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Retina  |   April 2012
DNA Methylation Is Associated with Altered Gene Expression in AMD
Author Affiliations & Notes
  • Allan Hunter
    From the F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania;
  • Paul A. Spechler
    From the F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania;
  • Alyssa Cwanger
    From the F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania;
  • Ying Song
    From the F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania;
  • Zhe Zhang
    Children's Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania;
  • Gui-shuang Ying
    Center for Preventive Ophthalmology and Biostatistics, University of Pennsylvania, Philadelphia, Pennsylvania; and
  • Anna K. Hunter
    Children's Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania;
  • Edwin deZoeten
    Children's Hospital of Colorado, Denver, Colorado.
  • Joshua L. Dunaief
    From the F.M. Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University of Pennsylvania, Philadelphia, Pennsylvania;
  • Corresponding author: Allan Hunter, MD, F.M. Kirby Center, 303 Stellar Chance Labs, 422 Curie Blvd, Philadelphia, PA 19104; [email protected]
Investigative Ophthalmology & Visual Science April 2012, Vol.53, 2089-2105. doi:https://doi.org/10.1167/iovs.11-8449
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      Allan Hunter, Paul A. Spechler, Alyssa Cwanger, Ying Song, Zhe Zhang, Gui-shuang Ying, Anna K. Hunter, Edwin deZoeten, Joshua L. Dunaief; DNA Methylation Is Associated with Altered Gene Expression in AMD. Invest. Ophthalmol. Vis. Sci. 2012;53(4):2089-2105. https://doi.org/10.1167/iovs.11-8449.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly. Evidence suggests oxidative stress plays a role in the disease. To assess the potential contribution of epigenetic regulation of antioxidant genes relevant to AMD pathogenesis, we evaluated DNA methylation, a tissue-specific genetic modulation that affects gene expression.

Methods: Using the Infinium HumanMethylation27 Illumina platform, we performed DNA bisulfite sequencing to compare the methylation status in postmortem retina pigment epithelium (RPE)/choroid between patients with AMD and age-matched controls. Gene expression was assessed with the Affymetrix Exon Array. TaqMan gene expression assays were used for relative quantification (RT-PCR) confirmation of the expression array results. Glutathione S-transferase isoform mu1 (GSTM1) and mu5 (GSTM5) promoter methylation was confirmed by CpG island bisulfite pyrosequencing. To assess protein levels and localization, we used Western analysis, immunohistochemistry, and immunofluorescence with murine and human samples.

Results: The mRNA levels of GSTM1 and GSTM5 were significantly reduced in AMD versus age-matched controls in RPE/choroid and neurosensory retina (NSR), which corresponded to hypermethylation of the GSTM1 promoter. mRNA and protein levels were decreased (RPE to a greater extent than NSR) in AMD postmortem samples, irrespective of age. Immunohistochemistry and immunofluorescence confirm the presence of the enzymes in the NSR and RPE.

Conclusions: Comparison of DNA methylation, together with mRNA levels, revealed significant differences between AMD versus normal retinas. The evidence presented suggests that GSTM1 and GSTM5 undergo epigenetic repression in AMD RPE/choroid, which may increase susceptibility to oxidative stress in AMD retinas.

Introduction
The retina's high oxygen saturation and easily oxidized polyunsaturated fatty acids (e.g., docosahexaenoate [DHA]) impart a great oxidative burden. 13 These oxidative insults can cause irreversible damage to retinal cellular machinery and function. 47 Mechanisms used to counter oxygen toxicity include compartmentalization, repair, removal of damaged macromolecules, and free radical elimination by “scavenger” molecules, such as vitamins or antioxidant enzymes or compounds. 8,9  
Although the cause of age-related macular degeneration (AMD) is not completely understood, there is evidence that oxidative stress is involved. Antioxidant vitamins can slow the progression in moderate to advanced AMD. 10 The retina has a high concentration of vitamin C, a natural antioxidant, which decreases after intense light exposure. 11,12 The choroidal arteries have the highest oxygen saturation in the body. 13,14 There is an increase in ω-(2-carboxyethyl)pyrrole protein adducts secondary to oxidation of DHA-containing lipids in the retinas in AMD versus controls. 15,16  
These oxidative insults cause an accumulation of free radical by-products. In addition, patients with AMD appear to have a reduced serum antioxidant potential, which is partially alleviated by vitamin supplementation. 16,17 Further, smoking, a potent oxidative insult, is a known risk factor for AMD. 1822  
More than solely damaging proteins and lipids, free radicals can influence chromatin structure. Oxygen radicals can cause point mutations, deletions, and rearrangements. 23,24 One result is altered sequence-specific-protein interactions. DNA methyl transferases (DNMTs), for example, when confronted with free radical induced DNA damage, change the methylation “profile” of DNA. Indeed, reactive oxygen species have been shown to cause both hyper- and hypomethylation of DNA. 25,26  
The modification of DNA by the addition of a methyl group to cytosine changes the electrostatic nature of chromatin. 27,28 DNA methylation, along with histone acetylation, deacetylation, or methylation, is a primary chemical modification that alters transcription factor–DNA affinity. Hypermethylation of promoter CpG islands and further upstream cis-regulatory CpG “island shores” have been linked with heterochromatin and gene silencing. 2934 The methylated cytosines of DNA act, primarily, by increasing electrostatic interactions with methyl-CpG-binding domain (MBD) proteins that act as transcriptional repressors through interactions with histone deacetylases (HDACs). 
Decreased expression of glutathione S-transferase phi (GSTP1) has been linked to DNA hypermethylation in certain cancers, and mRNA levels are diminished in AMD. 35,36 GSTP1 is a scavenger of reactive oxygen species and its absence could reduce protection from genome-damaging oxidants, resulting in increased vulnerability to further oxidative insults. 
Although DNA methylation is an inheritable, covalent epigenetic change, it is modifiable. “Silenced' expression can be increased with demethylation of the promoter region. 37,38 The demethylation activity of the spice curcumin and the phenol epigallocatechin-3-gallate, found in green tea, has been described. 3941 Green tea polyphenols inhibit DNMT1 in human prostate cancer cells, resulting in demethylation of the proximal GSTP1 promoter and increased expression of GSTP1. 42  
AMD phenotype discordance in monozygotic (MZ) twins helps clarify the potential impact of environment on AMD pathogenesis, given MZ twins' identical genetic background. Worse AMD phenotype (i.e., advanced stage of disease and fundoscopy with larger drusen size and/or pigment area) was associated with the MZ twin who smoked the most and had the lower dietary intake of vitamin D, betaine, and methionine. These modifiable environmental and dietary exposures have been shown to effect DNA methylation and epigenetic mechanisms. 43  
To determine if DNA methylation is involved in gene expression in AMD, we used microarray technology. Herein, we detected expression differences in AMD versus age-matched controls using the Affymetrix exon microarray in postmortem retina pigment epithelium (RPE)/choroid samples. Coupling expression results to DNA bisulfite sequencing with the Infinium HumanMethylation27 Illumina array (San Diego, CA) showed a significant methylation change of promoter CpG sites that corresponded to altered expression of 63 genes. 
Materials and Methods
Histopathologic Assessment
Whole human donor eyes were obtained from the National Disease Research Interchange (NDRI, Philadelphia, PA) and whole human RPE/choroid were from Christine Curcio (University of Alabama, Birmingham, AL). All specimens were obtained in accordance with institutional review board regulations and the provisions of the Declaration of Helsinki for research involving human tissues (Supplemental Table 1 ). Average postmortem tissue harvest time was 5.2 hours (range 2.5 to 9.0 hours). 
Specimen Preparation
The postmortem specimens were flash frozen upon tissue harvest and stored in −70°C at all times. Tissue preparation was done on dry ice. The anterior segments were removed. The posterior segment of the eye was cut into quarters centered upon the fovea and the horizontal section was placed through the horizontal raphe of the retina and the optic nerve center. The neurosensory retina (NSR) and RPE/choroid were then separated. 
DNA and RNA isolation
DNA isolation was performed with the DNeasy Blood & Tissue Kit (Qiagen, Inc., Germantown, MD) per manufacturer's protocol. The final DNA concentration was determined by nanodrop and diluted to 50 ng/μL. RNA was isolated with the RNeasy Mini Kit (Qiagen, Inc.) per manufacturer's protocol. The final RNA concentration was determined by nanodrop and diluted to 50 ng/μL. 
DNA Bisulfite Conversion
DNA was bisulfite converted using the EZ DNA Methylation kit (Zymo Research, Irvine, CA)per manufacturer's protocol. 
PCR of Bisulfite-Treated DNA to Confirm Conversion
Primers to confirm DNA bisulfite conversion were obtained from EZ DNA Methylation kit (Zymo Research) and PCR was done per manufacturer's protocol. 
RNA Analysis on Affymetrix Exon Array
Nanochips were run on Agilent (Santa Clara, CA) Bioanalyzer 2100 to determine the quantity and concentration of RNA. Ambion (Santa Clara, CA) WT Expression Kit for Affymetrix GeneChip Whole Transcript (WT) Expression Arrays, P/N 4425209 was used to generate sense-strand cDNA from 100 ng of total RNA.A 5.5-μg amount of sense-strand cDNA was fragmented and labeled using the Affymetrix (Santa Clara, CA) GeneChip WT Terminal Labeling and Hybridization Kit (PN 702880); 5 μg of fragmented and labeled sense-strand cDNA was hybridized to an Affymetrix HuEx1.0ST Array. Arrays were washed on an Affymetrix GeneChip Fluidics Station 450 using fluidics protocol FS450_0001 and scanned on Affymetrix GeneChip Scanner 3000. 
Gene-level measurements were obtained by applying the Robust Multichip Average (RMA) method implemented in the “affy” package of BioConductor to the raw data and using the custom library file generated by BRAINARRAY. The processed data set includes 23,536 unique Entrez genes. The gene-level data of all RPE samples were further normalized by the QSPLINE method also implemented in the “affy” package using autosomal genes. 
Illumina Infinium HumanMethylation27 Microarray
High-resolution methylation analyses of AMD eyes (n = 10) and normal eyes (n = 11) were conducted on the Illumina Infinium HumanMethylation27 microarray platform. This BeadChip assay measures methylation, given as a β value ranging from zero to one, at more than 27,000 CpG loci. Results were outputted by BeadStudio without normalization. Arrays were processed at the Center for Applied Genomics at Children's Hospital of Philadelphia according to the manufacturer's protocol. 
Array control probes were used to assess sample performance. Multivariate characteristics of array control probes were used to screen outliers. Sex chromosome loci (n = 1092) were excluded to avoid gender-specific methylation bias, resulting in a final dataset that consisted of 26,486 autosomal loci associated with 13,890 genes. Sequence context information such as CpG island status and transcription factor binding site proximity was extracted from tracks of the University of California Santa Cruz Genome Browser (http://genome.ucsc.edu/). 
Table 1.
 
A Total of 46 mRNAs from the Affymetrix Exon Microarray That Showed a Significant Correlation (P < 0.1) Between HCS and NDRI Samples and Also Had an FC of More Than 25% Between AMD and Control
Table 1.
 
