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Thomas Vo, Marilyn Chwa, Shari Atilano, Deepika Malik, Claudio Ramirez, Javier Cáceres del Carpio, S Michal Jazwinski, Miceli Michael, Baruch Kuppermann, Cristina Kenney; Comparison Study of IL-33 Gene Expression in Haplogroup H, J, L, and K Cybrids. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4988.
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Age-related macular degeneration (AMD) is a leading cause of vision loss among the elderly population. The mechanisms through which AMD develops have been investigated, and current studies have identified many genes to be associated with the onset of AMD. A recently discovered cytokine, interleukin-33 (IL-33), is related to the IL-1 superfamily and due to its pro-inflammatory properties, may have a possible association with the pathogenesis of AMD. IL-33 has been shown to play a critical role in several autoimmune and inflammatory diseases through interactions with the MAP-Kinase and NF-κB signaling pathways. This study utilizes cybrids (cytoplasmic hybrids), containing identical nuclei but different haplogroup mitochondrial DNA (mtDNA), to compare expression levels of IL-33. We hypothesize that haplogroups H, J, L, and K contain unique single nucleotide polymorphisms within the mtDNA that can affect nuclear gene expression levels.
In order to create the cybrids, ARPE-19 cells depleted of their mitochondria (Rho0) were fused with platelets from individuals with H, J, L, or K haplogroup backgrounds (n=3 each). RNA was isolated from these cybrids and cDNA synthesized for analyses with the Affymetrix Human U133 Plus 2.0 Array. Gene expressions were analyzed with pathway analysis software (INGENUITY Systems, Inc.). Our array findings of the H, J, K, and L cybrids were verified by quantitative PCR (Q-PCR) with primers specific for IL-33.
The Affymetrx array analyses showed that compared to H cybrids, the L cybrids and J cybrids had 5.7 fold and 3.5 fold lower expression levels of IL-33 but the K cybrids were similar (1.7 fold difference). As determined by Q-PCR, the gene expression levels of IL-33 for cybrids J, K, and L were measured relative to the expression in H cybrid. An analysis of the data showed decreased expression of the IL-33 gene in J cybrids (0.46 fold, p=0.004) and L cybrids (0.30 fold, p=0.002), but similar expression levels of IL-33 in the K cybrids and H cybrids (1.35 fold, p=0.40).
This study demonstrates that expression of the IL-33 gene varies in cybrids that have identical nuclei but different haplogroups defined by specific mtDNA variants. This is significant because it suggests that the mtDNA variants may mediate IL-33, a pro-inflammatory cytokine that may be important in the pathogenesis of AMD.
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