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N. V. Strunnikova, W. Chen, A. Maminishkis, C. Zhi, D. Stambolian, A. O. Edwards, A. Swaroop, G. Abecasis, P. Munson, S. S. Miller; Retinal Pigment Epithelial Signature Gene Function and Retinal Disease. Invest. Ophthalmol. Vis. Sci. 2010;51(13):493.
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The gene signature of the RPE distinguishes it from other cell types and we seek to understand the role of these genes in RPE physiology and pathophysiology.
A set of 154 RPE "signature" genes was determined using expression profiles of native human adult and fetal RPE, and fetal RPE cell cultures. The association of RPE signature genes with AMD was tested in a genome wide association study (GWAS), which included 2.5 million SNPs collected in 1150 controls and 2157 AMD cases. Proteins of interest were downregulated using shRNA lentiviral particles (Sigma). Expression and localization of signature proteins was determined by immunoblotting and immunofluorescence in primary cultures of human fetal retinal pigment epithelium.
A cross-sectional analysis of the RPE signature genes against an AMD GWAS dataset identified novel candidate genes potentially relevant to AMD. The TIMP3 gene (rs5754221, p = 5×10-5), GRAMD3 (rs4836255, p = 3×10-4), PITPNA (rs17821234, p = 4×10-4) and CHRNA3 (rs11072791, p = 6×10-4) were significantly associated. SNPs near 44 other signature genes show some association to AMD at p <0.01. Functional analysis of the 48 genes by DAVID revealed that 18 genes have a signal sequence at the N-terminus that targets these proteins to the ER. Mutations in signal sequences can cause protein misfolding, ER stress, and cell degeneration. In functional experiments, signature RPE gene products such as DCT were modified by shRNA in confluent monolayers. Down regulation of DCT significantly altered transepithelial resistance (TER) and the expression levels of proteins critical for RPE function (bestrophin, Kir7.1, NaK-ATPase, claudin 10, ezrin, ZO-1).
Our identification of RPE signature genes has uncovered highly expressed genes whose functions in RPE physiology are still unknown. This data set has also revealed novel candidates and pathways that could underlie retinal degenerative disease.
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