Cystinuria is a common inherited aminoaciduria that leads to recurrent cystine nephrolithiasis. Mutations in a gene encoding a renal amino acid transporter (SLC3A1) have been identified in patients with cystinuria establishing one molecular cause for the disease. To facilitate systematic screening of this gene for mutations, we have delineated the complete genomic organization of the SLC3A1 coding region using polymerase chain reaction strategies. The complete coding region of the gene is contained within a single yeast artificial chromosome clone and consists of 10 exons and 9 introns. Oligonucleotide primers capable of amplifying selected exons have been made and used in mutational analysis of DNA from 24 cystinuria probands. We illustrate the usefulness of this approach by identifying two novel SLC3A1 mutations. One novel mutation causes replacement of a highly conserved arginine residue (arginine-452) with tryptophan in the cytoplasmic loop between the putative third and fourth membrane spanning segments. A second previously unreported mutation results in replacement of a highly conserved tyrosine (tyrosine-461) residue with histidine in the same region of the protein. In addition, we detected three previously reported SLC3A1 mutations, R270X, 1500 +1/G toT, and M467T, the latter being present in ~20% of cystinuria chromosomes examined. Our findings provide a foundation for the development of more accessible diagnostic screening assays for detecting SLC3A1 mutations using patient genomic DNA, and also contribute to the emerging spectrum of cystinuria genotypes.
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