A Total of 46 mRNAs from the Affymetrix Exon Microarray That Showed a Significant Correlation (P < 0.1) Between HCS and NDRI Samples and Also Had an FC of More Than 25% Between AMD and Control
Gene_ID Chr Symbol HCS NDRI Name
Control AMD AMD-Control FC NSD Control AMD AMD-Control FC P Value
10,930 6 APOBEC2 4.749 6.450 1.701 3.250 3.154 6.245 6.665 0.420 1.338 0.028 Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 2
6228 5 RPS23 8.039 9.413 1.374 2.591 9.466 9.490 9.872 0.382 1.303 0.011 Ribosomal protein S23
4160 18 MC4R 3.121 4.339 1.217 2.325 3.855 4.673 5.494 0.821 1.767 0.045 Melanocortin 4 receptor
100,132,476 17 KRTAP4-7 7.174 8.324 1.150 2.219 3.846 7.532 7.886 0.353 1.278 0.099 Keratin-associated protein 4–7
100,129,026 NA 6.507 7.454 0.947 1.928 5.516 7.487 7.880 0.393 1.313 0.010 NA
731,157 5 LOC731157 4.550 5.289 0.740 1.670 3.731 5.198 5.650 0.452 1.368 0.025 Similar to HSPC101
730,032 2 LOC730032 5.110 5.663 0.553 1.467 3.468 5.143 5.629 0.486 1.400 0.012 Similar to RIKEN cDNA C230030N03
729,851 NA 3.784 4.330 0.546 1.460 3.521 4.207 4.731 0.524 1.438 0.036 NA
79,870 8 BAALC 6.009 6.509 0.499 1.414 6.270 6.931 7.365 0.435 1.352 0.079 Brain and acute leukemia, cytoplasmic
284,120 NA 5.413 5.768 0.355 1.279 3.973 5.973 6.507 0.534 1.448 0.067 NA
4482 8 MSRA 8.481 8.134 −0.347 −1.272 5.486 7.965 7.635 −0.330 −1.257 0.019 Methionine sulfoxide reductase A
340,371 8 NRBP2 9.481 9.098 −0.382 −1.303 3.880 8.832 8.389 −0.444 −1.360 0.022 Nuclear receptor binding protein 2
2844 9 GPR21 7.854 7.422 −0.432 −1.349 4.245 7.618 7.091 −0.527 −1.441 0.042 G protein–coupled receptor 21
100,131,601 16 LOC100131601 7.031 6.587 −0.444 −1.360 3.136 6.541 6.121 −0.420 −1.338 0.031 Similar to hCG1980470
29,065 8 DDEF1IT1 6.723 6.268 −0.455 −1.371 3.061 6.207 5.819 −0.388 −1.308 0.006 DDEF1 intronic transcript 1 (nonprotein coding)
100,131,231 9 LOC100131231 8.344 7.871 −0.473 −1.388 4.176 8.077 7.741 −0.336 −1.262 0.057 Hypothetical protein LOC100131231
58,496 6 LY6G5B 8.878 8.394 −0.484 −1.398 7.390 8.683 8.331 −0.352 −1.276 0.055 Lymphocyte antigen 6 complex, locus G5B
729,793 NA 7.227 6.714 −0.513 −1.427 4.324 6.689 6.271 −0.418 −1.336 0.047 NA
55,556 18 ENOSF1 8.999 8.484 −0.515 −1.429 3.166 8.554 8.113 −0.441 −1.357 0.010 Enolase superfamily member 1
645,771 13 RP11-385E5.2 3.482 2.937 −0.546 −1.460 3.453 3.571 3.171 −0.400 −1.320 0.015 Poly (ADP-ribose) polymerase family, member 4 pseudogene
642,367 NA 6.605 6.057 −0.548 −1.462 8.032 6.085 5.746 −0.339 −1.265 0.022 NA
100,129,743 2 RPL7P14 6.644 6.086 −0.558 −1.472 14.077 5.712 4.947 −0.766 −1.700 0.007 Ribosomal protein L7 pseudogene 14
6819 2 SULT1C2 8.329 7.725 −0.604 −1.520 5.642 7.492 6.955 −0.538 −1.452 0.036 Sulfotransferase family, cytosolic, 1C, member 2
728,655 6 HULC 5.293 4.674 −0.619 −1.536 3.010 5.123 4.677 −0.446 −1.362 0.008 Highly upregulated in liver cancer (nonprotein coding)
388,815 21 C21orf34 8.434 7.807 −0.627 −1.545 4.506 7.900 7.342 −0.558 −1.472 0.055 Chromosome 21 open reading frame 34
646,272 4 LOC646272 4.966 4.336 −0.630 −1.547 3.221 4.613 4.283 −0.329 −1.257 0.019 Similar to ubiquinol-cytochrome c reductase, complex III subunit VII
8038 10 ADAM12 7.512 6.861 −0.652 −1.571 5.172 6.763 6.400 −0.363 −1.286 0.091 ADAM metallopeptidase domain 12
100,130,696 NA 4.040 3.385 −0.655 −1.575 4.061 3.907 3.494 −0.413 −1.331 0.043 NA
727,819 NA 4.729 4.069 −0.660 −1.580 3.915 4.228 3.722 −0.506 −1.420 0.017 NA
51,134 12 CCDC41 5.660 4.955 −0.705 −1.630 5.269 5.475 5.143 −0.332 −1.259 0.019 Coiled-coil domain containing 41
10,693 17 CCT6B 4.769 4.037 −0.731 −1.660 5.336 4.632 4.244 −0.388 −1.308 0.046 Chaperonin containing TCP1, subunit 6B (zeta 2)
51,474 12 LIMA1 9.444 8.673 −0.771 −1.706 3.632 9.245 8.895 −0.350 −1.275 0.070 LIM domain and actin binding 1
100,131,993 13 LOC100131993 7.989 7.170 −0.819 −1.764 3.880 6.957 6.556 −0.401 −1.321 0.077 Similar to hCG2020760
2949 1 GSTM5 9.389 8.444 −0.946 −1.926 2.102 9.117 8.622 −0.495 −1.409 0.037 Glutathione S-transferase mu 5
28,516 14 TRDV3 5.083 4.076 −1.007 −2.010 4.140 4.386 3.926 −0.460 −1.376 0.069 T-cell receptor delta variable 3
5729 14 PTGDR 7.864 6.839 −1.024 −2.034 3.878 7.302 6.727 −0.575 −1.490 0.064 Prostaglandin D2 receptor (DP)
5935 X RBM3 9.893 8.865 −1.028 −2.040 6.851 9.693 8.963 −0.730 −1.659 0.100 RNA binding motif (RNP1, RRM) protein 3
10,561 1 IFI44 7.002 5.951 −1.051 −2.072 9.181 6.777 6.083 −0.694 −1.618 0.093 Interferon-induced protein 44
100,132,099 13 UNQ1829 6.932 5.777 −1.155 −2.227 4.265 6.116 5.494 −0.622 −1.539 0.038 FRSS1829
100,129,349 1 IFI44L 7.324 6.026 −1.299 −2.460 5.245 7.029 6.375 −0.654 −1.574 0.070 NA
440,482 18 ANKRD20A5 6.053 4.751 −1.302 −2.466 3.087 5.492 4.416 −1.076 −2.108 0.040 Ankyrin repeat domain 20 family, member A5
391,267 21 C21orf81 7.626 6.062 −1.563 −2.956 4.678 6.672 5.774 −0.898 −1.864 0.004 Ankyrin repeat domain 20 family, member A3 pseudogene
390,072 11 OR52N4 6.241 4.655 −1.586 −3.002 4.645 6.035 5.351 −0.685 −1.608 0.051 Olfactory receptor, family 52, subfamily N, member 4
284,232 13 LOC284232 5.678 4.034 −1.644 −3.125 5.628 4.465 3.538 −0.926 −1.901 0.001 Ankyrin repeat domain 20 family, member A2 pseudogene
80,867 6 HCG2P7 6.870 4.550 −2.320 −4.995 9.852 5.120 4.420 −0.700 −1.625 0.045 HLA complex group 2 pseudogene 7
2944 1 GSTM1 10.748 6.768 −3.980 −15.780 3.376 8.688 5.950 −2.738 −6.672 0.002 Glutathione S-transferase mu 1
Pyrosequencing
Human glutathione-S-transferase mu1 (GSTM1) methylation assays were developed to cover nine CG dinucleotides from −540 to −320 from the translational start site (ATG) based on Ensembl Gene ID Ensembl:ENSG00000134184. To sequence through every CpG site in this region, two PCR reactions and three pyrosequencing assays were designed and tested for PCR preferential amplification and quantitative pyrosequencing. The bisulfate-converted target sequences from each pyrosequencing reaction are listed in Supplemental Table 2
Table 2.
 
PCR Analysis (AMD/Control) of Individual RPE/Choroid mRNAs That Were Significantly Increased or Decreased in the Exon Microarray
Table 2.
 
