Low frequency mitochondrial DNA heteroplasmy SNPs in blood, retina, and [RPE+choroid] of age-related macular degeneration subjects

Abstract

Purpose Mitochondrial (mt) DNA damage is associated with age-related macular degeneration (AMD) and other human aging diseases. This study was designed to quantify and characterize mtDNA low-frequency heteroplasmy single nucleotide polymorphisms (SNPs) of three different tissues isolated from AMD subjects using Next Generation Sequencing (NGS) technology. Methods DNA was extracted from neural retina, [RPE+choroid] and blood from three deceased age-related macular degeneration (AMD) subjects. Entire mitochondrial genomes were analyzed for low-frequency heteroplasmy SNPs using NGS technology that independently sequenced both mtDNA strands. This deep sequencing method (average sequencing depth of 30,000; range 1,000–100,000) can accurately differentiate low-frequency heteroplasmy SNPs from DNA modification artifacts. Twenty-three ‘hot-spot’ heteroplasmy mtDNA SNPs were analyzed in 222 additional blood samples. Results Germline homoplasmy SNPs that defined mtDNA haplogroups were consistent in the three tissues of each subject. Analyses of SNPs with <40% heteroplasmy revealed the blood had significantly greater numbers of heteroplasmy SNPs than retina alone (p≤0.05) or retina+choroid combined (p = 0.008). Twenty-three ‘hot-spot’ mtDNA heteroplasmy SNPs were present, with three being non-synonymous (amino acid change). Four ‘hot-spot’ heteroplasmy SNPs (m.1120C>T, m.1284T>C, m.1556C>T, m.7256C>T) were found in additional samples (n = 222). Five heteroplasmy SNPs (m.4104A>G, m.5320C>T, m.5471G>A, m.5474A>G, m.5498A>G) declined with age. Two heteroplasmy SNPs (m.13095T>C, m.13105A>G) increased in AMD compared to Normal samples. In the heteroplasmy SNPs, very few transversion mutations (purine to pyrimidine or vice versa, associated with oxidative damage) were found and the majority were transition changes (purine to purine or pyrimidine to pyrimidine, associated with replication errors). Conclusion Within an individual, the blood, retina and [RPE+choroid] contained identical homoplasmy SNPs representing inherited germline mtDNA haplogroup. NGS methodology showed significantly more mtDNA heteroplasmy SNPs in blood compared to retina and [RPE+choroid], suggesting the latter tissues have substantial protection. Significantly higher heteroplasmy levels of m.13095T>C and m.13105A>G may represent potential AMD biomarkers. Finally, high levels of transition mutations suggest that accumulation of heteroplasmic SNPs may occur through replication errors rather than oxidative damage.

Mitochondrial DNA heteroplasmy analysis in keratoconus patients from China

“…As is reported by Geng et al (2019) , the heteroplasmic levels in peripheral blood leukocytes were closely associated with clinical manifestations and valuable for evaluating the clinical severity of the m.3243A>G mutation. Besides, Atilano et al (2021) found the m.13095T>C and m.13105A>G heteroplasmic levels were higher in age-related macular degeneration, with the higher heteroplasmic levels possibly representing potential biomarkers. In the present study, the association of heteroplasmic levels with KC was analyzed.…”

Inflammatory cell activity in treated neovascular age-related macular degeneration, a histologic case study

“…27 Lipid hydroperoxides are created when reactive oxygen species react with polyunsaturated fatty acids such as those in drusen. 28 Of note drusen are several micro-meters away from hundreds of mitochondria in the basal RPE cell body, 29 and RPE mitochondrial DNA is damaged in AMD. 30 By contrast, SDD starts far from mitochondria of both RPE and photoreceptor inner segments at the apical RPE.…”

The Complicated Nature of Somatic mtDNA Mutations in Aging

“…Mutations in POLG, the nuclear encoded mtDNA replicative polymerase, are also associated with familial PD, albeit likely from mtDNA depletion, providing orthogonal evidence supporting a link between mtDNA maintenance and neurodegeneration (Luoma et al, 2007). In addition to neurodegenerative diseases, increased mtDNA mutation loads have also been reported in a number of non-neurodegenerative diseases, including diabetes (Nomiyama et al, 2002), sarcopenia (Herbst et al, 2007;Shah et al, 2009), macular degeneration (Kenney et al, 2010;Atilano et al, 2021), heart disease (Matam et al, 2014), and ulcerative colitus (Baker et al, 2019), suggesting widespread, but tissue specific effects.…”