EXPRESSION CLONING OF A GENE EFFECTING HDL CHOLESTEROL

描述

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.