Cloning of caninemyocilincDNA and molecular analysis of themyocilingene in Shiba Inu dogs

Abstract

To identify canine myocilin cDNA and compare its sequence in glaucomatous and nonglaucomatous Shiba Inu dogs with closed and open iridocorneal angles (ICAs). Total RNA was extracted from the ciliary body of the eyes of a healthy Beagle, and the canine myocilin gene was cloned and sequenced. Of the Shiba Inu dogs tested, five were glaucomatous with closed ICA, three were nonglaucomatous with open ICA, and two were nonglaucomatous with closed ICA. The genomic DNA of these dogs was extracted from peripheral blood leukocytes. The exons of the canine myocilin gene were amplified using the polymerase chain reaction (PCR), and sequenced. The frequency of mutation in canine myocilin DNA was verified in these dogs by using the myocilin cDNA of a Beagle. The canine myocilin cDNA was 1452 bp long and contained the entire open reading frame encoding 483 amino acids. A leucine zipper-like motif and olfactomedin-like domain were conserved in the amino acid residues. The presence of sequence variants in the genomic DNA of Shiba Inu dogs was independent of the occurrence of glaucoma and ICA grading. Myocilin RNA was detected in the ciliary body and trabecular meshwork (TM) of a Beagle. The myocilin sequence of Shiba Inu dogs suggests that myocilin mutations are unlikely to play a significant role in the pathogenesis of primary closed-angle glaucoma in this breed. However, several mutations in the myocilin gene in exon 1 of Shiba Inu dogs may predispose them to an obstruction in the anterior aqueous outflow.