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P. P. Karunadharma, C. L. Nordgaard, T. W. Olsen, D. A. Ferrington; Mitochondrial DNA Damage in Human Retinal Pigment Epithelium With Age-Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2351.
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Converging evidence from multiple labs, including our previous proteome analyses of human donor retinal pigment epithelium (RPE), implicate mitochondrial dysfunction in the pathogenesis of AMD. Proteomic results suggest that potential functional defects in mitochondrial structure and bioenergetics (Nordgaard et al., IOVS 2006, 2008) could result from mutations or deletions of mitochondrial DNA (mtDNA). The current study tests the hypothesis that mtDNA mutations and deletions will increase at early stages of AMD compared to age-matched controls.
Human donor eyes obtained from the Minnesota Lions Eye Bank were classified to four progressive stages of AMD using the Minnesota Grading System (Olsen and Feng, IOVS 2003). Genomic DNA was isolated from the macular region of the RPE. The relative amount of mtDNA lesions were quantified using quantitative polymerase chain reaction (QPCR) with primers designed to amplify specific mitochondrial regions. Decreased amplification in QPCR is due to mtDNA lesions or deletions. A small region of 220-bp was amplified for each sample to measure the total amount of mtDNA. Amplified product was quantified using Quant-iTTM PicoGreen® dsDNA dye. Verification of expected product size and the presence of deletions were examined on agarose gels.
The yield of genomic DNA from the RPE macula was 111 ± 8 ng per donor. Amplification of mtDNA fragments using conditions optimized for each primer set showed that 3-5 ng of genomic DNA was sufficient to produce the desired product. Based on the migration on agarose gels, amplicons of correct size were produced for each primer set. In donors with AMD, amplification of the region containing the common deletion produced multiple bands smaller than the expected size suggesting the presence of multiple deletions. Younger donors (negative controls) showed only a single product at the expected mass.
QPCR showed mtDNA lesions and deletions in donors with AMD, suggesting a potential link between mtDNA damage and AMD pathogenesis.
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