The Dalmatian is unique among dogs in that it excretes uric acid rather than allantoin, the normal byproduct of purine metabolism. Thus, the Dalmatian is a natural model for the human conditions hyperuricosuria and hyperuricemia (i.e., elevated levels of uric acid in the urine and blood, respectively). Tissue transplantation experiments have demonstrated that the defect is intrinsic to the liver, consistent with an inborn error in purine metabolism. Although the defect is fixed within the Dalmatian population, a multigenerational Pointer x Dalmatian backcross family segregates a single locus responsible for the autosomal recessive disorder. The proposed research will identify the gene responsible for hyperuricosuria/hyperuricemia (huu) in the Dalmatian breed. A genome scan has been performed which demonstrated linkage of huu to CFA 3 with a LOD score of 6.5. Further fine structure mapping using microsatellites derived from the canine genome sequence localized huu to a 3.3 Mb interval flanked by recombination breakpoints. Based on the recent availability of the canine genome sequence, there are twenty four candidate genes in this interval. Interestingly, none of these genes have previously identified functions in purine metabolism or urate transportation, confirming that this work will yield novel insights into hyperuricosuria. A three tiered approach will be taken to identify the mutation responsible for hyperuricosuria in the Dalmatian. First, SNPs obtained from the canine genome sequence will be evaluated for linkage disequilibrium in the Dalmatian breed. The area of LD may exclude additional candidate genes as well as establish our expectations for potential disease-causing alleles. Second, candidate genes from the critical interval that are expressed in the canine liver will be determined using RT-PCR. At the same time, expression levels will be compared between Dalmatians and non-Dalmatians using Northern Blots to identify any qualitative or quantitative differences in expression. Based on the results of the three approaches, we will begin sequencing cDNA from the remaining candidates as well as performing Southern blot analysis to identify any large rearrangements. Putative mutation-causing alleles will be evaluated by verification of homozygosity in the Dalmatian breed, lack of homozygosity in other dog breeds, as well as appropriate functional studies of the mutation. Identification of the gene and mutation that cause high levels of uric acid in the Dalmatian dog will further establish it as a model system for the human condition. Uric acid levels are a contributing factor for many human diseases including gout, kidney stones, cardiovascular and renal disease. The elucidation of a novel gene function should provide new avenues of investigation for the treatment of the human condition. ? ? ?
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