CLONING AND FUNCTIONAL CHARACTERIZATION OF INHERITED NEURODEGENERATIVE DISORDERS

遗传性神经退行性疾病的克隆和功能表征

• 批准号: 2345084
• 负责人: D A TAGLE
• 金额: --
• 依托单位: NATIONAL HUMAN GENOME RESEARCH INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2345084
• 关键词: Huntington's disease; Niemann Pick disease; artificial chromosomes; ataxia telangiectasia; child (0-11); gene expression; gene mutation; gene targeting; genetic disorder; genetic mapping; genetic markers; genetically modified animals; human genetic material tag; human subject; human tissue; laboratory mouse; model design /development; molecular cloning; neural degeneration; nucleic acid sequence; pathologic process; transfection /expression vector

This project entails the identification of genes responsible for a variety of human inherited disorders. These include development and refinement of methodologies for gene mapping and isolation, and the characterization and functional analyses of these genes for a better understanding of their pathogenic mechanisms that will lead to therapy and intervention. Using positional cloning strategies, our laboratory participated in the identification the Huntington Disease (HD) gene and the ataxia-telangiectasia gene (A-T). HD is a progressive, neurodegenerative disorder with onset in midlife. and the gene contains a polymorphic (CAG)n trinucleotide repeat which is pathogenic when the repeats expand to 42 or more. Subcellular expression and localization of the normal and expanded versions of the full length cDNA constructs are being examined in baculovirus and mammalian expression systems. In addition, mouse YAC transgenic lines are being developed by microinjecting a YAC containing the entire HD gene that has been retrofitted with an expanded CAG repeat into the pronucleus of fertilized eggs. A-T is an autosomal recessive disorder characterized by progressive cerebellar degeneration, oculocutaneous telangiectasia, immunodeficiency, radiosensitivity, susceptibility to cancer and chromosomal instability. Current research efforts for A-T involves the function of homologous sequences in S. cerevisae; generation of antisera specific for the A-T protein; characterization of the expression pattern and localization of the protein product; and generation of knockout mouse models. A current positional cloning project involves the identification of the defective gene for Niemann-Pick type C. NPC is an autosomal-recessive, neurovisceral lipid storage disorder marked by neurological decline in patients which is highlighted by supranuclear gaze palsy and is typically accompanied by progressive cognitive impairment, ataxia, dystonia, dysphagia, dysarthria, seizures, cataplexy and dementia. Recombinant studies point to a localization at 18q11-q12, more specifically in a 2 cM interval between markers D18S44 and D18S480. We are currently in the process of refining the genetic map by searching for additional polymorphic markers and assembling YAC and cosmid contigs for the region. Candidate genes are being isolated by cDNA selection and exon trapping. Additionally, entire YACs are being introduced into NPC cells to check for correction of the cellular defect. Taking advantage of an NPC mouse model, backcrosses are being performed to further delimeate the affected locus in mice and identify the synthenic region in humans. A novel method for gene identification is also underway and this involves development of a YAC fragmentation vector that includes 3' exon trapping cassette, yeast selectable marker, Alu sequences for targeting, yeast telomere sequences. Acentric and centric versions on the vector are being made with Alu sequences also found in both orientations.

该项目需要鉴定负责一个基因， 各种人类遗传性疾病。 其中包括发展和 完善基因定位和分离的方法， 这些基因的特征和功能分析，以更好地 了解他们的致病机制，这将导致治疗 和干预。利用定位克隆策略，我们的实验室 参与了亨廷顿病（HD）基因的鉴定， 共济失调-毛细血管扩张症基因（A-T）。HD是一种渐进的， 中年发病的神经退行性疾病。 这个基因包含 多态性（CAG）n三核苷酸重复，当 重复序列扩展到42个或更多。的亚细胞表达和定位 全长cDNA构建体的正常和扩增形式是 在杆状病毒和哺乳动物表达系统中进行检测。 在 此外，小鼠YAC转基因系正在开发中， 显微注射含有整个HD基因的YAC， 用扩增的CAG重复序列改造受精卵的原核， 鸡蛋 A-T是一种常染色体隐性遗传疾病，其特征在于： 进行性小脑变性，眼皮肤毛细血管扩张， 免疫缺陷、放射敏感性、对癌症的易感性以及 染色体不稳定性 目前对A-T的研究工作涉及 同源序列在S.抗血清的产生 对A-T蛋白具有特异性;表达模式的表征 和蛋白产物的定位;以及产生敲除小鼠 模型目前的一个定位克隆项目涉及识别 尼曼-皮克C型缺陷基因的。 NPC是 常染色体隐性遗传，神经内脏脂质沉积障碍， 神经功能下降的患者，这是突出的核上 凝视麻痹，通常伴有渐进性认知障碍， 损伤、共济失调、肌张力障碍、吞咽困难、构音障碍、癫痫发作、紧张症 和痴呆症。重组研究指出定位于18 q11-q12， 更具体地，在标记D18 S44和D18 S480之间的2cM间隔中。 我们目前正在通过搜索， 用于额外的多态性标记和组装YAC和粘粒重叠群 为该地区。通过cDNA选择分离候选基因， 外显子捕获 此外，整个YAC正在引入NPC 检查细胞缺陷的纠正。 利用 在NPC小鼠模型中，正在进行回交，以进一步 在小鼠中去除受影响的位点并鉴定致病区域 在人类身上。一种新的基因鉴定方法也在进行中， 这涉及开发一种YAC片段化载体， 外显子捕获盒，酵母选择标记，Alu序列 靶向，酵母端粒序列。 非中心和中心版本 载体是用Alu序列制成，Alu序列也存在于 方向。