PCR Analysis (AMD/Control) of Individual RPE/Choroid mRNAs That Were Significantly Increased or Decreased in the Exon Microarray
Gene ID FC (AMD:Control RPE) P Value
FC confirmation by qPCR of mRNAs originally quantified by the exon microarray
 GSTM1 0.02 < 0.05
 GPR21 0.03 < 0.05
 LOC10013 0.05 < 0.05
 c21orf34 0.33 < 0.05
 GSTM5 0.34 < 0.05
 RPS23 0.41 NS
 PTGDR 0.46 < 0.05
 IF144 0.51 < 0.05
 CCT6B 0.63 < 0.05
 LY6G5B 0.63 < 0.05
 ADAM12 0.65 < 0.05
 ARSG 0.65 < 0.05
 CCDC41 0.73 < 0.05
 NRBP2 0.76 < 0.05
 ANGPTL2 0.78 NS
 MSRA 0.79 NS
 ENOSF1 0.83 NS
 SULT1C2 0.86 NS
 LIMA1 0.89 NS
 BAALC 1.62 < 0.05
 MC4R 2.22 < 0.05
 APOBEC2 2.97 < 0.05
 ALOX15B 3.36 < 0.05
 AANAT 3.91 < 0.05
qPCR quantification (AMD versus controls) of mRNAs involved in epigenetic modulation
 MBD1 0.98 NS
 MBD2 0.92 NS
 MBD3 1.14 NS
 MeCP2 1.02 NS
 TRDMT1 0.61 NS
 HAT1 0.83 NS
 HDAC9 0.69 NS
 HNMT 0.53 NS
Human glutathione-S-transferase mu5 (GSTM5) methylation assays were developed to cover 32 CG dinucleotides −577 to −16 from the translational start site (ATG) based on Ensembl Gene ID Ensembl:ENSG00000134201. To sequence through every CpG site in this region, three PCR reactions and five pyrosequencing assays were designed and tested for PCR preferential amplification and quantitative pyrosequencing. Bisulfite-converted target sequences from each pyrosequencing reaction are listed in Supplemental Table 2: ADS1741 is for GSTM1 promoter and ADS1746 to 1748 are for GSTM5 promoter. 
Bisulfite conversion was carried out as stated previously. PCR was performed with 0.2 μM of each primer and one of the PCR primers was biotinylated to purify the final PCR product using Sepharose beads. The PCR product was bound to streptavidin sepharose HP (Amersham Biosciences, Uppsala, Sweden), and the sepharose beads containing immobilized PCR product were purified, washed, and denatured using a 0.2-M NaOH solution and rewashed using the Pyrosequencing Vacuum Prep Tool (Pyrosequencing, Qiagen), as recommended by the manufacturer. Then, 0.5 μM Pyrosequencing primer was annealed to the purified single-stranded PCR product; 10 μL of the PCR products were sequenced by Pyrosequencing PSQ96 HS System (Pyrosequencing, Qiagen) following the manufacturer's instructions. Methylation status of each locus was analyzed individually as a T/C SNP using QCpG software (Pyrosequencing, Qiagen). 
Quantitative Real-Time RT-PCR
Quantitative PCR (qPCR) was done using the TaqMan Custom Array (Applied Biosystems, Carlsbad, CA). RNA isolation/quantification and synthesis of cDNA were done as described. 44 Gene expression assays (TaqMan; Applied Biosystems) were obtained and used for PCR analysis. Genes are listed in Table 2. Eukaryotic 18S rRNA (Hs99999901_s1) served as an internal control. qPCR was performed and results analyzed as described. 44  
Immunohistochemistry
Human postmortem globes were prepared as described. 44 Immunohistochemistry was performed on 10-μm-thick sections, as described. 45 The sections were bleached with the Delicate Melanin Bleach Kit for Special Stains and Immunohistochemistry (IHC) (Polysciences, Inc., Warrington, PA) per manufacturer's protocol. Primary antibodies were rabbit anti-GSTM1 (Abcam, Cambridge, MA) at 1:2500 dilution and rabbit anti-GSTM5 (Abcam) at 1:2500 dilution. Control sections were treated identically except for the omission of primary antibodies. Sections were analyzed by bright field microscopy with identical exposure parameters using the Eclipse 80i microscope (Nikon, Melville, NY) with NIS Elements software (Nikon). 
Immunofluorescence
Human postmortem globes were prepared as described. 44 Immunofluorescence was performed on 10-μm-thick sections, as described. 46 Primary antibodies were rabbit anti-GSTM1 (Abcam, Cambridge, MA) at 1:2500 dilution. Primary antibody reactivity was detected using fluorophore-labeled secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA). Control sections and section analysis were done as described above. 
Western Analysis
Human postmortem NSR samples were dissected and processed for Western analysis as described. 44 Membranes were incubated overnight at 4°C with rabbit anti-GSTM1 (Abcam) at 1:1000 dilution and rabbit anti-GSTM5 (Abcam) antibody at 1:1000 dilution. After washes, membranes were incubated, developed, and imaged as described. 44  
Statistical Analysis
Methylation and gene expression microarray data were analyzed in R statistical software environment v2.11.1 (http://www.r-project.org). The difference between normal and AMD was represented as fold change (FC) and the number of AMD sample standard deviations (NSD) was calculated. Pearson correlation of FCs of the histologically confirmed samples (HCS) and NDRI samples was calculated. One-sided t-test was applied to the NDRI samples. Pearson correlation of β difference of the HCS and NDRI samples was calculated. One-sided t-test was also applied to the NDRI samples to validate the group differences of HCS samples. 
Comparison of demographic characteristics between AMD cases and normal controls was performed by Fisher's exact test for categorical characteristics, and two-group t-test and Wilcoxon rank sum test for continuous characteristics. 
qPCR data in AMD and normal control groups were summarized by mean ± SE and compared using the two-group t-test. The Western quantification data and mRNA data were summarized by median (minimum, maximum) and compared between AMD and normal controls using Wilcoxon rank sum test owing to the skewed distribution of data. Two-sided P less than 0.05 was considered statistically significant. These analyses were performed with statistical software GraphPad (GraphPad Software, Inc. San Diego, CA) and SAS v9.2 (SAS Institute Inc., Cary, NC). 
Results
Analysis of RPE/Choroid mRNA Levels in AMD Versus Controls by Microarray
Microarray analysis of AMD versus control RPE/choroid samples included 23,536 unique Entrez genes (Supplemental Table 3 ). The FC and NSD between control and AMD were listed. Analysis was carried out on two separate groups of specimens and results sequentially compared. The first group was the HCS. These were classified as described previously (Supplemental Table 1). The second set was from the NDRI, classified by medical history and confirmed by gross examination of the retina as described previously (Supplemental Table 1). A total of 885 genes among the HCS eyes with FC and NSD greater than 25% and 3.0, respectively, were selected for comparison with NDRI samples. There were 46 genes with P values less than 0.1 and FC more than 25% (Table 1). The antioxidant gene GSTM1 was reduced more than fivefold in AMD versus controls, whereas GSTM5 was twofold reduced. Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 2 (APOBEC2) was increased in AMD samples. 
Quantitative PCR Confirmation of mRNA Levels
Most microarray exon expression results listed in Table 1 were confirmed by qPCR (Table 2). All relative expression changes (i.e., increased or decreased) as determined by exon microarray were confirmed by qPCR, except for 40S ribosomal protein S23 (RPS23, listed in bold type). Six genes tested by qPCR did not reach statistical significance for mRNA level changes. 
Analysis of Promoter Methylation Determined by Bisulfite Microarray Sequencing in RPE/Choroid
Microarray analysis of AMD versus control RPE/choroid samples included 28,328 CpG dinucleotides spanning 14,495 genes; 561 CpG sites of HCS samples were selected with more than 5% difference in methylation (β value) in AMD versus control. This was compared with NDRI samples. Pearson correlation between HCS versus NDRI for the 561CpG sites was 0.229 (P = 4.3e−8). One sided t-test was applied to the 15 NDRI samples (Supplemental Table 4) and 67 CpG sites with more than 5% methylation differences between AMD versus control were selected with P less than 0.1 (Table 3). GSTM1 along with 2% of CpG sites were removed before any analysis owing to specimen-independent microarray platform read variation. CpG measurements of all RPE samples were further normalized by QSPLINE method using autosomal sites (Supplemental Table 4). 
Table 3.
 
67 CpG Sites Determined by Bisulfite Microarray Sequencing of RPE/Choroid That Showed a Significant Correlation (P < 0.1) Between HCS and NDRI Samples and Also Had a 5% Methylation Difference Between AMD and Control
Table 3.
 
