CAPACITATIVE CALCIUM ENTRY DEFECT IN IMMUNE DEFICIENCY

描述(改编自申请者的描述)：本试验的目的 该项目是为了确定两名患有严重肺炎的患者的分子缺陷。 联合免疫缺陷综合征。这些病人是他们亲生的兄弟 父母是近亲，因此该缺陷很可能是隐性突变 在单个基因中。患者的T细胞表现出明显的缺陷。 容量性(存储操作)钙离子进入；因此，他们无法 激活钙调神经磷酸酶调节的转录因子NFAT(核因子 激活的T细胞)，并显示出明显的转录缺陷 细胞因子基因。潜在的分子缺陷可能涉及一个亚单位 电容性钙离子进入通道(CRAC)，或信号的组成部分 通向CRAC航道开放的通道。商店经营的机制 Ca2+进入本质上是未知的，但在许多IF中可能是保守的 不是所有非兴奋性细胞类型，因此我们的研究将是一般性的 引起了细胞生物学家和免疫学家的兴趣。 研究人员将采取两种主要方法来鉴定基因产物 这是患者T细胞的缺陷：(I)通过以下方式捕获突变基因 逆转录病毒插入亲本T细胞。杂合子来源的T细胞系 父母将接受全基因组的逆转录病毒插入(基因捕获) 使用一种逆转录病毒载体，这样它就可以从它的 基因组中整合的位置。在商店运营中有缺陷的突变T细胞 钙离子的进入将从感染细胞的群体中分离出来。单元格 将分析哪些逆转录病毒切除可以逆转突变的表型 确定整合地点。通过这一过程鉴定出的基因(S)是 亲本细胞是野生型等位基因的有力候选者 杂合子。(2)互补克隆。微细胞介导的染色体 将使用转移(MMCT)方法来识别人类染色体区域 补充性钙离子进入缺陷的患者T 细胞。利用光谱核型和微卫星标记进行缺失定位 将被用来进一步缩小涉及到的染色体区域 将对该区域的缺陷和候选基因进行评估。

DESCRIPTION (Adapted from applicant's description): The aim of this pilot project is to identify the molecular defect in two patients with a severe combined immunodeficiency (SCID) syndrome. The patients are brothers born to consanguineous parents, thus the defect is likely to be a recessive mutation in a single gene. The patients' T cells exhibit a pronounced defect in capacitative (store-operated) Ca2+ entry; in consequence, they are unable to activate the calcineurin-regulated transcription factor NFAT (nuclear factor of activated T cells) and show a marked deficiency in transcription of cytokine genes. The underlying molecular defect likely involves a subunit of the capacitative Ca2+ entry channel (CRAC), or a component of the signaling pathway that leads to CRAC channel opening. The mechanisms of store-operated Ca2+ entry are essentially unknown but are likely to be conserved in many if not all non-excitable cell types, and our studies will therefore be of general interest to cell biologists as well as immunologists. The investigators will take two major approaches to identify the gene product that is defective in the patients' T cells: (i) Trapping the mutant gene by retroviral insertion in parental T cells. T cell lines from the heterozygous parents will be subjected to genome-wide retroviral insertion (gene-trapping) using a retroviral vector designed so that it can be cleanly excised from its site of integration in the genome. Mutant T cells defective in store-operated Ca2+ entry will be isolated from the population of infected cells. Cells in which retroviral excision reverts the mutant phenotype will be analyzed to determine the site of integration. Gene(s) identified by this process are strong candidates for the wildtype allele for which the parental cells are heterozygous. (ii) Complementation cloning. A microcell-mediated chromosome transfer (MMCT) approach will be used to identify human chromosomal regions that complement the capacitative calcium entry defect in the patients' T cells. Deletion mapping using spectral karyotyping and microsatellite markers will be used to further narrow down the chromosomal region involved in the defect and candidate genes in this region will be evaluated.