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: 2622821
• 负责人: GRETCHEN P EBERHART
• 金额: $ 8.43万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 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 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2622821
• 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 (摘自申请者摘要)高密度脂蛋白(HDL)是 对预防心脏病很重要，但它的主要来源 高密度脂蛋白(HDL-C)的遗传变异性尚不确定。低高密度脂蛋白，-C 由于载脂蛋白AI(Apo AI)的缺陷已被描述，但大多数 低高密度脂蛋白胆固醇患者载脂蛋白A正常。高密度脂蛋白胆固醇水平极低的受试者 有正常的载脂蛋白AI提供机会来发现额外的基因(S) 可能会对高密度脂蛋白胆固醇产生重大影响。已经有两个这样的主题 确认身份。1例(VF)有典型的丹吉尔病：伴有角膜的低密度脂蛋白胆固醇 浑浊、橙色扁桃体和神经病变，而第二个(SF)仅表现为 低高密度脂蛋白胆固醇丹吉尔病和SF中的载脂蛋白A1已被证明是 结构上是正常的。成纤维细胞胆固醇外流对载脂蛋白的影响 人工智能在两个先证者中都被发现是有缺陷的。外周细胞与 胆固醇外流减少可能会降低向高密度脂蛋白输送胆固醇的能力 颗粒，导致较低的高密度脂蛋白。 先证者的基因缺陷(S)将通过表达克隆来寻找 策略。将开发一种基于它们细胞异常的分析方法 并用于筛选瞬时转染的正常成纤维细胞c DNA文库 进入先证者的成纤维细胞。获得的克隆(S)将在 将对两个先证者的成纤维细胞以及他们的成纤维细胞中的rRNA进行筛选 用Northern blotting和RT-PCR来确定相应的突变 是存在的。开发的化验方法也将用于基因检测 一种基于大型数据库的位置克隆项目的互补分析 丹吉尔有血缘关系。一旦通过任何一种策略获得一个基因(S)，功能性的 对新发现的蛋白质的研究将继续进行。 应聘者是一名完成内分泌学分专业培训的内科医生 在马萨诸塞州综合医院(MGH)，在此期间胆固醇 对SF的转运缺陷进行了鉴定。概述的研究将会完成 几乎，一个拥有大量资源的机构致力于培养 研究，由The Night主任梅森·弗里曼博士赞助 脂代谢单位。候选人还将在咨询意见的指导下 委员会由Brian Seed博士、Hank Kronenberg博士、James Gusella博士和 丹尼斯·布朗。这个小组提供了表达克隆、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 该计划旨在为应聘者提供必要的技能 应用分子和细胞技术解决生物多样性问题的学术生涯 脂类代谢。