67 CpG Sites Determined by Bisulfite Microarray Sequencing of RPE/Choroid That Showed a Significant Correlation (P < 0.1) Between HCS and NDRI Samples and Also Had a 5% Methylation Difference Between AMD and Control
Gene ID Chr Symbol CpG Loc TSS Coordinate Distance to TSS HCS NDRI
Control AMD AMD-Control NSD Control AMD AMD-Control P Value
117,194 11 MRGPRX2 cg22051636 19,038,166 19,038,804 638 0.271 0.804 0.534 19.988 0.691 0.802 0.112 0.073
3716 1 JAK1 cg15997411 65,124,972 65,124,574 −398 0.711 0.879 0.168 10.090 0.857 0.878 0.020 0.050
84,221 21 C21orf56 cg07747299 46,428,480 46,428,729 249 0.516 0.143 −0.373 8.264 0.218 0.128 −0.089 0.025
84,699 19 CREB3L3 cg23777956 4,104,671 4,104,629 42 0.487 0.267 −0.220 6.970 0.346 0.300 −0.046 0.054
9724 13 UTP14C cg24167928 51,496,903 51,496,828 75 0.856 0.918 0.062 6.303 0.871 0.894 0.023 0.041
51,179 1 HAO2 cg03762535 119,713,003 119,712,925 78 0.564 0.637 0.073 6.194 0.632 0.669 0.038 0.090
132,724 4 TMPRSS11B cg19510180 68,794,175 68,794,004 −171 0.661 0.736 0.074 6.161 0.681 0.709 0.027 0.076
4481 8 MSR1 cg01668126 16,095,111 16,094,595 −516 0.867 0.802 −0.065 6.126 0.859 0.836 −0.022 0.028
284,114 17 TMEM102 cg14782678 7,280,445 7,279,486 959 0.511 0.455 −0.056 6.011 0.488 0.469 −0.019 0.049
134,864 6 TAAR1 cg15582891 133,008,721 133,008,835 114 0.573 0.731 0.158 6.005 0.726 0.754 0.028 0.030
50,514 9 DEC1 cg26981881 116,943,245 116,943,918 −673 0.761 0.828 0.067 5.359 0.807 0.836 0.029 0.020
29,974 10 ACF cg03817621 52,315,405 52,315,441 36 0.727 0.844 0.117 4.866 0.789 0.815 0.025 0.061
55,856 6 THEM2 cg16381688 24,773,926 24,775,254 −1328 0.680 0.753 0.073 4.286 0.713 0.738 0.025 0.065
10,507 9 SEMA4D cg22496652 91,284,445 91,284,431 −14 0.707 0.625 −0.082 4.263 0.672 0.648 −0.024 0.070
6374 4 CXCL5 cg04559909 75,083,589 75,083,280 −309 0.438 0.581 0.143 4.173 0.554 0.593 0.039 0.092
2532 1 DARC cg23507131 157,440,780 157,441,134 −354 0.698 0.622 −0.076 4.088 0.677 0.652 −0.025 0.048
5478 7 PPIA cg17269548 44,802,815 44,802,777 38 0.179 0.255 0.076 3.998 0.163 0.207 0.045 0.011
1232 3 CCR3 cg11126313 46,259,266 46,258,692 574 0.785 0.846 0.061 3.991 0.800 0.845 0.044 0.003
83,876 18 MRO cg27318546 46,599,904 46,600,366 462 0.193 0.128 −0.064 3.829 0.191 0.165 −0.026 0.069
221,823 7 PRPS1L1 cg00911873 18,033,988 18,034,011 23 0.812 0.863 0.051 3.773 0.786 0.842 0.055 0.010
6613 17 SUMO2 cg19776090 70,690,552 70,690,693 141 0.378 0.430 0.053 3.752 0.352 0.376 0.025 0.039
337,977 21 KRTAP21-1 cg22373097 31,050,931 31,049,567 −1364 0.575 0.749 0.174 3.568 0.715 0.787 0.072 0.042
3694 2 ITGB6 cg21105318 160,764,766 160,764,836 70 0.825 0.895 0.070 3.547 0.857 0.881 0.024 0.049
84,218 17 TBC1D3 cg14532417 33,601,782 33,602,396 614 0.813 0.737 −0.076 3.492 0.783 0.751 −0.032 0.069
63,895 18 FAM38B cg21165219 10,688,044 10,687,814 −230 0.744 0.800 0.055 3.417 0.794 0.821 0.026 0.061
4719 2 NDUFS1 cg06868758 206,733,636 206,732,432 −1204 0.551 0.470 −0.081 3.409 0.456 0.434 −0.022 0.054
54,103 7 LOC54103 cg26594488 76,873,584 76,873,361 −223 0.789 0.858 0.069 3.255 0.797 0.839 0.042 0.023
2044 4 EPHA5 cg13701273 66,218,375 66,218,104 −271 0.136 0.191 0.055 3.120 0.146 0.195 0.049 0.057
260,436 4 C4orf7 cg25600236 71,125,801 71,126,404 −603 0.650 0.750 0.099 3.103 0.746 0.777 0.030 0.061
1184 X CLCN5 cg20062122 49,720,482 49,720,896 −414 0.842 0.892 0.050 3.063 0.855 0.880 0.025 0.081
390,212 11 GPR152 cg00587613 66,976,799 66,976,776 −23 0.877 0.827 −0.050 3.051 0.857 0.837 −0.020 0.069
79,861 10 TUBAL3 cg07803864 5,436,998 5,436,795 −203 0.748 0.805 0.057 3.039 0.767 0.794 0.028 0.100
10,148 19 EBI3 cg16592658 4,180,887 4,180,540 347 0.748 0.661 −0.087 3.018 0.745 0.712 −0.033 0.032
7531 17 YWHAE cg25299176 1,250,091 1,250,267 176 0.115 0.210 0.096 3.017 0.126 0.176 0.049 0.079
126,433 19 FBXO27 cg11402505 44,215,276 44,215,038 −238 0.183 0.124 −0.059 3.000 0.155 0.130 −0.025 0.033
1041 6 CDSN cg24735489 31,196,331 31,196,202 −129 0.729 0.638 −0.091 2.933 0.716 0.692 −0.024 0.038
351 21 APP cg00542846 26,465,416 26,465,003 −413 0.178 0.234 0.056 2.891 0.210 0.262 0.052 0.059
84,221 21 C21orf56 cg10296238 46,429,602 46,428,729 −873 0.515 0.298 −0.217 2.885 0.385 0.265 −0.120 0.053
3784 11 KCNQ1 cg16465939 2,510,986 2,439,259 71,727 0.149 0.200 0.051 2.847 0.209 0.228 0.019 0.056
339,500 1 ZNF678 cg26683023 225,817,515 225,817,867 −352 0.740 0.794 0.054 2.815 0.757 0.797 0.040 0.009
166,647 4 GPR125 cg26631477 22,126,293 22,126,770 477 0.173 0.245 0.072 2.771 0.168 0.197 0.029 0.054
196,472 12 FAM71C cg04282622 98,565,053 98,565,662 −609 0.742 0.801 0.059 2.726 0.790 0.823 0.033 0.042
81,493 1 SYNC1 cg05342835 32,933,378 32,933,460 82 0.585 0.452 −0.133 2.668 0.529 0.478 −0.050 0.033
8061 11 FOSL1 cg18818531 65,424,853 65,424,573 −280 0.697 0.595 −0.101 2.649 0.650 0.611 −0.039 0.065
388,818 21 KRTAP26-1 cg18822544 30,614,336 30,614,478 142 0.760 0.674 −0.086 2.611 0.727 0.697 −0.030 0.057
8369 6 HIST1H4G cg23540745 26,355,112 26,355,184 72 0.648 0.717 0.068 2.592 0.647 0.680 0.033 0.050
114,035 21 C21orf81 cg14384940 14,274,661 14,274,636 −25 0.339 0.459 0.121 2.439 0.399 0.497 0.098 0.021
120,065 11 OR5P2 cg13410437 7,774,741 7,775,065 324 0.828 0.886 0.058 2.412 0.882 0.907 0.025 0.024
127,943 1 FCRLM2 cg27495845 159,959,712 159,959,081 631 0.502 0.573 0.072 2.404 0.572 0.617 0.045 0.026
81,793 4 TLR10 cg23855121 38,461,333 38,460,984 −349 0.629 0.684 0.055 2.376 0.663 0.690 0.027 0.072
4848 12 CNOT2 cg10464585 68,922,758 68,923,489 −731 0.218 0.155 −0.064 2.329 0.202 0.161 −0.041 0.100
148,646 1 C1orf188 cg15731815 6,191,847 6,191,507 340 0.170 0.275 0.105 2.313 0.218 0.285 0.068 0.078
284,424 19 C19orf30 cg03996793 4,720,537 4,720,152 385 0.157 0.245 0.088 2.284 0.210 0.252 0.042 0.072
3150 21 HMGN1 cg13791713 39,642,786 39,642,917 131 0.264 0.317 0.053 2.279 0.265 0.291 0.026 0.067
23,524 16 SRRM2 cg06736444 2,741,794 2,742,655 −861 0.317 0.481 0.164 2.262 0.362 0.399 0.037 0.090
3827 3 KNG1 cg12454167 187,917,754 187,917,814 −60 0.492 0.331 −0.162 2.235 0.352 0.305 −0.047 0.071
3624 7 INHBA cg16415646 41,709,526 41,709,231 −295 0.806 0.865 0.059 2.235 0.819 0.851 0.032 0.046
163,589 1 TDRD5 cg09656934 177,828,123 177,827,648 475 0.214 0.276 0.062 2.189 0.250 0.282 0.032 0.069
27,004 14 TCL6 cg05023540 95,186,723 95,187,268 −545 0.735 0.675 −0.060 2.181 0.734 0.702 −0.032 0.063
3212 17 HOXB2 cg09313705 43,977,490 43,977,391 −99 0.275 0.370 0.095 2.151 0.314 0.344 0.030 0.092
64,174 16 DPEP2 cg04774694 66,590,771 66,590,857 86 0.757 0.696 −0.061 2.124 0.728 0.708 −0.020 0.073
5369 5 PMCHL1 cg12530080 22,177,396 22,178,218 −822 0.683 0.741 0.057 2.119 0.725 0.750 0.024 0.066
9541 2 CIR cg14138171 174,969,892 174,968,689 −1203 0.719 0.781 0.062 2.092 0.676 0.753 0.077 0.017
22,901 17 ARSG cg15308737 63,814,923 63,815,191 −268 0.820 0.879 0.059 2.090 0.817 0.853 0.036 0.055
140,685 20 BTBD4 cg21291985 61,907,479 61,907,300 −179 0.592 0.660 0.068 2.065 0.582 0.616 0.033 0.093
359 12 AQP2 cg12650635 48,630,730 48,630,796 −66 0.804 0.737 −0.066 2.054 0.796 0.773 −0.023 0.046
10,974 10 C10orf116 cg12261786 88,717,810 88,718,168 −358 0.426 0.377 −0.050 2.033 0.404 0.377 −0.027 0.006
2949 1 GSTM5 cg04987894 110,056,139 110,056,388 −249 0.1119 0.1685 0.0566 0.5664 0.1496 0.2330 0.0834 0.0335
Stepwise Comparison of Methylation Changes Corresponding to Expression Changes in RPE Array Results
Microarray analysis comparing AMD versus control retinas indicate differences in RPE mRNA levels that corresponded to methylation changes within the corresponding promoter sequences (Tables 13, Supplemental Tables 3 and 4). Genes were selected on the basis of an exon microarray NSD greater than 1.25 and a CpG methylation difference greater than 2% (Table 4). Of the possible 26,486 CpG-gene pairs, 63 genes were selected. 
Table 4.
 
A Total of 63 Genes With Exon Microarray Absolute Expression Change (FC) >1.25 in Both HCS and NDRI, and Bisulfite Microarray Sequencing Methylation Difference >2%
Table 4.
 
A Total of 63 Genes With Exon Microarray Absolute Expression Change (FC) >1.25 in Both HCS and NDRI, and Bisulfite Microarray Sequencing Methylation Difference >2%
Gene ID Chr Symbol CpG CpG Location TSS Coordinate Distance to TSS Expression
HCS
Control AMD AMD-Control FC NSD
15 17 AANAT cg09382492 71,975,276 71,975,246 30 6.065 6.380 0.315 1.244 6.991
8038 10 ADAM12 cg13488201 128,067,313 128,067,055 −258 7.512 6.861 −0.652 −1.571 5.172
247 17 ALOX15B cg15799267 7,883,131 7,883,127 4 6.418 6.932 0.514 1.428 0.641
23,452 9 ANGPTL2 cg11213150 128,924,278 128,924,865 587 7.586 7.175 −0.412 −1.330 9.087
314 17 AOC2 cg19317715 38,250,104 38,250,135 −31 5.799 6.849 1.049 2.070 0.833
10,930 6 APOBEC2 cg22375610 41,129,139 41,128,991 148 4.749 6.450 1.701 3.250 3.154
22,901 17 ARSG cg15308737 63,814,923 63,815,191 −268 7.865 7.792 −0.072 −1.051 0.655
284,424 19 C19orf30 cg03996793 4,720,537 4,720,152 385 5.757 6.016 0.259 1.197 0.898
10,842 7 C7orf16 cg23216015 31,693,179 31,693,372 −193 5.561 5.717 0.156 1.115 1.139
389,799 9 C9orf171 cg25344672 134,275,028 134,275,432 −404 5.898 6.162 0.264 1.200 1.342
27,091 17 CACNG5 cg06226384 62,303,813 62,303,913 −100 5.926 6.857 0.931 1.906 4.184
283,316 12 CD163L1 cg13986618 7,487,248 7,488,015 767 6.769 6.400 −0.369 −1.292 1.348
1184 X CLCN5 cg20062122 49,720,482 49,720,896 −414 7.999 7.677 −0.322 −1.250 0.638
119,587 10 CPXM2 cg09619146 125,641,024 125,641,490 466 9.596 9.303 −0.293 −1.225 0.628
1400 4 CRMP1 cg03544320 5,945,592 5,945,686 94 6.865 7.163 0.298 1.230 1.126
8451 13 CUL4A cg16155588 112,909,934 112,911,087 −1153 7.789 7.510 −0.280 −1.214 5.573
54,849 16 DEF8 cg25193494 88,543,505 88,542,652 853 8.086 8.420 0.333 1.260 1.425
126,433 19 FBXO27 cg11402505 44,215,276 44,215,038 −238 7.318 7.882 0.564 1.478 2.439
26,157 7 GIMAP2 cg20663831 150,014,087 150,013,727 360 5.760 5.930 0.169 1.125 1.459
51,659 16 GINS2 cg19890739 84,281,040 84,280,081 −959 5.896 6.010 0.114 1.082 0.607
55,105 1 GPATCH2 cg01727899 215,872,498 215,871,032 −1466 7.782 7.446 −0.336 −1.262 4.095
390,212 11 GPR152 cg00587613 66,976,799 66,976,776 −23 6.655 6.966 0.311 1.241 2.311
9402 22 GRAP2 cg03840259 38,626,982 38,627,032 −50 6.862 6.208 −0.654 −1.574 3.604
2949 1 GSTM5 cg04987894 110,056,139 110,056,388 −249 9.389 8.444 −0.946 −1.926 2.102
3149 X HMGB3 cg05935584 149,902,481 149,902,421 60 6.304 5.785 −0.519 −1.433 1.479
3624 7 INHBA cg16415646 41,709,526 41,709,231 −295 6.375 6.790 0.416 1.334 5.121
3664 1 IRF6 cg23283495 208,046,402 208,046,102 −300 5.971 6.498 0.527 1.441 2.399
55,600 1 ITLN1 cg08356693 159,121,824 159,121,584 −240 3.566 3.718 0.153 1.112 1.386
199,834 1 LCE4A cg17542385 150,948,603 150,948,176 427 5.804 6.322 0.518 1.432 3.224
84,856 10 LOC84856 cg00042156 42,290,848 42,290,967 −119 7.633 7.828 0.195 1.145 1.205
147,172 17 LRRC37B2 cg06488505 25,958,201 25,958,802 −601 6.213 6.374 0.161 1.118 1.452
2872 19 MKNK2 cg21030400 2,003,564 2,002,233 −1331 9.279 9.592 0.313 1.242 1.718
23,209 22 MLC1 cg05861567 48,865,813 48,866,041 228 6.762 7.026 0.264 1.201 1.667
4481 8 MSR1 cg01668126 16,095,111 16,094,595 −516 6.023 6.753 0.730 1.659 1.180
4481 8 MSR1 cg16303562 16,094,704 16,094,595 −109 6.023 6.753 0.730 1.659 1.180
4641 17 MYO1C cg00597076 1,342,630 1,342,745 115 9.737 9.593 −0.144 −1.105 1.087
55,264 21 NA cg13033054 32,870,432 32,870,062 −370 6.818 7.002 0.184 1.136 2.240
55,849 X NA cg19963797 110,811,123 110,811,069 54 4.242 4.080 −0.161 −1.118 1.175
4837 11 NNMT cg14209518 113,671,846 113,671,745 101 7.739 8.395 0.655 1.575 1.085
26,532 19 OR10H3 cg25843439 15,713,574 15,713,203 371 5.922 5.218 −0.705 −1.630 2.782
5016 1 OVGP1 cg22997415 111,772,543 111,771,922 −621 6.261 6.016 −0.245 −1.185 2.707
9796 8 PHYHIP cg05947740 22,145,723 22,145,549 −174 6.382 6.892 0.510 1.424 1.475
9271 12 PIWIL1 cg13861644 129,388,239 129,388,567 −328 5.519 5.138 −0.381 −1.302 2.035
5368 8 PNOC cg03642518 28,230,922 28,230,568 354 6.377 6.577 0.200 1.149 1.945
5446 7 PON3 cg24750391 94,864,147 94,863,598 −549 7.154 6.277 −0.877 −1.836 1.329
5478 7 PPIA cg17269548 44,802,815 44,802,777 38 6.990 6.808 −0.181 −1.134 0.996
5522 4 PPP2R2C cg07867360 6,526,057 6,524,911 −1146 6.081 6.476 0.396 1.315 1.343
5935 X RBM3 cg12251508 48,317,941 48,317,780 161 9.893 8.865 −1.028 −2.040 6.851
166,863 4 RBM46 cg22496683 155,922,060 155,921,950 110 5.157 4.750 −0.407 −1.326 1.006
27,316 X RBMX cg14642832 135,790,803 135,790,605 −198 7.708 7.907 0.199 1.148 1.090
55,511 X SAGE1 cg19856594 134,803,587 134,803,451 136 3.323 3.438 0.115 1.083 3.568
65,012 12 SLC26A10 cg12883767 56,299,376 56,299,960 −584 6.590 6.338 −0.251 −1.190 0.866
6817 16 SULT1A1 cg18530748 28,542,345 28,542,367 22 7.438 7.196 −0.242 −1.183 2.310
6855 X SYP cg10818284 48,943,549 48,943,605 56 7.245 7.044 −0.201 −1.150 1.093
6872 X TAF1 cg23986186 70,502,270 70,502,839 −569 8.135 8.003 −0.132 −1.096 6.151
84,218 17 TBC1D3F cg14532417 33,601,782 33,602,396 614 3.271 6.520 3.248 9.503 8.660
79,875 15 THSD4 cg04616566 69,807,614 69,807,942 −328 8.330 7.995 −0.335 −1.262 1.852
11,011 17 TLK2 cg23181434 57,909,890 57,910,136 −246 6.464 6.142 −0.323 −1.251 0.994
6399 X TRAPPC2 cg24352688 13,661,648 13,662,648 1000 4.961 4.689 −0.272 −1.208 1.076
10,346 11 TRIM22 cg12461141 5,667,230 5,667,664 −434 8.773 8.213 −0.560 −1.474 2.868
10,009 X ZBTB33 cg13128531 119,268,412 119,268,635 −223 7.937 8.092 0.154 1.113 0.965
64,429 10 ZDHHC6 cg17872476 114,195,644 114,196,662 1018 7.956 7.574 −0.383 −1.304 2.066
7542 11 ZFPL1 cg19507591 64,606,853 64,608,270 −1417 8.253 8.387 0.134 1.097 1.316
Table 4.
 
