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M. C. Kenney, S. R. Atilano, M. Chwa, D. S. Boyer, D. Hwang, P. Coskun, D. C. Wallace, A. B. Nesburn, N. Udar; Variations in the Control Region of the Mitochondrial DNA in Age-Related Macular Degeneration Retinas. Invest. Ophthalmol. Vis. Sci. 2008;49(13):249.
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To analyze the control region (CR) of the mitochondrial DNA (mtDNA) from Age-related macular degeneration (AMD) retinas compared to normal retinas.
Retinas were isolated from 10 normal eyes (mean age 79.9 ± 3.8, range 52 to 97) and 11 AMD eyes (mean age 81.5 ± 2.6, range 69-93). Total DNA was extracted and LX-PCR amplification was performed with primers specific for mtDNA. The ratio of mtDNA to nuclear DNA (nDNA) was measured by semi-quantitative PCR of mtND2 and r18s. The mtDNA CR was amplified by PCR (15,704 to 770 nts), treated with ExoSAP-IT and then sequenced at the UCLA Sequencing and Genotyping Core, Los Angeles, CA. Sequences were analyzed using the Mutation Surveyor program, (Softgenetics, State College, PA) and the Fisher exact test using GraphPad Prism software. Immunohistochemistry was performed on paraffin embedded AMD and normal eyes using a monoclonal antibody to ATP synthase (complex V) and a polyclonal antibody to 8-hydroxydeoxyguanidine (8-OH-dG). Nuclei were visualized with DAPI stain.
Smaller-sized LX-PCR mtDNA bands (<16 and >1 kb) were quantified and the normal and AMD retinas had 8.73 ± 1.47 versus 10.89 ± 0.82 bands per individual (p=0.25). The mtND2/r18s ratios for normal and AMD retinal samples were similar to each other (1.34 ± 0.27 versus 1.37 ± 0.49, p=0.87). The PCR product for the mtDNA CR was 1,634 nucleotide pairs (nt). Sequencing showed 4.63 versus 9.87 SNPs per individual in the normal retinas compared to the AMD retinas. One SNP was significantly more common in the AMD retinas compared to normal retinas OR=11.66, p<0.026. The T414C and A189G SNPs were not found in AMD retinas. There was increased staining of 8-OH-gD in the outer segment region of the AMD retinas.
The CR functions in the replication and transcription of mtDNA. The AMD retinas showed an increased number of SNPs in the mtDNA CR compared to normal retinas. In addition, the AMD retinas had higher levels of 8-OH-gD indicating oxidative stress in the outer segments. The increased levels of SNPs may represent sites for "hot spots" in this critical region of the mtDNA.
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