There is distention of the gall bladder with abundant luminal accumulations of mucus PF01367338 interspersed with scant amounts of bile. The mucosa of the gall bladder is lined by moderately hyperplastic columnar epithelial cells with accentuation of the normal folds by accumulations of mucus. Within the lamina propria and the tunica muscularis there are occasional multifocal to perivascular accumulations of lymphocytes and rare plasma cells. Hematoxylin

and eosin staining. Bar = 250 μm. Sequencing of Canine ABCB 4 Sequencing of all exons (1 to 26) of canine ABCB4 was performed on genomic DNA from cheek swab samples (Shetland Sheepdogs) or from archived liver tissue (affected dogs that Selleck MK-1775 were not Shetland Sheepdogs). A single base pair insertion (G) was identified in exon 12 (Figure 2) in 14 of 15 affected Shetland Sheepdogs, 1 of 21 unaffected Shetland Sheepdogs, and 3 affected dogs of other breeds (Cairn Terrier, Cocker Spaniel,

and Pomeranian). The insertion mutation (ABCB 4 1583_1584G) is significantly associated (P < 0.0001) with the diagnosis of gallbladder mucocele in Shetland Sheepdogs, with an odds ratio of 280 (95% CI 12.7-12,350). In other dog breeds, ABCB 4 1583_1584G is also significantly associated with the diagnosis of gallbladder mucocele (P < QNZ in vivo 0.0006). The frame shift generated by the insertion results in 4 premature stop codons within exon 12. The full canine ABCB 4 gene contains 26 exons which encode essential

structural elements that characterize ABC transporters: two ATP binding domains and two substrate binding sites. Essential structural elements of ABCB4 normally contained within exon 12 and subsequent exons include both ATP binding sites and a substrate binding site. Figure 2 Electropherograms for wildtype and mutant canine ABCB 4. The insertion is indicated by an arrow. A missense mutation in exon 15 of canine ABCB 4 was identified in the one affected Shetland Sheepdog that did not harbor ABCB 4 1583_1584G. This SNP results in a nonhomologous amino acid substitution (alanine to serine) in exon 15 which may affect tertiary protein structure. However, this mutation was also present in 9 of the 21 unaffected Shetland Sheepdogs and 10 of the 15 affected Shetland Sheepdogs, so its significance enough is unclear. No obvious differences were apparent in disease severity or biochemical parameters in the affected dogs with the mutation in exon 15. Confirmation of Insertion by Allele Specific PCR To confirm the presence of ABCB 4 1583_1584G as well as determine the genotype of each dog, allele specific primers were designed and used to amplify the region of interest in exon 12 (Figure 3). All dogs harboring the insertion were heterozygous at the mutant allele suggesting a dominant mode of inheritance with incomplete penetrance. None of the dogs in the study were homozygous for the mutant allele. Genotype frequencies are shown in Table 3.