Extended
Table 4.
 
Extended
Expression Methylation
NDRI HCS NDRI
Control AMD AMD-Control FC P Value Control AMD AMD-Control NSD Control AMD AMD-Control P Value
6.265 6.561 0.296 1.228 0.044 0.513 0.567 0.054 1.099 0.519 0.546 0.027 0.234
6.763 6.400 −0.363 −1.286 0.091 0.190 0.240 0.049 1.149 0.178 0.207 0.028 0.131
6.348 6.553 0.205 1.153 0.090 0.492 0.361 −0.131 4.936 0.411 0.387 −0.024 0.208
7.211 7.048 −0.163 −1.119 0.245 0.451 0.401 −0.050 0.945 0.413 0.377 −0.036 0.135
6.646 7.177 0.531 1.444 0.290 0.534 0.463 −0.071 1.127 0.506 0.482 −0.024 0.152
6.245 6.665 0.420 1.338 0.028 0.297 0.373 0.076 1.966 0.338 0.358 0.020 0.230
8.595 8.364 −0.231 −1.173 0.257 0.820 0.879 0.059 2.090 0.817 0.853 0.036 0.055
5.838 6.018 0.180 1.133 0.095 0.157 0.245 0.088 2.284 0.210 0.252 0.042 0.072
5.477 5.616 0.139 1.101 0.035 0.657 0.569 −0.088 0.535 0.649 0.613 −0.036 0.023
6.030 6.178 0.149 1.108 0.100 0.587 0.498 −0.089 5.956 0.549 0.522 −0.027 0.108
7.007 7.596 0.589 1.504 0.127 0.582 0.517 −0.065 1.466 0.577 0.554 −0.023 0.076
6.596 6.280 −0.316 −1.245 0.158 0.657 0.711 0.054 1.024 0.665 0.710 0.045 0.021
7.205 6.895 −0.310 −1.239 0.151 0.842 0.892 0.050 3.063 0.855 0.880 0.025 0.081
8.668 8.193 −0.476 −1.391 0.164 0.172 0.237 0.065 1.590 0.229 0.251 0.022 0.024
7.930 8.297 0.366 1.289 0.257 0.193 0.314 0.121 1.340 0.164 0.298 0.135 0.050
7.618 7.403 −0.215 −1.161 0.003 0.355 0.399 0.045 0.875 0.373 0.401 0.028 0.155
7.921 8.088 0.167 1.123 0.056 0.475 0.588 0.113 1.453 0.539 0.576 0.037 0.203
7.464 7.598 0.134 1.098 0.204 0.183 0.124 −0.059 3.000 0.155 0.130 −0.025 0.033
5.465 5.831 0.366 1.289 0.184 0.292 0.240 −0.052 1.139 0.362 0.332 −0.030 0.269
6.047 6.189 0.143 1.104 0.279 0.531 0.615 0.084 1.515 0.630 0.659 0.029 0.147
7.795 7.550 −0.244 −1.184 0.075 0.749 0.796 0.047 2.008 0.794 0.837 0.043 0.012
6.777 7.019 0.243 1.183 0.074 0.877 0.827 −0.050 3.051 0.857 0.837 −0.020 0.069
6.169 5.984 −0.185 −1.137 0.206 0.648 0.571 −0.077 1.078 0.621 0.602 −0.019 0.235
9.117 8.622 −0.495 −1.409 0.037 0.112 0.169 0.057 0.566 0.150 0.233 0.083 0.017
5.614 5.386 −0.228 −1.171 0.144 0.562 0.332 −0.231 0.707 0.284 0.251 −0.033 0.426
6.775 7.008 0.233 1.175 0.126 0.806 0.865 0.059 2.235 0.819 0.851 0.032 0.046
6.321 6.462 0.141 1.103 0.140 0.064 0.118 0.054 1.254 0.149 0.169 0.020 0.095
3.559 3.754 0.196 1.145 0.003 0.710 0.763 0.053 1.032 0.751 0.783 0.033 0.127
5.935 6.071 0.135 1.098 0.072 0.704 0.655 −0.049 1.222 0.662 0.637 −0.025 0.224
7.253 7.414 0.161 1.118 0.166 0.716 0.649 −0.066 1.345 0.647 0.597 −0.050 0.035
6.684 6.845 0.161 1.118 0.210 0.714 0.765 0.051 1.881 0.729 0.753 0.024 0.216
9.194 9.322 0.128 1.093 0.118 0.730 0.669 −0.060 0.673 0.741 0.682 −0.059 0.004
6.719 7.101 0.382 1.303 0.046 0.476 0.407 −0.070 1.094 0.508 0.490 −0.018 0.242
6.306 6.881 0.575 1.490 0.135 0.867 0.802 −0.065 6.126 0.859 0.836 −0.022 0.028
6.306 6.881 0.575 1.490 0.135 0.713 0.659 −0.053 0.752 0.714 0.696 −0.018 0.102
9.390 9.071 −0.319 −1.247 0.134 0.564 0.492 −0.073 2.492 0.596 0.578 −0.018 0.256
6.883 7.030 0.148 1.108 0.077 0.639 0.486 −0.154 2.903 0.538 0.509 −0.029 0.281
4.381 4.228 −0.153 −1.112 0.136 0.371 0.169 −0.201 1.317 0.168 0.131 −0.038 0.328
7.846 8.138 0.292 1.224 0.281 0.587 0.532 −0.055 1.457 0.582 0.557 −0.025 0.171
5.096 4.559 −0.537 −1.451 0.004 0.374 0.422 0.048 0.750 0.411 0.448 0.036 0.164
6.048 5.905 −0.143 −1.104 0.143 0.775 0.820 0.046 2.560 0.771 0.794 0.023 0.087
6.554 6.749 0.196 1.145 0.166 0.241 0.300 0.059 3.392 0.266 0.286 0.021 0.119
5.339 5.002 −0.337 −1.263 0.076 0.554 0.671 0.117 1.169 0.713 0.789 0.076 0.179
6.299 6.498 0.199 1.148 0.191 0.562 0.476 −0.086 1.840 0.534 0.504 −0.029 0.146
6.062 5.889 −0.174 −1.128 0.289 0.156 0.210 0.054 1.269 0.195 0.255 0.060 0.001
7.078 6.865 −0.213 −1.159 0.164 0.179 0.255 0.076 3.998 0.163 0.207 0.045 0.011
6.562 6.823 0.261 1.198 0.146 0.769 0.724 −0.045 1.292 0.778 0.744 −0.034 0.070
9.693 8.963 −0.730 −1.659 0.100 0.344 0.202 −0.142 0.840 0.174 0.154 −0.020 0.407
4.725 4.078 −0.647 −1.566 0.014 0.586 0.632 0.046 0.571 0.595 0.641 0.045 0.174
7.454 7.660 0.206 1.153 0.087 0.451 0.243 −0.208 0.895 0.209 0.185 −0.024 0.414
3.361 3.505 0.144 1.105 0.008 0.642 0.687 0.045 0.580 0.677 0.708 0.031 0.182
6.451 6.237 −0.214 −1.160 0.102 0.736 0.691 −0.045 0.987 0.767 0.745 −0.022 0.095
6.811 6.580 −0.231 −1.173 0.019 0.132 0.081 −0.051 3.120 0.117 0.092 −0.025 0.122
9.124 8.774 −0.350 −1.275 0.297 0.539 0.294 −0.245 0.811 0.255 0.237 −0.019 0.455
8.060 7.921 −0.139 −1.101 0.025 0.364 0.241 −0.124 2.505 0.258 0.229 −0.030 0.262
7.072 7.206 0.134 1.098 0.374 0.813 0.737 −0.076 3.492 0.783 0.751 −0.032 0.069
8.196 7.948 −0.247 −1.187 0.244 0.688 0.734 0.047 1.616 0.645 0.708 0.063 0.189
6.790 6.574 −0.216 −1.161 0.118 0.198 0.246 0.048 1.748 0.217 0.246 0.029 0.065
5.141 4.975 −0.166 −1.122 0.236 0.666 0.606 −0.061 0.673 0.596 0.576 −0.019 0.344
8.399 8.191 −0.208 −1.155 0.248 0.379 0.451 0.072 1.406 0.465 0.487 0.022 0.179
8.224 8.452 0.228 1.171 0.099 0.326 0.201 −0.125 0.995 0.190 0.167 −0.023 0.360
7.725 7.461 −0.263 −1.200 0.020 0.449 0.603 0.154 3.004 0.479 0.520 0.040 0.262
8.184 8.326 0.142 1.104 0.068 0.654 0.783 0.130 3.533 0.684 0.704 0.020 0.200
Reduced Levels of GSTM1 and GSTM5 mRNA Levels in AMD NSR and RPE/Choroid
Microarray analysis comparing the AMD versus control retinas suggested a possible difference in mRNA levels of GSTM1 and GSTM5. This corresponded to hypermethylation of the promoter sequence of GSTM5 (Table 4). qPCR was performed on all NSR and RPE samples from human postmortem specimens (Fig. 1). There was significantly less GSTM1 and GSTM5 mRNA in AMD eyes versus controls (P < 0.05). 
Figure 1.
 
