FC-8 Microarray analysis of differential gene expression between genetically normal, heterozygous and homozygous affected Norfolk terrier dogs with a heritable keratin 10 defect

A heritable cornification defect resulting from a keratin 10 mutation has been identified in Norfolk terrier dogs. Affected dogs have marked scaling and hyperpigmentation, and develop erosions following mild trauma. The morphologic lesions are similar to epidermolytic hyperkeratosis in humans. The mutation is recessive, and heterozygous dogs lack cutaneous lesions. A diagnostic test for heterozygosity has been developed. Total RNA was extracted from 6-mm skin biopsy samples from two genetically normal, two affected and two heterozygous dogs. Reverse transcription with P33-labeled dCTP generated a cDNA probe that was hybridized to a commercially available cDNA array designed for dermatologic research. This array contained approximately 4400 human cDNAs, and cross-hybridization strategies were developed to allow canine cDNA to hybridize to the membrane. The average background intensity was subtracted from the raw intensity value for each gene. Genes with a corrected value less than one were eliminated from further analysis. Each gene was then normalized to the median intensity of the remaining genes. A gene was considered upregulated or downregulated if its relative expression was greater than two or less than two, respectively. Fifty-three genes were upregulated in both heterozygous and affected dogs over normal. Five genes were downregulated in affected dogs when compared to normal. No downregulated genes were identified in heterozygotes. No differential regulation was apparent between affected and heterozygous dogs. These results indicate that despite a normal phenotype, the decreased keratin 10 production associated with heterozygosity leads to many of the same alterations in gene expression identified in affected dogs. Funding: Morris Animal Foundation, AAVD Postdoctoral Fellowship.