Cloning and characterization of canine PAX6 and evaluation as a candidate gene in a canine model of aniridia

PURPOSE: Mutations in PAX6 cause human aniridia. The small eye (sey) mouse represents an animal model for aniridia. However, no large animal model currently exists. We cloned and characterized canine PAX6, and evaluated PAX6 for causal associations with inherited aniridia in dogs. METHODS: Canine PAX6 was cloned from a canine retinal cDNA library using primers designed from human and mouse PAX6 consensus sequences. An RH3000 radiation hybrid panel was used to localize PAX6 within the canine genome. Genomic DNA was extracted from whole blood of dogs with inherited aniridia, and association testing was performed using markers on CFA18. Fourteen PAX6 exons were sequenced and scanned for mutations, and a Southern blot was used to test for large deletions. RESULTS: Like the human gene, canine PAX6 has 13 exons and 12 introns, plus an alternatively spliced exon (5a). PAX6 nucleotide and amino acid sequences were highly conserved between dog, human, and mouse. The canine PAX6 cDNA sequence determined in this study spans 2 large gaps present in the current canine genomic sequence. Radiation hybrid mapping placed canine PAX6 on CFA18 in a region with synteny to HSA11p13. Exon-scanning revealed single nucleotide polymorphisms, but no pathological mutations, and Southern blot analysis revealed no differences between normal and affected animals. CONCLUSIONS: Canine PAX6 was cloned and characterized, and results provide sequence information for gaps in the current canine genome sequence. Canine PAX6 nucleotide and amino acid sequences, as well as gene organization and map location, were highly homologous with that of the human gene. PAX6 was evaluated in dogs with an inherited form of aniridia, and sequence analysis indicated no pathological mutations in the coding regions or splice sites of aniridia-affected dogs, and Southern blot analysis showed no large deletions.