MEMBRANE DEFECT IN THE SLO SYNDROME

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DESCRIPTION (provided by applicant): The Smith-Lemli-Opitz (SLOS) syndrome is an often lethal, autosomal recessive birth defect characterized by widespread neurological, skeletal and anatomical abnormalities including cleft palate in afflicted subjects. This disorder has been shown to be caused by an inheritable metabolic defect in which the enzyme that catalyzes the final step in cholesterol biosynthesis, DHCR7, is genetically deficient. In SLOS, blood and tissue levels of cholesterol are greatly suppressed while the levels of the principle precursor to cholesterol synthesis, 7 dehydrocholesterol (7-DHC) are greatly elevated. Because cholesterol is absolutely required for the normal biosynthesis of cell membranes as well as steroid and sex hormones, it is thought that the suppressed cholesterol synthesis in SLOS patients underlies the widespread tissue and organ malformations. While the metabolic defect underlying SLOS has been defined, its impact on cell function has not been determined. In skin fibroblasts obtained from SLOS patients, we have generated preliminary data demonstrating that membrane calcium permeability is markedly augmented while membrane-bound Na+/7K+ATPase activity, folate uptake and IP3 signaling is markedly suppressed. Furthermore, employing X-ray diffraction analyses in synthetic membranes prepared to mimic SLOS membranes, we observed a highly atypical membrane structure. We have developed the hypothesis that in SLOS patients, the deficiency in cholesterol biosynthesis leads to the production of cell membranes that are defective in their composition, dynamics and function, and that this membrane defect contributes to the cellular pathobiology in these patients. In this study, Aim 1 will determine whether defects exist in composition, structure and fluidity of the cell plasma membrane of skin fibroblasts obtained from SLOS patients and DHCR7 transgene knockout (k/o) mice. Aim 2 will determine whether these membrane defects correlate with impaired membrane function (folate uptake, Ca++ permeability, Na+/K+ATPase activity and IP3 signaling) and cell proliferation. Aim 3 will determine the degree to which cell and membrane functions can be corrected by restoring the membrane sterols back to normal levels. This project employs biophysical, biochemical and cell biology approaches to study fibroblasts obtained from SLOS patients and DHCR7 k/o mice in an attempt to shed light on the cellular basis underlying this profoundly debilitating disease which has no cure. By defining the membrane defects in SLOS we will advance our understanding of cell and molecular basis of this disease. In addition, we expect to validate the DHCR7 k/o mouse model of SLOS and thereby provide greater opportunity for us and others to study SLOS in the hope of providing clinical relief to patients faced with this disease.