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: 2900985
• 负责人: GRETCHEN P EBERHART
• 金额: $ 11.47万
• 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 2003-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2900985
• 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 （改编自申请人摘要）高密度脂蛋白（HDL）是 重要的是防止心脏病，但主要来源， HDL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）缺陷， 低HDL-C患者的载脂蛋白A1正常。 正常的apo AI提供了鉴定额外基因的机会， 可能对HDL-C有显著影响。 两个这样的主题已经 鉴定 1例（VF）患有典型的Tangier病：低HDL-C伴角膜 混浊，橙子扁桃体和神经病变，而第二个（SF）仅表现出 低HDL-C。 丹吉尔病和SF中的载脂蛋白A1已被证明是 结构正常。 成纤维细胞对载脂蛋白的胆固醇流出 AI在两个先证者中都被发现有缺陷。 周边细胞 胆固醇流出减少， 颗粒，导致低HDL-C。 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 注入先证者的成纤维细胞 获得的克隆将在 先证者的成纤维细胞和来自其成纤维细胞的rRNA将被筛选 用北方印迹和RT-PCR来确定相应的突变是否 存在。 开发的检测方法也将被用作遗传学检测。 互补试验的位置克隆项目的基础上，大 丹吉尔家族。 一旦通过任一策略获得基因，功能性突变将被抑制。 将继续对新发现的蛋白质进行研究。 候选人是完成内分泌专科培训的内科医生 在马萨诸塞州总医院（MGH）， 确定了SF中的运输缺陷。 将完成概述的研究 在NIGH，一个拥有大量资源的机构，致力于培养 在NIGH主任Mason Freeman博士的赞助下， 脂质代谢单位。 候选人还将受到一份咨询报告的指导， 委员会由Brian Seed博士，Hank Kronenberg，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，职业发展 计划包括在麻省理工学院的教学工作，参加现场课程， 研讨会和50%的时间在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， 学术生涯应用分子和细胞技术的问题 脂质代谢