MEMBRANE DEFECT IN THE SLO SYNDROME

描述（由申请人提供）：Smith-Lemli-Opitz （SLOS）综合征是一种通常致命的常染色体隐性出生缺陷，其特征是患者普遍存在神经、骨骼和解剖异常，包括腭裂。这种疾病已被证明是由一种遗传性代谢缺陷引起的，在这种代谢缺陷中，催化胆固醇生物合成最后一步的酶DHCR7基因缺陷。在SLOS中，血液和组织中的胆固醇水平被大大抑制，而胆固醇合成的主要前体7-脱氢胆固醇（7- dhc）的水平则大大升高。由于胆固醇对于细胞膜、类固醇和性激素的正常生物合成是绝对必需的，因此人们认为，sls患者中胆固醇合成的抑制是广泛存在的组织和器官畸形的基础。虽然SLOS背后的代谢缺陷已被明确，但其对细胞功能的影响尚未确定。在从sls患者身上获得的皮肤成纤维细胞中，我们获得的初步数据表明，膜钙通透性显著增强，而膜结合的Na+/7K+ atp酶活性、叶酸摄取和IP3信号明显抑制。此外，利用x射线衍射分析制备的模拟sls膜的合成膜，我们观察到高度非典型的膜结构。我们已经提出了这样的假设，即在SLOS患者中，胆固醇生物合成的缺乏导致了细胞膜在组成、动力学和功能上的缺陷，而这种膜缺陷导致了这些患者的细胞病理。在本研究中，Aim 1将确定从sls患者和DHCR7转基因敲除（k/o）小鼠中获得的皮肤成纤维细胞的细胞膜组成、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膜功能受损（叶酸摄取、钙离子渗透性、Na+/K+ atp酶活性和IP3信号传导）和细胞增殖相关。目的3将确定在何种程度上细胞和膜功能可以通过恢复细胞膜固醇恢复到正常水平而得到纠正。该项目采用生物物理、生化和细胞生物学方法，研究从sls患者和DHCR7 k/o小鼠身上获得的成纤维细胞，试图揭示这种严重使人衰弱且无法治愈的疾病的细胞基础。通过定义sls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，我们希望验证sls的DHCR7 k/o小鼠模型，从而为我们和其他人研究sls提供更大的机会，以期为面临该疾病的患者提供临床救济。

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.