MOLECULAR GENETIC BASIS OF CYSTINURIA

产品描述：（直接取自申请）该项目的总体目标是确定胱氨酸尿症三种亚型的分子遗传学基础，并确定基因缺陷与临床症状之间的关系。该项目将测试这样的假设，即基于生理和生化测定的胱氨酸尿症的经典亚型具有涉及不同基因突变的独特遗传起源，并且所表达的胱氨酸尿症的严重程度取决于哪个基因突变以及基因内的特定突变。突变已被发现在SLC 3A 1基因在约一半的患者与I型胱氨酸尿症检查。II型和III型胱氨酸尿症的分子缺陷尚不清楚。目的1通过对非洲爪蟾卵母细胞19 q13区域的定位克隆和肾cDNA文库的表达筛选，验证低亲和力胱氨酸转运蛋白突变是III型胱氨酸尿症发病的假设。目的2将使用在用SLC 3A 1基因转染的COS-7细胞中的表达克隆来筛选肾cDNA文库中的50 kDa亚基，来检验II型胱氨酸尿症是由高亲和力胱氨酸转运蛋白的50 kDa亚基突变引起的假设。目的3将检验I型胱氨酸尿症是至少两种基因缺陷的结果的假设，其中之一是在SLC 3A 1基因中，该基因编码高亲和力转运蛋白的78 kDa亚基。我们将筛选SLC 3A 1基因，使用RNase切割试验和RFLP分析识别突变，然后测序以表征突变。未鉴定出SLC 3A 1突变的I型胱氨酸尿症患者将进行连锁研究以排除SLC 3A 1基因位点的参与，然后进行全基因组搜索以鉴定新的染色体位点。目的4将测试的假设，胱氨酸尿症的病理生理学可以预测仅基于基因组异常，这方面的知识可以作为基础，制定治疗性治疗。

Description: (Taken directly from the application) The overall goal of this project is to define the molecular genetic basis of the three sub-types of cystinuria and to determine relationships between gene defects and clinical symptoms. The project will test the hypothesis that the classical sub-types of cystinuria, based on physiological and biochemical determinations, have unique genetic origins involving mutations in different genes, and that the severity of cystinuria expressed is dependent upon which gene is mutated and the specific mutation within the gene. Mutations have been found in the SLC3A1 gene in about half of the patients with Type I cystinuria examined. The molecular defect(s) in Types II an III cystinuria are unknown. Aim 1 will test the hypothesis that Type III cystinuria is due to mutations in the low affinity cystine transporter using positional cloning of chromosomal region 19q13 to identify the gene defect and expression screening of a renal cDNA library in Xenopus oocytes in order to clone the low affinity transporter in. Aim 2 will test the hypothesis that Type II cystinuria is caused by mutations in the 50 kDa subunit of the high affinity cystine transporter using expression cloning in COS-7 cells transfected with the SLC3A1 gene to screen a renal cDNA library for the 50 kDa subunit. Aim 3 will test the hypothesis that Type I cystinuria is the result of at least two gene defects, one of which is in the SLC3A1 gene which codes for a 78 kDa subunit of the high affinity transporter. We will screen the SLC3A1 gene to identify mutations using RNase cleavage assays and RFLP analysis followed by sequencing to characterize mutations. Type I cystinurics with no identified SLC3A1 mutations will be subjected to linkage studies to rule out involvement of the SLC3A1 gene locus, followed by a genome wide search to identify a new chromosomal locus. Aim 4 will test the hypothesis that the pathophysiology of cystinuria can be predicted based solely upon genomic abnormalities and that this knowledge may be used as the basis for the formulation of therapeutic treatment.