Cystinuria is an autosomal recessive disease in which there is excessive excretion of cystine in the urine, with precipitation of urinary cystine stones. Patients with this disorder may have renal colic, urinary tract obstruction, secondary urinary tract infection, or, if untreated, renal insufficiency. By the beginning of this reporting period we had mapped a cystinuria susceptibility gene to the short arm of chromosome 2, and had identified mutations in the SLC3A1 gene (which is located in the appropriate chromosomal region) in one American cystinuria family. Another group had independently identified 6 other SLC3A1 mutations in cystinuria. During the last year our goals have been to screen our panel of 17 cystinuria families for additional mutations, and to elucidate the genomic structure of SLC3A1. Including the 2 mutations reported last year, we have now identified a total of 5 cystinuria-associated SLC3A1 mutations. This includes: a) a frameshift mutation and b) a large deletion in a non-Jewish American family; c) a C-to-T nonsense mutation at cDNA position 808 in 1 Israeli Druze and 4 Ashkenazi Jewish families (this mutation was found in 8 of 8 Ashkenazi carrier chromosomes examined); d) a C-to- A transversion at cDNA position 383 in a Persian and a Yemenite Jewish family; and e) an A-to-G transition in the splice donor site of exon 4, causing skipping of this exon, in a Moslem Arab family. Studies of the genomic structure of SLC3A1 were based on our identification a P1/PAC contig from the appropriate chromosomal region. SLC3A1 contains 10 exons, and spans a total of approximately 45 kb of genomic DNA. We have constructed an EcoRI restriction map of the region, and have developed PCR primers from introns that allow exon amplification from genomic DNA, thus obviating the need for RT-PCR on illegitimate transcripts from lymphoid cells. We have also sequenced 650 bp upstream from the initiation codon, and have identified several regulatory elements in the promoter region. Finally, we have obtained evidence that, in several Libyan Jewish families, the cystinuria susceptibility gene is not SLC3A1. This is based on the following observations on these families: a) for 5/5 individ-uals tested (3 of them from inbred families), we were unable to find mutations in the SLC3A1 cDNA; b) by urine amino acid analysis, we have confirmed the affection status of all individuals; c) using an intragenic microsatellite that we identified, we have found definite SLC3A1 recombinants; and d) hap-lotype analysis with other newly identified flanking genetic markers is also incompatible with SLC3A1 causing cystinuria in these families. Future studies will be directed toward the mapping and eventual cloning of a second cystinuria susceptibility gene.