Graph showing relative quantification of GSTM1 and GSTM5 mRNA levels by qPCR in AMD versus controls. GSTM1 mRNA levels were decreased in AMD samples in both NSR (A) and RPE (B) versus controls, P < 0.05 by two-sample t-test. GSTM5 mRNA levels were decreased in both NSR (C) and RPE (D), P < 0.05.
Figure 1.
 
Graph showing relative quantification of GSTM1 and GSTM5 mRNA levels by qPCR in AMD versus controls. GSTM1 mRNA levels were decreased in AMD samples in both NSR (A) and RPE (B) versus controls, P < 0.05 by two-sample t-test. GSTM5 mRNA levels were decreased in both NSR (C) and RPE (D), P < 0.05.
Reduced GSTM1 and GSTM5 Protein in AMD Retinas
Western analysis of NSR was used to assess GSTM1 and GSTM5 protein levels in AMD (n = 7) versus controls (n = 7) (Fig. 2). A single band was detected with both anti-GSTM1 and anti-GSTM5 antibodies. Normalized to α-tubulin, the AMD eyes had a significant reduction in the amount of GSTM1 and GSTM5 (P < 0.05). 
Figure 2.
 
Western analysis of retinal NSR extracts from human samples. GSTM1 levels were decreased in AMD versus control (A) with median levels of 0.67 vs. 1.15 (AMD versus control), total n = 14 (7 AMD versus 7 control) (B). GSTM5 levels were decreased in AMD NSR (C) with median levels of 0.49 vs. 1.0 (AMD versus control), total n = 14 (D). Wilcoxon rank sum test was used because of the skewed distribution of data, P < 0.05.
Figure 2.
 
Western analysis of retinal NSR extracts from human samples. GSTM1 levels were decreased in AMD versus control (A) with median levels of 0.67 vs. 1.15 (AMD versus control), total n = 14 (7 AMD versus 7 control) (B). GSTM5 levels were decreased in AMD NSR (C) with median levels of 0.49 vs. 1.0 (AMD versus control), total n = 14 (D). Wilcoxon rank sum test was used because of the skewed distribution of data, P < 0.05.
IHC Localization of GSTM1 and GSTM5
To assess retinal localization of GSTM1 and GSTM5, control and AMD postmortem macular sections were immunostained. Sections were bleached before immunostaining to facilitate visualization of the immunolabel within the melanin-rich RPE (Fig. 3). In the normal and AMD retinas, GSTM1 and GSTM5 were present primarily within the apical aspect of RPE, the nerve fiber layer, the outer plexiform layer, and in the outer segments of the photoreceptors. Although IHC is semiquantitative at best, macula sections of AMD subjects had lighter immunostain in some RPE cells (Figs. 3E, 3H). Immunofluorescence detected similar GSTM1 retinal localization in the albino mouse compared with human samples (Fig. 3F). 
Figure 3.
 
Photomicrographs of retina sections immunostained with either anti-GSTM1 or anti-GSTM5 antibodies. (A) Left: AMD retina immunostained with anti-GSTM1. Right: No primary antibody. (B) Higher magnification of A. (C) Normal human macula labeled with anti-GSTM1. (D) Higher magnification. (E) AMD retina labeled with anti-GSTM1 at higher magnification. Arrow shows diminished labeling intensity in RPE cells. (F) Albino Balb/c mouse retina stained with anti-GSTM1 (red fluorescence). Nuclei stained with DAPI (blue). (G) AMD retina immunostained with anti-GSTM5. (H) Higher magnification. (I) Normal human macula labeled with anti-GSTM5. (J) Higher magnification. Scale bar: 100 μm.
Figure 3.
 
Photomicrographs of retina sections immunostained with either anti-GSTM1 or anti-GSTM5 antibodies. (A) Left: AMD retina immunostained with anti-GSTM1. Right: No primary antibody. (B) Higher magnification of A. (C) Normal human macula labeled with anti-GSTM1. (D) Higher magnification. (E) AMD retina labeled with anti-GSTM1 at higher magnification. Arrow shows diminished labeling intensity in RPE cells. (F) Albino Balb/c mouse retina stained with anti-GSTM1 (red fluorescence). Nuclei stained with DAPI (blue). (G) AMD retina immunostained with anti-GSTM5. (H) Higher magnification. (I) Normal human macula labeled with anti-GSTM5. (J) Higher magnification. Scale bar: 100 μm.
Bisulfite Pyrosequencing of GSTM1 and GSTM5 Promoter Confirm Hypermethylation of the GSTM1 Promoter
RPE GSTM1 was significantly hypermethylated in AMD versus controls (Table 5). By comparing the methylation status of the promoter to relative quantification of RPE mRNA for GSTM1 and GSTM5, a significant correlation of total GSTM1 promoter hypermethylation to decreased expression of both isoforms is evident (Table 6). NSR samples, in contrast, while showing a trend toward hypermethylation of GSTM1 promoter in AMD, did not show a significant difference in total promoter methylation compared with controls (Table 5) and there did not appear to be a significant correlation of total NSR GSTM1 promoter hypermethylation to decreased expression of either isoform, except in comparing maximum methylation percentage (Table 7). 
Table 5.
 
Comparison of Percent Methylation Data Between AMD and Controls: Comparison of Bisulfite Pyrosequencing Percent Methylation Average, Median, Maximum, and Minimum for GSTM1 and GSTM5 Promoter for NSR and RPE
Table 5.
 
Comparison of Percent Methylation Data Between AMD and Controls: Comparison of Bisulfite Pyrosequencing Percent Methylation Average, Median, Maximum, and Minimum for GSTM1 and GSTM5 Promoter for NSR and RPE
AMD Cases (n = 10) Controls (n = 11) P Value*
n Mean (SD) n Mean (SD)
GSTM1 NSR
 CPG average 5 37.7 (5.07) 9 32.9 (7.91) 0.25
 CPG median 5 35.4 (10.1) 9 28.0 (10.0) 0.21
 CPG maximum 5 68.3 (7.07) 9 59.5 (15.7) 0.26
 CPG minimum 5 8.22 (4.84) 9 10.1 (5.48) 0.53
GSTM1 RPE
 CPG average 10 22.9 (2.37) 10 16.2 (7.47) 0.04
 CPG median 10 19.2 (5.88) 10 12.6 (7.67) 0.04
 CPG maximum 10 38.6 (7.51) 10 31.1 (13.4) 0.14
 CPG minimum 10 7.83 (3.72) 10 5.59 (3.79) 0.20
GSTM5 NSR
 CPG average 5 51.7 (3.11) 9 52.5 (3.02) 0.62
 CPG median 5 53.2 (6.32) 9 54.3 (3.01) 0.72
 CPG maximum 5 86.5 (4.44) 9 87.9 (6.09) 0.66
 CPG minimum 5 16.4 (9.42) 9 18.0 (6.26) 0.70
GSTM5 RPE
 CPG average 10 22.4 (6.33) 10 25.7 (5.64) 0.24
 CPG median 10 20.9 (6.38) 10 24.4 (5.75) 0.21
 CPG maximum 10 48.8 (11.8) 10 50.8 (9.45) 0.68
 CPG minimum 10 8.81 (2.96) 10 9.88 (4.58) 0.54
Table 6.
 
The Correlation Between RPE GSTM1 Promoter Methylation and GSTM1 and GSTM5 Gene Expression: Pearson and Spearman Correlation of Percent Methylation (Individual CpG Dinucleotides, Average, Media, Maximum, and Minimum) and GSTM1 and GSTM5 Relative Gene Expression Determined by qPCR for RPE/Choroid
Table 6.
 
The Correlation Between RPE GSTM1 Promoter Methylation and GSTM1 and GSTM5 Gene Expression: Pearson and Spearman Correlation of Percent Methylation (Individual CpG Dinucleotides, Average, Media, Maximum, and Minimum) and GSTM1 and GSTM5 Relative Gene Expression Determined by qPCR for RPE/Choroid
GSTM1 Methylation Location Correlation Coefficient and P ValueWith GSTM1 Gene Expression Correlation Coefficient and P Value With GSTM5 Gene Expression
Pearson Correlation Spearman Correlation Pearson Correlation Spearman Correlation
CpG__1 −0.01669 −0.05904 −0.11474 −0.23509
0.9443 0.8047 0.64 0.3326
CpG__2 −0.47005 −0.45366 −0.34243 −0.04386
0.0365 0.0445 0.1513 0.8585
CpG__3 0.25108 0.21285 0.04744 −0.14035
0.2856 0.3676 0.8471 0.5666
CpG__4 −0.38924 −0.53911 −0.1975 −0.06842
0.0898 0.0142 0.4177 0.7808
CpG__5 −0.6437 −0.29363 −0.54658 −0.22807
0.0022 0.2089 0.0155 0.3477
CpG__6 −0.58249 −0.2245 −0.53872 −0.29825
0.007 0.3413 0.0173 0.2149
CpG__7 −0.22386 −0.07224 −0.33567 −0.56491
0.3427 0.7621 0.16 0.0117
CpG__8 −0.53213 −0.42958 −0.59881 −0.63509
0.0157 0.0587 0.0067 0.0035
CpG__9 −0.51406 −0.26256 −0.57479 −0.43333
0.0204 0.2634 0.01 0.0638
CpG_average −0.57789 −0.55464 −0.59848 −0.54386
0.0076 0.0111 0.0068 0.0161
CpG_media −0.40782 −0.47852 −0.45723 −0.58596
0.0743 0.0328 0.049 0.0084
CpG_maximum −0.5434 −0.49716 −0.44711 −0.08947
0.0133 0.0257 0.0549 0.7157
CpG_minimum −0.10095 −0.11186 −0.34811 −0.60351
0.6719 0.6387 0.1442 0.0062
Table 7.
 
