MEMBRANE DEFECT IN THE SLO SYNDROME

描述（由申请人提供）：Smith-Lemli-Opitz（SLOS）综合征是一种常致死的常染色体隐性出生缺陷，其特征为受试者中广泛的神经、骨骼和解剖异常，包括腭裂。这种疾病已被证明是由遗传性代谢缺陷引起的，其中催化胆固醇生物合成最后一步的酶DHCR 7是遗传缺陷的。在SLOS中，胆固醇的血液和组织水平被极大地抑制，而胆固醇合成的主要前体7-脱氢胆固醇（7-DHC）的水平被极大地升高。由于胆固醇是细胞膜以及类固醇和性激素的正常生物合成所必需的，因此认为SLOS患者中胆固醇合成受到抑制是广泛的组织和器官畸形的基础。虽然SLOS的代谢缺陷已被确定，但其对细胞功能的影响尚未确定。在从SLOS患者获得的皮肤成纤维细胞中，我们已经产生了初步数据，表明膜钙渗透性显着增强，而膜结合Na+/7 K + ATP酶活性，叶酸摄取和IP 3信号传导显着抑制。此外，采用X-射线衍射分析在合成膜制备模仿SLOS膜，我们观察到一个高度非典型的膜结构。我们提出了一个假设，即在SLOS患者中，胆固醇生物合成的缺陷导致细胞膜的产生，这些细胞膜的组成、动力学和功能都有缺陷，这种膜缺陷有助于这些患者的细胞病理学。在本研究中，目的1将确定从SLOS患者和DHCR 7转基因敲除（k/o）小鼠获得的皮肤成纤维细胞的细胞质膜的组成、结构和流动性是否存在缺陷。目的2将确定这些膜缺陷是否与受损的膜功能（叶酸摄取、Ca++渗透性、Na+/K+ ATP酶活性和IP 3信号传导）和细胞增殖相关。目标3将确定通过将膜固醇恢复到正常水平可以纠正细胞和膜功能的程度。该项目采用生物物理，生物化学和细胞生物学方法研究从SLOS患者和DHCR 7 k/o小鼠中获得的成纤维细胞，试图揭示这种无法治愈的严重衰弱性疾病的细胞基础。通过明确SLOS的膜缺陷，我们将进一步了解这种疾病的细胞和分子基础。此外，我们期望验证SLOS的DHCR 7 k/o小鼠模型，从而为我们和其他人研究SLOS提供更大的机会，希望为面临这种疾病的患者提供临床缓解。

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.