of GA cases will be pursued. Patient tissues will be analyzed at the DNA, RNA and protein levels. The specific nature of each defect, be it gene deletion, rearrangement, point mutation or other changes will be identified. Subtle gene changes such as point mutations will be tested functionally for their effects on OAT in order to confirm type of mutation revealed (by us and others), the frequency of its occurrence in all of our cases will be established and compared with clinical findings, to determine if conclusions can be drawn concerning the type of mutation and the disease phenotype that results from it. Specific Aim 3. Investigation into the pathophysiological mechanisms of GA. The precise mechanism by which the OAT defects lead to the chorioretinal degeneration, or even which cell types are primarily affected, are still not known, largely due to unavailability of eye tissue from patients and lack of an animal model. Ongoing projects will be continued in which attempts are being made to reproduce the OAT deficiency state of GA through inactivation of endogenous OAT in vitro and in vivo. The in vitro approach involves transfer of antisense expression OAT gene into cultured cells of various types including retinal pigment epithelium, and biochemical analysis of the effects of OAT inactivation. in vivo OAT inactivation, achieved by a combination of the antisense and transgenic approaches, is an attempt to construct a mouse model of GA which will be analyzed at multiple levels. Elucidation of the pathophysiological basis of GA will not only help in understanding this disease but, in view of the similarity of GA to other retinal degenerative diseases such as retinitis pigmentosa (RP), may also shed some light on possible mechanisms involved in RP. Finally, elucidaiton of the molecular and pathophysiological mechanisms of GA will render this disease one of the best models of hereditary ocular diseases for consideration of gene replacement therapy.

说明：（申请人摘要）。 这一广泛的长期目标 这项工作是为了了解遗传性视网膜病变的分子基础， 退行性疾病，包括疾病基因的鉴定， 证明基因缺陷的具体性质，并阐明 所涉及的病理生理机制，所有这些都可能使 基因替代疗法在未来 在各种继承的 视网膜变性疾病，导致失明的人，回旋萎缩 (GA)作为一个独特的模型，因为知识的基础， 生化缺陷，即线粒体的普遍缺乏 酶鸟氨酸氨基转移酶（OAT），使我们能够采取 相对简单的方法来解决这种疾病的基因， 水平 特异性目的1和2 OAT基因的分析及其在GA中的表达 例：基因缺陷与疾病表型的相关性。 GA是一个 在临床水平上表现出相当大的异质性的疾病。 我们和其他人对遗传性贫血病例的分子遗传学分析揭示了 非常复杂的画面，其中不是一个，而是各种不同的 OAT基因失活的机制导致疾病。 几乎所有的情况 迄今为止的分析不仅提供了有关 疾病，但也对基因表达的基本机制， 调控 因此，将对GA案例进行分析。 将在DNA、RNA和蛋白质水平上分析患者组织。 的 每种缺陷的特定性质，无论是基因缺失、重排、点 将识别突变或其他变化。 细微的基因变化， 将在功能上测试点突变对OAT的影响， 为了确认（我们和其他人）揭示的突变类型， 它在我们所有案件中发生的频率将被确定， 与临床结果进行比较，以确定是否可以得出结论 关于突变的类型和疾病表型， 了 具体目标3。 探讨脑梗死的病理生理机制 佐治亚州 OAT缺陷导致 脉络膜视网膜变性，甚至哪些细胞类型是主要的 受影响，仍然不知道，主要是由于眼睛组织的不可用性 缺乏动物模型。 正在进行的项目将 继续进行，试图重现OAT缺陷 通过体外和体内内源性OAT的失活来改变GA的状态。 体外方法包括转移反义表达OAT基因 培养成各种类型的细胞，包括视网膜色素上皮细胞， 和OAT失活影响的生化分析。 体内OAT 灭活，通过反义和转基因的组合实现， 方法，是试图构建一个小鼠模型的遗传算法，这将是 在多个层面上进行分析。 阐明病理生理学基础 这不仅有助于理解这种疾病，而且，鉴于 GA与其他视网膜变性疾病如视网膜炎的相似性 色素沉着症（RP），也可能揭示了一些可能的机制， 在RP中。 最后，阐明了分子和病理生理 GA的机制将使这种疾病的最佳模型之一， 遗传性眼病的基因替代治疗的考虑。

项目成果

Heterogeneity and uniqueness of ornithine aminotransferase mutations found in Japanese gyrate atrophy patients.

• DOI: 10.3109/02713689609003464
• 发表时间: 1996
• 期刊: Current eye research
• 影响因子: 2
• 作者: Y. Mashima;T. Shiono;M. Tamai;G. Inana

Rapid and efficient molecular analysis of gyrate atrophy using denaturing gradient gel electrophoresis.

使用变性梯度凝胶电泳对脑回萎缩进行快速有效的分子分析。

• 发表时间: 1994
• 期刊: Investigative ophthalmology & visual science
• 影响因子: 4.4
• 作者: Mashima,Y;Shiono,T;Inana,G
• 通讯作者: Inana,G

A deletion in the ornithine aminotransferase gene in gyrate atrophy.

脑回萎缩中鸟氨酸转氨酶基因缺失。

• 发表时间: 1992
• 期刊: The Journal of biological chemistry
• 作者: Akaki,Y;Hotta,Y;Mashima,Y;Murakami,A;Kennaway,NG;Weleber,RG;Inana,G
• 通讯作者: Inana,G

A single-base change at a splice acceptor site in the ornithine aminotransferase gene causes abnormal RNA splicing in gyrate atrophy.

鸟氨酸转氨酶基因剪接受体位点的单碱基变化会导致脑回萎缩中 RNA 剪接异常。

• DOI: 10.1007/bf00220086
• 发表时间: 1992
• 期刊: Human genetics
• 影响因子: 5.3
• 作者: Mashima,Y;Weleber,RG;Kennaway,NG;Inana,G
• 通讯作者: Inana,G

Nonsense-codon mutations of the ornithine aminotransferase gene with decreased levels of mutant mRNA in gyrate atrophy.

• 发表时间: 1992
• 期刊: American journal of human genetics
• 影响因子: 9.8
• 作者: Y. Mashima;Akira Murakami;R. Weleber;N. Kennaway;L. Clarke;Takashi Shiono;G. Inana