The Correlation Between NSR GSTM1 Methylation and GSTM1 and GSTM5 Gene Expression: Pearson and Spearman Correlation of Percent Methylation (Individual Cpg Dinucleotides, Average, Media, Maximum, and Minimum) and GSTM1 and GSTM5 Relative Gene Expression Determined by qPCR for NSR
Table 7.
 
The Correlation Between NSR GSTM1 Methylation and GSTM1 and GSTM5 Gene Expression: Pearson and Spearman Correlation of Percent Methylation (Individual Cpg Dinucleotides, Average, Media, Maximum, and Minimum) and GSTM1 and GSTM5 Relative Gene Expression Determined by qPCR for NSR
GSTM1 Methylation Location Correlation Coefficient and P Value With GSTM1 Gene Expression Correlation Coefficient and P Value With GSTM5 Gene Expression
Pearson Correlation Spearman Correlation Pearson Correlation Spearman Correlation
CpG__1 0.08844 −0.56037 0.34053 0.13986
0.796 0.073 0.2788 0.6646
CpG__2 −0.75036 −0.73349 −0.57685 −0.66434
0.0078 0.0102 0.0496 0.0185
CpG__3 0.17865 −0.15034 0.30995 0.2662
0.5992 0.659 0.3269 0.403
CpG__4 −0.47443 −0.60046 −0.25531 −0.59895
0.1404 0.0508 0.4232 0.0396
CpG__5 −0.27526 −0.26879 −0.22223 −0.12587
0.4127 0.4242 0.4876 0.6967
CpG__6 −0.24482 0.07289 −0.21525 0.04895
0.4681 0.8313 0.5017 0.8799
CpG__7 0.38965 0.55125 0.26116 0.44755
0.2362 0.0788 0.4123 0.1446
CpG__8 0.20985 0.1139 0.20004 0.23077
0.5357 0.7388 0.5331 0.4705
CpG__9 −0.26344 −0.21868 −0.18274 −0.06993
0.4338 0.5183 0.5697 0.829
CpG_average −0.38165 −0.4328 −0.16966 −0.16783
0.2468 0.1836 0.5981 0.6021
CpG_media −0.30005 −0.48747 −0.04052 −0.00699
0.37 0.1283 0.9005 0.9828
CpG_maximum −0.5863 −0.6287 −0.49273 −0.59441
0.058 0.0383 0.1036 0.0415
CpG_minimum 0.57967 0.48747 0.48633 0.51138
0.0616 0.1283 0.1089 0.0893
There was no difference in total methylation of GSTM5 promoter in AMD versus controls for NSR and RPE samples (Table 5). Total GSTM5 promoter methylation did not correlate with expression changes of that immediate downstream isoform (data not shown). 
Discussion
The gene expression pattern was altered in RPE/choroid of AMD versus control, as determined by exon microarray and confirmed for more than 20 genes by qPCR. Changes in the percent methylation of specific cytosines within promoters of 63 genes with altered expression profiles were identified. GSTM1 and GSTM5 mRNA levels in NSR and RPE were decreased in postmortem AMD samples versus controls, and confirmed by qPCR (Fig. 1). RPE mRNA levels of each isoform were reduced to a greater extent than levels in NSR for AMD versus control. Similarly, there was a decrease of both protein isoforms in NSR extracts quantified by Western analysis (Fig. 2). 
GST mu class was previously immunolocalized to Müller cells and rod outer segments in rats. 49 We found a similar distribution of GSTM1 and GSTM5 by immunohistochemistry in human retinas within the NSR and also detected both isoforms within the RPE (Fig. 3). GSTM1 also localizes in the murine retinas in a similar manner (Fig. 3F). 
Because there was a slight age-bias toward older patients in our AMD cohort (Supplemental Table 5 ), we compared relative quantification of GSTM1 and GSTM5 mRNA levels to donor age and found no statistically significant age-dependent decline by both Pearson or Spearman correlation calculations (Supplemental Table 6 ). The demographic characteristics of the samples used for protein quantification showed no significant difference in mean age or gender (Supplemental Table 7 ). There was a Caucasian race predominance for all samples with only one sample being non-Caucasian, consistent with the higher prevalence of AMD in Caucasians. 
DNA hypermethylation of the GSTM1 promoter in RPE/choroid appears to coincide with diminished levels of both GSTM1 and GSTM5 transcripts. In addition to modulating expression of the adjoining gene, methylation of the GSTM1 promoter may influence expression of the downstream gene, GSTM5. The GSTM1 promoter may act as a CpG island shore for the downstream GSTM5. 33 Usually, these epigenetic changes occur without comitant genetic lesions (i.e., deletions or nulls). 35 If somatic mutation occurs at high frequency (i.e., adenomatosis polyposis coli in colorectal tumorigenesis), the promoter usually maintains a hypomethylated state. Additionally, epigenetic silencing is often an “early event” in the natural history of disease and hypermethylation of silent loci is observed before full-blown disease progression. 5055 Epigenetic changes in plausible pathogenetic loci, therefore, are likely antecedent to disease “onset.” 
There are four known allelic variants of GSTM1, one of which is a deletion resulting in loss of function, which has been reported in up to 50% of the human population. 56,57 Information regarding allelic variants of the GSTMs is available in the ophthalmic literature. 5863 In our sample set, we did not find any homozygous deletions for GSTM1 (data not shown). 
To increase the validity and minimize Poisson errors from our microarray analyses, we used two distinct sample cohorts. There was a modest correlation among microarray candidate genes listed in Tables 1 and 3 between HCS and NDRI sample sources. It is likely that a higher degree of statistical significance could have been obtained with a larger sample size and a larger cohort of histologically confirmed and staged AMD cases. The lack of histological classification in NDRI samples is a potential source of AMD misclassification and a limitation of the study. 
The comparison of the expression exon microarray to the bisulfite DNA sequencing microarray data revealed 63 genes where methylation changes in promoter sequence correlated with changes in mRNA. Apolipoprotein B mRNA editing enzyme, APOBEC2, a member of the cytidine deaminase family of DNA/RNA editing enzymes, showed elevated transcription in RPE of AMD versus controls, which corresponded to hypermethylation. Increased methylation of promoter sequences are noted to cause elevated expression in certain “reduced-repressor” of promoter examples within the literature. 33 Hyperapolipoproteinemia B has been implicated in the pathogenesis of AMD in several human investigations and a recent description of an AMD-like transgenic murine model that hyperexpresses apoB10064–67; however, any effect of this mRNA editing enzyme on ApoB mRNA has not been elucidated. 68,69 APOBEC1, a cytidine deaminase family member, has been shown to “edit” APOB mRNA but the physiologic role of APOBEC2 is still unknown. 70 Interestingly, APOBEC2 is induced by TGF-β signaling in a mammalian myoblastic cell line.3,71 TGFβ has been shown to upregulate transcriptional factors associated with epithelial-mesenchymal transition in RPE cells in human choroidal neovascularization in AMD. 72  
We examined the relative quantification of mRNAs encoding several proteins involved in epigenetic modulation to determine if the changes in DNA methylation in AMD were attributable to a more generalized disruption of epigenetic mechanisms. mRNAs of MBD1-3 proteins, methyl CpG binding protein 2, an MBD gene family member, tRNA aspartic acid methyltransferase, histone acetyltransferase, HDAC, and histamine N-methyltransferase were not significantly different in AMD versus control (Table 2, bottom). This suggests that the hypermethylation of the GSTM1 promoter in AMD is not a result of a global dysfunction or loss of function of epigenetic control mechanisms within the retina. 
Retinal GSTs are likely to have a significant role in protection against oxidative insult.36,73–80 Pharmacologic augmentation of the body's antioxidant defenses, therefore, may prove to be protective in AMD pathogenesis, especially in people with low GSTM1 and GSTM5 expression. 
In conclusion, we report here the first evidence of a potential decrease of specific GSTM isoenzymes in human postmortem ophthalmic specimens in relation to a retinal disease, AMD. The function and location of these isoenzymes suggest that this decline in antioxidant function could lead to increased oxidative stress in AMD eyes and contribute to AMD pathogenesis. The hypermethylation of the GSTM1 promoter coincides with diminished expression of two immediate downstream genes, GSTM1 and GSTM5, suggesting that these methylation events could contribute to AMD risk or pathogenesis. 
Supplementary Materials
References
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Footnotes
 Supported by unrestricted core grant K12 EY015398 from Research to Prevent Blindness.
Footnotes
 Disclosure: A. Hunter, None; P.A. Spechler, None; A. Cwanger, None; Y. Song, None; Z. Zhang, None; G.-S. Ying, None; A.K. Hunter, None; E. deZoeten, None; J.L. Dunaief, None
Figure 1.
 
Graph showing relative quantification of GSTM1 and GSTM5 mRNA levels by qPCR in AMD versus controls. GSTM1 mRNA levels were decreased in AMD samples in both NSR (A) and RPE (B) versus controls, P < 0.05 by two-sample t-test. GSTM5 mRNA levels were decreased in both NSR (C) and RPE (D), P < 0.05.
Figure 1.
 
Graph showing relative quantification of GSTM1 and GSTM5 mRNA levels by qPCR in AMD versus controls. GSTM1 mRNA levels were decreased in AMD samples in both NSR (A) and RPE (B) versus controls, P < 0.05 by two-sample t-test. GSTM5 mRNA levels were decreased in both NSR (C) and RPE (D), P < 0.05.
Figure 2.
 
Western analysis of retinal NSR extracts from human samples. GSTM1 levels were decreased in AMD versus control (A) with median levels of 0.67 vs. 1.15 (AMD versus control), total n = 14 (7 AMD versus 7 control) (B). GSTM5 levels were decreased in AMD NSR (C) with median levels of 0.49 vs. 1.0 (AMD versus control), total n = 14 (D). Wilcoxon rank sum test was used because of the skewed distribution of data, P < 0.05.
Figure 2.
 
Western analysis of retinal NSR extracts from human samples. GSTM1 levels were decreased in AMD versus control (A) with median levels of 0.67 vs. 1.15 (AMD versus control), total n = 14 (7 AMD versus 7 control) (B). GSTM5 levels were decreased in AMD NSR (C) with median levels of 0.49 vs. 1.0 (AMD versus control), total n = 14 (D). Wilcoxon rank sum test was used because of the skewed distribution of data, P < 0.05.
Figure 3.
 
Photomicrographs of retina sections immunostained with either anti-GSTM1 or anti-GSTM5 antibodies. (A) Left: AMD retina immunostained with anti-GSTM1. Right: No primary antibody. (B) Higher magnification of A. (C) Normal human macula labeled with anti-GSTM1. (D) Higher magnification. (E) AMD retina labeled with anti-GSTM1 at higher magnification. Arrow shows diminished labeling intensity in RPE cells. (F) Albino Balb/c mouse retina stained with anti-GSTM1 (red fluorescence). Nuclei stained with DAPI (blue). (G) AMD retina immunostained with anti-GSTM5. (H) Higher magnification. (I) Normal human macula labeled with anti-GSTM5. (J) Higher magnification. Scale bar: 100 μm.
Figure 3.
 
