CORE--PATIENT MUTATION ANALYSIS

核心——患者突变分析

• 批准号: 6202583
• 负责人: EILEEN REMOLD-O'DONNELL
• 金额: $ 42.66万
• 依托单位: IMMUNE DISEASE INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6202583
• 关键词: Wiskott Aldrich syndrome; biomedical facility; blood cells; cell line; cell population study; cryopreservation; gene expression; gene mutation; human tissue; lymphocyte; nucleic acid sequence; phenotype; single strand conformation polymorphism; tissue /cell culture; tissue resource /registry; western blottings

The Patient Mutation Analysis Core, is a blood processing, tissue culture and mutation analysis core. In this facility, we collect and process peripheral blood of WAS and XLT patients including quantifying and phenotyping blood cell subpopulations, isolation of mononuclear cells and lymphocytes, cryopreservation, and generation, maintenance and characterization of EBV-transformed B-cell lines that will be used in investigations in Projects 1 and 2. The Core contributes to diagnosis of WAS in newly referred patients by performing platelet sizing and, where family history is lacking, heterozygosity analysis of female relatives. The Core identifies the WAS gene mutation of diagnosed patients by isolating DNA and amplifying exon regions, analyzing amplified regions by single chain conformation polymorphism analysis (SCCP) followed be sequencing of the abnormal region. The Core characterizes and quantifies the mutated WASP protein expressed in patient cells by a combination of Western blotting and Phosphor-Imaging. The Core will determine whether addition of protease inhibitors, a potential therapeutic treatment, will increase the level of WASP protein in cultured cells of some patients. Finally the Core maintains a database of patient blood samples and cell lines as well as clinical and molecular information on participating patients and a separate database of published WAS defects.

患者突变分析核心是血液处理、组织培养 和突变分析核心。 在该设施中，我们收集并处理 WAS 和 XLT 患者的外周血，包括定量和 对血细胞亚群进行表型分析、分离单核细胞和 淋巴细胞、冷冻保存、生成、维持和 EBV 转化 B 细胞系的表征，该细胞系将用于 项目 1 和 2 中的调查。核心有助于诊断 通过进行血小板大小测定，对新转诊的患者进行 WAS，其中 缺乏家族史、女性亲属的杂合性分析。 Core通过以下方式识别确诊患者的WAS基因突变 分离 DNA 并扩增外显子区域，通过分析扩增区域 单链构象多态性分析（SCCP） 异常区域的测序。 核心表征和量化 突变的 WASP 蛋白通过以下组合在患者细胞中表达 蛋白质印迹和荧光成像。 核心将决定是否 添加蛋白酶抑制剂（一种潜在的治疗方法）将 增加某些患者培养细胞中WASP蛋白的水平。 最后，核心维护患者血液样本和细胞的数据库 线路以及参与的临床和分子信息 患者和已发布的 WAS 缺陷的单独数据库。