Abstract
Purpose :
Canine cone-rod dystrophy (cord1) is the first canine inherited retinal disease model in which a multigenic etiology has been shown. A homozygous mutation in RPGRIP1 was identified as the primary cause of disease and a homozygous ~22kb deletion in MAP9 was later identified as a modifier that accelerates the disease. Both gene products are known to localize to the connecting cilia of rods and cones; however the effect of the mutant MAP9 protein in disease modification remains unknown. This study aimed to further characterize wild type (WT) and mutant MAP9 using the canine cord1 model.
Methods :
RNA was extracted from MAP9 WT and mutant canine retinas followed by reverse transcription. Sanger sequencing of MAP9 cDNA was analyzed with Sequencher. To study the expressed protein, WT and mutant MAP9 cDNA were subcloned into an expression vector and transfected into cultured mammalian cells. Western blot analysis was done using the cell lysates. Transfected cells were fixed for immunocytochemistry (ICC) and co-labelled with cellular markers to assess subcellular localization of WT and mutant MAP9. Images were captured via light and confocal microscopy.
Results :
The mutant MAP9 sequence was found to have 99.2% and 98.9% homology to WT at the cDNA and amino acid levels, respectively. Due to a unique partially duplicated tandem MAP9 pseudogene, the overall exon structure of MAP9 was conserved in the mutant as in the WT consisting of 14 exons. Western analysis revealed protein bands of comparable sizes (100kDa), suggesting the presence of full MAP9 products in both mutant and WT. Moreover, preliminary ICC imaging found predominantly cytoplasmic expressions of both WT and mutant MAP9.
Conclusions :
Despite the extensive deletion in MAP9, the mutant MAP9 exhibits the complete exon structure with the 3’ region being formed by a redundant MAP9 partial pseudogene. The full-length cDNA and amino acid sequences are retained with high homologies to the WT, conferring a potentially functional MAP9. In spite of the structural conservation, to account for the phenotypically observed modifier effect of mutant MAP9, molecular interactions between RPGRIP1, MAP9, and other players of the cilia complex are further investigated to better understand the pathogenesis of cord1 and other cilia-associated retinopathies.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.