MOLECULAR GENETIC BASIS OF CYSTINURIA

描述：（直接取自申请书）本项目的总体目标是明确胱氨酸尿三亚型的分子遗传学基础，确定基因缺陷与临床症状之间的关系。该项目将验证以下假设：基于生理和生化测定，胱氨酸尿的经典亚型具有独特的基因起源，涉及不同基因的突变，胱氨酸尿的严重程度取决于哪个基因发生突变以及基因内的特定突变。在大约一半的I型胱氨酸尿患者中发现了SLC3A1基因突变。II型和III型胱氨酸尿的分子缺陷尚不清楚。目的1将验证III型胱氨酸尿症是由低亲和力胱氨酸转运体突变引起的假设，利用染色体19q13区定位克隆鉴定基因缺陷并筛选爪蟾卵母细胞肾cDNA文库表达，从而克隆出低亲和力胱氨酸转运体。目的2将通过在转染SLC3A1基因的COS-7细胞中进行表达克隆，筛选50 kDa亚基的肾脏cDNA文库，验证II型胱氨酸尿是由高亲和力胱氨酸转运体50 kDa亚基突变引起的假设。Aim 3将检验I型胱氨酸尿是至少两个基因缺陷的结果的假设，其中一个是SLC3A1基因，该基因编码高亲和力转运蛋白的一个78 kDa亚基。我们将筛选SLC3A1基因，使用RNase裂解试验和RFLP分析鉴定突变，然后进行测序以表征突变。未发现SLC3A1突变的I型胱氨酸血症将进行连锁研究，以排除SLC3A1基因位点的参与，然后进行全基因组搜索以确定新的染色体位点。目的4将测试假设，胱氨酸尿的病理生理可以预测仅基于基因组异常，这一知识可能被用作制定治疗方案的基础。

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.