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Mohamed Tarek, Javier Cáceres del Carpio, Claudio Ramirez, Payam Falatoonzadeh, Shari Atilano, Deepika Malik, S Michal Jazwinski, Miceli Michael, Baruch Kuppermann, Cristina Kenney; Comparison Between Haplogroup H and L Cybrids in ROS/RNS Production and Gene Expression Further step in understanding the pathogenesis of Age-related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6195. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Mitochondrial DNA haplogroups can be classified according to accumulation of specific single nucleotide polymorphisms (SNPs). The H haplogroup is of European origin and protective for AMD, the L haplogroup which is of African origin shows only early AMD changes. Our work is to investigate the differences in Reactive Oxygen/Nitrogen Species (ROS/RNS) production and gene expression profiles of cybrids (cytoplasmic hybrids) that have either the H haplogroup or the L haplogroup.
ARPE-19 cells lacking mitochondrial DNA (mtDNA) (Rho0) were fused with platelets from individuals with either H or L haplogroup mtDNA as determined by PCR analysis. After culture for 24 hours, ROS/RNS levels were determined with 2`, 7`-dichlorodihydrofluorescein diacetate dye assay. Experiments were repeated twice and performed in 8 wells per sample. RNA was obtained from the H cybrids (n=3) and L cybrids (n=3) and the gene expression patterns were determined using the Affymetrix Human Genome U133 plus 2.0 array. The pathway profiles were analyzed with the INGENUITY systems program. Quantitative PCR (Q-PCR) was performed using primers for complement component 3 (C3) and complement factor H (CFH) genes for the different H and L cybrids.
Cybrids with the haplogroup L mtDNA produced 25.79±6.21 % lower levels of ROS/RNS compared to haplogroup H cybrids (p=0.026). Complement pathway genes were expressed differently, the haplogroup H cybrids showed higher expression levels in C1S, C3, CFH, C4BPB, CFHR4, and ITGB2 genes by 2.9, 5.5, 2, 3.8, 17.8 and 3.4 folds respectively, while the C1QC, C4A, C8B and CFP genes were expressed at higher levels in the haplogroup L cybrids (3.2, 3.3, 6.2 and 3 folds respectively). By Q-PCR analyses, the L cybrids had a 0.66-fold expression level for the CFH gene (p=0.03) and 0.17-fold level for the C3 gene (p=0.0004) compared to the H cybrids.
Our findings demonstrate that cybrids which are identical except for their mtDNA, have different levels of expression for genes involved in the alternative complement pathway. Our results are significant because they show that mtDNA variants (haplogroups) can mediate not only energy production but also the expression of genes for major cellular pathways involved in inflammation.
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