Dataset Information

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.

SUBMITTER: Atilano SR

PROVIDER: S-EPMC7846006 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Atilano Shari R SR Udar Nitin N Satalich Timothy A TA Udar Viraat V Chwa Marilyn M Kenney M Cristina MC

PloS one 20210129 1

<h4>Purpose</h4>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.<h4>Methods</h4>DNA was extracted from neural retina, [RPE+choroid] and blood from three deceased age-related macular degeneration (AMD ...[more]

PMID: 33513185

Similar Datasets

Project description:Purpose: Age-related degeneration (AMD) is a major cause of blindness in developed countries. The molecular pathogenesis of early events in AMD is poorly understood. We investigated differential gene expression in samples of human retinal pigment epithelium (RPE)/choroid from early AMD and control maculas using exon-based arrays. Methods: Gene expression levels in nine early AMD and nine control human donor eyes were assessed using Affymetrix Human Exon ST 1.0 arrays. Two controls did not pass quality control and were removed. Differentially expressed genes were annotated using DAVID, and gene set enrichment analysis (GSEA) was performed on RPE-specific and endothelium-associated gene sets. CFH genotype was also assessed and differential expression was analyzed with respect to high AMD risk (YH/HH) and low AMD risk (YY) genotypes. Results: Seventy-five genes were identified as differentially expressed (raw p-value < 0.01; >50% fold change, mean log2 expression level in AMD or control M-bM-^IM-% median of all average gene expression values); however, no genes were significant (adj. p-value < 0.01) after correction for multiple hypothesis testing. Of 52 genes with decreased expression in AMD (fold change < 0.5; raw p-value < 0.01), 18 genes were identified by DAVID analysis as associated with vision or neurological processes. GSEA of RPE-associated and endothelium-associated genes revealed a significant decrease in genes typically expressed by endothelial cells in the early AMD group compared to controls, consistent with previous histologic and proteomic studies. Analysis with respect to CFH genotype indicated decreased expression of ADAMTS9 in eyes with high-risk genotypes (fold change = -2.61; raw p-value = 0.0008). Conclusions: GSEA results suggest that RPE transcripts are preserved or elevated in early AMD, concomitant with loss of endothelial cell marker expression. These results are consistent with the notion that choroidal endothelial cell dropout occurs early in the pathogenesis of AMD. Using AltAnalyze (ver. 2.0.7 beta), we analyzed nine early AMD and nine control eyes using Affymetrix Human Exon ST 1.0 arrays. Following initial processing in AltAnalyze, two control arrays were identified as potential outliers by tests implement in arrayQualityMetrics, a package for R.

Project description:Purpose: Age-related degeneration (AMD) is a major cause of blindness in developed countries. The molecular pathogenesis of early events in AMD is poorly understood. We investigated differential gene expression in samples of human retinal pigment epithelium (RPE)/choroid from early AMD and control maculas using exon-based arrays. Methods: Gene expression levels in nine early AMD and nine control human donor eyes were assessed using Affymetrix Human Exon ST 1.0 arrays. Two controls did not pass quality control and were removed. Differentially expressed genes were annotated using DAVID, and gene set enrichment analysis (GSEA) was performed on RPE-specific and endothelium-associated gene sets. CFH genotype was also assessed and differential expression was analyzed with respect to high AMD risk (YH/HH) and low AMD risk (YY) genotypes. Results: Seventy-five genes were identified as differentially expressed (raw p-value < 0.01; >50% fold change, mean log2 expression level in AMD or control ≥ median of all average gene expression values); however, no genes were significant (adj. p-value < 0.01) after correction for multiple hypothesis testing. Of 52 genes with decreased expression in AMD (fold change < 0.5; raw p-value < 0.01), 18 genes were identified by DAVID analysis as associated with vision or neurological processes. GSEA of RPE-associated and endothelium-associated genes revealed a significant decrease in genes typically expressed by endothelial cells in the early AMD group compared to controls, consistent with previous histologic and proteomic studies. Analysis with respect to CFH genotype indicated decreased expression of ADAMTS9 in eyes with high-risk genotypes (fold change = -2.61; raw p-value = 0.0008). Conclusions: GSEA results suggest that RPE transcripts are preserved or elevated in early AMD, concomitant with loss of endothelial cell marker expression. These results are consistent with the notion that choroidal endothelial cell dropout occurs early in the pathogenesis of AMD.

Project description:Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmentosa, preferentially affect distinct topographic regions of the retina. While much is known about the distribution of cell types in the retina, the distribution of molecular components across the posterior pole of the eye has not been well-studied. To investigate regional difference in molecular composition of ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq. RNA from temporal, macular, and nasal retina and RPE/choroid from four human donor eyes was extracted, poly-A selected, fragmented, and sequenced as 100 bp read pairs. Digital read files were mapped to the human genome and analyzed for differential expression using the Tuxedo software suite. Retina and RPE/choroid samples were clearly distinguishable at the transcriptome level. Numerous transcription factors were differentially expressed between regions of the retina and RPE/choroid. Photoreceptor-specific genes were enriched in the peripheral samples, while ganglion cell and amacrine cell genes were enriched in the macula. Within the RPE/choroid, RPE-specific genes were upregulated at the periphery while endothelium associated genes were upregulated in the macula. Consistent with previous studies, BEST1 expression was lower in macular than extramacular regions. The MAK gene was expressed at lower levels in macula than in extramacular regions, but did not exhibit a significant difference between nasal and temporal retina. The regional molecular distinction is greatest between macula and periphery and decreases between different peripheral regions within a tissue. Datasets such as these can be used to prioritize candidate genes for possible involvement in retinal diseases with regional phenotypes.

Dataset Information

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

Purpose

Methods

Results

Conclusion

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets