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Hui Cai, Gregory S Hageman, Rando Allikmets; Mitochondrial DNA Heteroplasmy in RPE of AMD Patients. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):827.
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© ARVO (1962-2015); The Authors (2016-present)
Oxidative stress has been associated with aging and implicated in etiology of AMD. Mitochondria are more vulnerable to oxidative stress than any other organelle due to less efficient DNA repair mechanism. They accumulate somatic and acquired changes in their genome (mtDNA) due to excessive stress and with age. We had hypothesized, also based on a pilot study, that accumulation of somatic variation (heteroplasmy) in mtDNA is associated with AMD. Here we used RPE/choroid samples from maculae of donor eyes from a cohort AMD patients and matched controls to assess the extent of mtDNA heteroplasmy acquired with age and disease.
Genomic DNA were isolated from RPE/choroid trephine punches from macular region of donor eyes of 46 AMD patients and 46 matched by age and ethnicity controls. mtDNA were sequenced with Illumina’s TruSeq Custom Amplicon mtDNA ultra-deep sequencing technology with a minimum of 3000X coverage. Sequence data were annotated and genetic variation (homo- and heteroplasmy) was identified and analyzed by NextGene (SoftGenetics) software. Identified variants were compared to published mtDNA variants [MITOMAP.org: A Human Mitochondrial Genome Database, updated Nov 6, 2014] and between the case and control groups.
The minimum depth of the reliably observed heteroplasmy was set at 2%. In order to assess possible clinically meaningful variants, the more stringent heteroplasmy threshold was set at 40%. This filtering resulted in 74 variants in 46 AMD samples which were not present in any control samples. From these, 58 variants, including 14 non-synonymous, are in t/rRNA sequences and in coding regions of the mtDNA genome, specifically in NADH subunits 2, 5 and 6, CYTB, COX1, 3, and ATP 6 and 8 genes. These genes are involved in the Krebs cycle and electron transport chain reaction of aerobic respiration in mitochondria. 25 variants have not been reported before and 3 variants have been associated with human diseases, including Leber hereditary optic neuropathy and non-insulin-dependent diabetes mellitus. Significantly more heteroplasmic variants were in tRNA sequences of AMD samples (7) than in control samples (3), which may suggest an effect on mitochondrial protein synthesis in AMD.
Ultra-deep sequencing of mtDNA is an effective tool to identify heteroplasmy. Many cases of mtDNA heteroplasmy were present in AMD patients and not in controls. Some of these variants may play a role in AMD etiology.
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