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Claudio Ramirez, Marilyn Chwa, Shari Atilano, Deepika Malik, Javier Cáceres del Carpio, Mohamed Tarek, S Michal Jazwinski, Miceli Michael, Baruch Kuppermann, Cristina Kenney; COMPARISON OF THE GENE EXPRESSION BY HAPLOGROUPS H AND J: IMPLICATIONS FOR AMD (AGE RELATED MACULAR DEGENERATION). Invest. Ophthalmol. Vis. Sci. 2013;54(15):4985.
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© ARVO (1962-2015); The Authors (2016-present)
AMD is a leading cause of vision loss in the elderly population. Mitochondrial genetics is an important area of research that may help us understand the predisposition for AMD between and within racial groups.Previous studies have shown that the mtDNA haplogroup J is associated with AMD, while the H haplogroup is protective. However, the functional consequences of this difference are not understood. We have used cybrids (cytoplasmic hybrids) to study the characteristics and biochemical differences between various haplogroups. Our hypothesis is that cybrids, dissimilar only in their mtDNA haplogroup will behave differently in vitro.
Cybrids were created by introducing the mitochondria from human individuals platelets into a host cell line (ARPE-19) that was devoid of mitochondrial DNA (Rho0). Therefore, all cybrids carry the same nuclear genes but vary only in their mitochondrial content. Cybrid cultures H and J were pelleted, the RNA was isolated and then quantified. RNA samples were reverse transcribed into cDNA. The RNAs from the three H and the three J haplogroups cybrid cultures were combined into a single sample each for analyses with the Affymetrix Human U133 Plus 2.0 Array. The gene expression results were analyzed with pathway analysis software (INGENUITY Systems). Q-PCR was performed using primers for genes associated with inflammation (TGFA.TGFB2, and IL6) and apoptosis (RARA1, BBC-3 and BCL2L13).
The array analyses showed that H and J cybrids had altered expression of nuclear genes involved in inflammation and apoptotic pathways. Q-PCR analyses showed that J cybrids had decreased expression levels for TGFA (0.43 fold, p=0.03), RARA (0.57 fold, p=0.007), and BLC2L13 (0.56 fold, p=0.005). There were no significant changes in expression levels for TGFB2 (0.86 fold, p=0.41), IL-6 (0.38 fold, p=0.09), and BBC-3 (0.70 fold, p=0.11) in the H versus J cybrids.
This study demonstrates that cybrids may be a useful tool to study the effects of mtDNA variants on gene expression. Our findings suggest that mtDNA haplogroup differences have functional consequences. This data may have implications for understanding the association between haplogroup J and its increased risk for AMD.
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