EXPRESSION CLONING OF A GENE EFFECTING HDL CHOLESTEROL

影响 HDL 胆固醇的基因的表达克隆

• 批准号: 6182736
• 负责人: GRETCHEN P EBERHART
• 金额: $ 6.72万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-15 至 2002-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6182736
• 关键词: clinical research; family genetics; genetic disorder; genetic mapping; high density lipoproteins; human genetic material tag; human subject; hypocholesterolemia; molecular cloning; polymerase chain reaction

DESCRIPTION (Adapted from applicants' abstract) High density lipoproteins (HDL) are important in protecting against heart disease, yet the major source of genetic variability in HDL cholesterol(HDL-C) is undetermined. Low HDL,-C due to defects in apolipoprotein AI(apo AI) have been described, but most patients with low HDL-C have normal apo Al. Subjects with very low HDL-C who have normal apo AI offer an opportunity to identify additional gene(s) which may have significant impact on HDL-C. Two such subjects have been identified. One(VF) has classic Tangier disease: low HDL-C with corneal clouding, orange tonsils and neuropathy, while the second(SF) exhibits only low HDL-C. Apo Al in Tangier disease and in SF has been shown to be structurally normal. Cholesterol efflux from fibroblasts in response to apo AI has been found to be defective in both probands. Peripheral cells with decreased cholesterol efflux could be less able to donate cholesterol to HDL particles, causing lower HDL-C. The probands' gene defect(s) will be pursued by an expression cloning strategy. An assay based on their cellular abnormality will be developed and used to screen a normal fibroblast cDNA library transiently transfected into the probands' fibroblasts. The clone(s) obtained will be tested in both probands' fibroblasts, and rRNA from their fibroblasts will be screened with Northern Blotting and RT-PCR to determine if a corresponding mutation is present. The assay developed will also be used as a genetic complementation assay for a positional cloning project based on a large Tangier kindred. Once a gene(s) is obtained by either strategy, functional studies of the newly identified protein will be pursued. The candidate is an internist completing Endocrinology subspecialty training at the Massachusetts General Hospital (MGH), during which the cholesterol transport defect in SF was identified. The research outlined will be done at NIGH, an institution with immense resources devoted to fostering research, under the sponsorship of Dr. Mason Freeman, director of the NIGH Lipid Metabolism unit. The candidate also will be guided by an advisory committee made up of Drs. Brian Seed, Hank Kronenberg, James Gusella and Dennis Brown. This group offers expertise in expression cloning, positional cloning and cell biology. In addition to research, the career development plan involves didactic work at MIT, participation in on-site courses and seminars, and 50% time in clinical care of lipid disorders. The entire program is designed to provide the candidate the skills necessary for an academic career applying molecular and cellular techniques to problems of lipid metabolism.

描述 （改编自申请人的摘要）高密度脂蛋白（HDL）是 对预防心脏病很重要，但也是心脏病的主要来源 高密度脂蛋白胆固醇（HDL-C）的遗传变异性尚未确定。 低HDL，-C 由于载脂蛋白 AI (apo AI) 的缺陷已被描述，但大多数 HDL-C 低的患者的 apo Al 正常。 HDL-C 极低的受试者 具有正常的 apo AI 提供了识别其他基因的机会 可能对 HDL-C 有显着影响。 已经有两个这样的课题 确定。 一名 (VF) 患有经典丹吉尔病：角膜 HDL-C 低 浑浊、橙色扁桃体和神经病变，而第二个（SF）仅表现出 低 HDL-C。 Apo Al 在丹吉尔病和 SF 中已被证明 结构正常。 成纤维细胞响应载脂蛋白流出胆固醇 两名先证者的人工智能均被发现存在缺陷。 外周细胞具有 胆固醇流出减少可能会降低向高密度脂蛋白提供胆固醇的能力 颗粒，导致 HDL-C 降低。 先证者的基因缺陷将通过表达克隆来追踪 战略。 将开发一种基于细胞异常的检测方法 并用于筛选瞬时转染的正常成纤维细胞cDNA文库 进入先证者的成纤维细胞。 获得的克隆将在 将筛查先证者的成纤维细胞及其成纤维细胞的 rRNA 使用 Northern Blotting 和 RT-PCR 来确定是否存在相应的突变 存在。 开发的检测方法也将用作遗传检测 基于大序列的定位克隆项目的互补分析 丹吉尔亲戚。 一旦通过任一策略获得基因，功能 将继续对新发现的蛋白质进行研究。 候选人是一名完成内分泌专科培训的内科医生 在马萨诸塞州总医院 (MGH) 期间，胆固醇 SF 中的运输缺陷被发现。 将完成概述的研究 在 NIGH，一个拥有大量资源致力于培养 研究由 NIGH 主任 Mason Freeman 博士赞助 脂质代谢单位。 候选人还将受到咨询的指导 委员会由博士组成。布莱恩·席德 (Brian Seed)、汉克·克罗嫩伯格 (Hank Kronenberg)、詹姆斯·古塞拉 (James Gusella) 和 丹尼斯·布朗. 该小组提供表达克隆、定位 克隆和细胞生物学。 除了研究之外，还有职业发展 计划涉及麻省理工学院的教学工作、参与现场课程和 研讨会，50% 的时间用于血脂紊乱的临床护理。 整个 该计划旨在为候选人提供必要的技能 应用分子和细胞技术解决问题的学术生涯 脂质代谢。