MOLECULAR GENETIC BASIS OF CYSTINURIA

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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.