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Emily S. Charlson, Jie Wu, Donald J Brown, James V Jester; Exon usage and splicing variation in mouse age-related meibomian gland dysfunction (ARMGD).. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified.
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© ARVO (1962-2015); The Authors (2016-present)
Dry eye disease is a common age-related condition frequently associated with ARMGD. We have previously shown aging mice develop atrophy of meibomian glands similar to dry eye patients with ARMGD. Further study of mouse and human ARMGD also identified altered expression of peroxisome proliferator activated receptor gamma (PPARγ) that regulates lipid synthesis along with other changes in meibocyte differentiation and proliferation. Recent whole transcriptome approaches found a number of pathways altered in mice ARMGD, however, alternative splicing events were not interrogated. Our purpose here is to discover and functionally characterize alternative exon usage in aging vs. young mouse MGs.
Tarsal plates containing MGs with overlying conjunctiva were harvested from four groups of C57B16 mice, males and females aged 3 months or 2 years. RNA was isolated, cDNA transcribed and sequenced on the Illumina HiSeq 2500 platform. Reads were quality controlled, mapped to the reference genome, and quantified at the exon level using Tophat2. Exon counts per group were normalized by sequencing depth and gene expression. Differential exon usage was quantified and datasets compared using DEXSeq. DAVID analyses were used for functional annotation of GO and KEGG pathways.
Over 212 million high-quality end reads were aligned. Principle component analysis identified age as the major driver of sample diversity with 78% of the variance explained by PC1. Highly expressed exons more often found in young mice belonged to keratinocyte differentiation, keratinization, and fatty acid synthesis GO families among others, as well as adherens junction KEGG pathways. Mirror analysis in old mice showed altered expression of PPAR signaling in addition to fatty acid and lipid biosynthesis. DEXSeq comparison of differential exon usage with FDR controlled by the Benjamini-Hochberg method at 10% identified significant under-usage of 42 exons affecting 36 genes, and over-usage of 84 exons affecting 56 genes (padj range from <0.1 to 4.26 x 10 -38) in aging mice belonging to cell-cell adhesion pathways.
These results characterize exon usage associated with mouse ARMGD. This analysis identified age-related splicing variation within keratinocyte differentiation, lipid synthesis and PPAR signaling pathways, among others that may play an important role in meibomian gland atrophy, ARMGD and evaporative dry eye disease.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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