Photomicrographs of retina sections immunostained with either anti-GSTM1 or anti-GSTM5 antibodies. (A) Left: AMD retina immunostained with anti-GSTM1. Right: No primary antibody. (B) Higher magnification of A. (C) Normal human macula labeled with anti-GSTM1. (D) Higher magnification. (E) AMD retina labeled with anti-GSTM1 at higher magnification. Arrow shows diminished labeling intensity in RPE cells. (F) Albino Balb/c mouse retina stained with anti-GSTM1 (red fluorescence). Nuclei stained with DAPI (blue). (G) AMD retina immunostained with anti-GSTM5. (H) Higher magnification. (I) Normal human macula labeled with anti-GSTM5. (J) Higher magnification. Scale bar: 100 μm.
Table 1.
 
A Total of 46 mRNAs from the Affymetrix Exon Microarray That Showed a Significant Correlation (P < 0.1) Between HCS and NDRI Samples and Also Had an FC of More Than 25% Between AMD and Control
Table 1.
 
A Total of 46 mRNAs from the Affymetrix Exon Microarray That Showed a Significant Correlation (P < 0.1) Between HCS and NDRI Samples and Also Had an FC of More Than 25% Between AMD and Control
Gene_ID Chr Symbol HCS NDRI Name
Control AMD AMD-Control FC NSD Control AMD AMD-Control FC P Value
10,930 6 APOBEC2 4.749 6.450 1.701 3.250 3.154 6.245 6.665 0.420 1.338 0.028 Apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 2
6228 5 RPS23 8.039 9.413 1.374 2.591 9.466 9.490 9.872 0.382 1.303 0.011 Ribosomal protein S23
4160 18 MC4R 3.121 4.339 1.217 2.325 3.855 4.673 5.494 0.821 1.767 0.045 Melanocortin 4 receptor
100,132,476 17 KRTAP4-7 7.174 8.324 1.150 2.219 3.846 7.532 7.886 0.353 1.278 0.099 Keratin-associated protein 4–7
100,129,026 NA 6.507 7.454 0.947 1.928 5.516 7.487 7.880 0.393 1.313 0.010 NA
731,157 5 LOC731157 4.550 5.289 0.740 1.670 3.731 5.198 5.650 0.452 1.368 0.025 Similar to HSPC101
730,032 2 LOC730032 5.110 5.663 0.553 1.467 3.468 5.143 5.629 0.486 1.400 0.012 Similar to RIKEN cDNA C230030N03
729,851 NA 3.784 4.330 0.546 1.460 3.521 4.207 4.731 0.524 1.438 0.036 NA
79,870 8 BAALC 6.009 6.509 0.499 1.414 6.270 6.931 7.365 0.435 1.352 0.079 Brain and acute leukemia, cytoplasmic
284,120 NA 5.413 5.768 0.355 1.279 3.973 5.973 6.507 0.534 1.448 0.067 NA
4482 8 MSRA 8.481 8.134 −0.347 −1.272 5.486 7.965 7.635 −0.330 −1.257 0.019 Methionine sulfoxide reductase A
340,371 8 NRBP2 9.481 9.098 −0.382 −1.303 3.880 8.832 8.389 −0.444 −1.360 0.022 Nuclear receptor binding protein 2
2844 9 GPR21 7.854 7.422 −0.432 −1.349 4.245 7.618 7.091 −0.527 −1.441 0.042 G protein–coupled receptor 21
100,131,601 16 LOC100131601 7.031 6.587 −0.444 −1.360 3.136 6.541 6.121 −0.420 −1.338 0.031 Similar to hCG1980470
29,065 8 DDEF1IT1 6.723 6.268 −0.455 −1.371 3.061 6.207 5.819 −0.388 −1.308 0.006 DDEF1 intronic transcript 1 (nonprotein coding)
100,131,231 9 LOC100131231 8.344 7.871 −0.473 −1.388 4.176 8.077 7.741 −0.336 −1.262 0.057 Hypothetical protein LOC100131231
58,496 6 LY6G5B 8.878 8.394 −0.484 −1.398 7.390 8.683 8.331 −0.352 −1.276 0.055 Lymphocyte antigen 6 complex, locus G5B
729,793 NA 7.227 6.714 −0.513 −1.427 4.324 6.689 6.271 −0.418 −1.336 0.047 NA
55,556 18 ENOSF1 8.999 8.484 −0.515 −1.429 3.166 8.554 8.113 −0.441 −1.357 0.010 Enolase superfamily member 1
645,771 13 RP11-385E5.2 3.482 2.937 −0.546 −1.460 3.453 3.571 3.171 −0.400 −1.320 0.015 Poly (ADP-ribose) polymerase family, member 4 pseudogene
642,367 NA 6.605 6.057 −0.548 −1.462 8.032 6.085 5.746 −0.339 −1.265 0.022 NA
100,129,743 2 RPL7P14 6.644 6.086 −0.558 −1.472 14.077 5.712 4.947 −0.766 −1.700 0.007 Ribosomal protein L7 pseudogene 14
6819 2 SULT1C2 8.329 7.725 −0.604 −1.520 5.642 7.492 6.955 −0.538 −1.452 0.036 Sulfotransferase family, cytosolic, 1C, member 2
728,655 6 HULC 5.293 4.674 −0.619 −1.536 3.010 5.123 4.677 −0.446 −1.362 0.008 Highly upregulated in liver cancer (nonprotein coding)
388,815 21 C21orf34 8.434 7.807 −0.627 −1.545 4.506 7.900 7.342 −0.558 −1.472 0.055 Chromosome 21 open reading frame 34
646,272 4 LOC646272 4.966 4.336 −0.630 −1.547 3.221 4.613 4.283 −0.329 −1.257 0.019 Similar to ubiquinol-cytochrome c reductase, complex III subunit VII
8038 10 ADAM12 7.512 6.861 −0.652 −1.571 5.172 6.763 6.400 −0.363 −1.286 0.091 ADAM metallopeptidase domain 12
100,130,696 NA 4.040 3.385 −0.655 −1.575 4.061 3.907 3.494 −0.413 −1.331 0.043 NA
727,819 NA 4.729 4.069 −0.660 −1.580 3.915 4.228 3.722 −0.506 −1.420 0.017 NA
51,134 12 CCDC41 5.660 4.955 −0.705 −1.630 5.269 5.475 5.143 −0.332 −1.259 0.019 Coiled-coil domain containing 41
10,693 17 CCT6B 4.769 4.037 −0.731 −1.660 5.336 4.632 4.244 −0.388 −1.308 0.046 Chaperonin containing TCP1, subunit 6B (zeta 2)
51,474 12 LIMA1 9.444 8.673 −0.771 −1.706 3.632 9.245 8.895 −0.350 −1.275 0.070 LIM domain and actin binding 1
100,131,993 13 LOC100131993 7.989 7.170 −0.819 −1.764 3.880 6.957 6.556 −0.401 −1.321 0.077 Similar to hCG2020760
2949 1 GSTM5 9.389 8.444 −0.946 −1.926 2.102 9.117 8.622 −0.495 −1.409 0.037 Glutathione S-transferase mu 5
28,516 14 TRDV3 5.083 4.076 −1.007 −2.010 4.140 4.386 3.926 −0.460 −1.376 0.069 T-cell receptor delta variable 3
5729 14 PTGDR 7.864 6.839 −1.024 −2.034 3.878 7.302 6.727 −0.575 −1.490 0.064 Prostaglandin D2 receptor (DP)
5935 X RBM3 9.893 8.865 −1.028 −2.040 6.851 9.693 8.963 −0.730 −1.659 0.100 RNA binding motif (RNP1, RRM) protein 3
10,561 1 IFI44 7.002 5.951 −1.051 −2.072 9.181 6.777 6.083 −0.694 −1.618 0.093 Interferon-induced protein 44
100,132,099 13 UNQ1829 6.932 5.777 −1.155 −2.227 4.265 6.116 5.494 −0.622 −1.539 0.038 FRSS1829
100,129,349 1 IFI44L 7.324 6.026 −1.299 −2.460 5.245 7.029 6.375 −0.654 −1.574 0.070 NA
440,482 18 ANKRD20A5 6.053 4.751 −1.302 −2.466 3.087 5.492 4.416 −1.076 −2.108 0.040 Ankyrin repeat domain 20 family, member A5
391,267 21 C21orf81 7.626 6.062 −1.563 −2.956 4.678 6.672 5.774 −0.898 −1.864 0.004 Ankyrin repeat domain 20 family, member A3 pseudogene
390,072 11 OR52N4 6.241 4.655 −1.586 −3.002 4.645 6.035 5.351 −0.685 −1.608 0.051 Olfactory receptor, family 52, subfamily N, member 4
284,232 13 LOC284232 5.678 4.034 −1.644 −3.125 5.628 4.465 3.538 −0.926 −1.901 0.001 Ankyrin repeat domain 20 family, member A2 pseudogene
80,867 6 HCG2P7 6.870 4.550 −2.320 −4.995 9.852 5.120 4.420 −0.700 −1.625 0.045 HLA complex group 2 pseudogene 7
2944 1 GSTM1 10.748 6.768 −3.980 −15.780 3.376 8.688 5.950 −2.738 −6.672 0.002 Glutathione S-transferase mu 1
Table 2.
 
PCR Analysis (AMD/Control) of Individual RPE/Choroid mRNAs That Were Significantly Increased or Decreased in the Exon Microarray
Table 2.
 
PCR Analysis (AMD/Control) of Individual RPE/Choroid mRNAs That Were Significantly Increased or Decreased in the Exon Microarray
Gene ID FC (AMD:Control RPE) P Value
FC confirmation by qPCR of mRNAs originally quantified by the exon microarray
 GSTM1 0.02 < 0.05
 GPR21 0.03 < 0.05
 LOC10013 0.05 < 0.05
 c21orf34 0.33 < 0.05
 GSTM5 0.34 < 0.05
 RPS23 0.41 NS
 PTGDR 0.46 < 0.05
 IF144 0.51 < 0.05
 CCT6B 0.63 < 0.05
 LY6G5B 0.63 < 0.05
 ADAM12 0.65 < 0.05
 ARSG 0.65 < 0.05
 CCDC41 0.73 < 0.05
 NRBP2 0.76 < 0.05
 ANGPTL2 0.78 NS
 MSRA 0.79 NS
 ENOSF1 0.83 NS
 SULT1C2 0.86 NS
 LIMA1 0.89 NS
 BAALC 1.62 < 0.05
 MC4R 2.22 < 0.05
 APOBEC2 2.97 < 0.05
 ALOX15B 3.36 < 0.05
 AANAT 3.91 < 0.05
qPCR quantification (AMD versus controls) of mRNAs involved in epigenetic modulation
 MBD1 0.98 NS
 MBD2 0.92 NS
 MBD3 1.14 NS
 MeCP2 1.02 NS
 TRDMT1 0.61 NS
 HAT1 0.83 NS
 HDAC9 0.69 NS
 HNMT 0.53 NS
Table 3.
 
67 CpG Sites Determined by Bisulfite Microarray Sequencing of RPE/Choroid That Showed a Significant Correlation (P < 0.1) Between HCS and NDRI Samples and Also Had a 5% Methylation Difference Between AMD and Control
Table 3.
 
67 CpG Sites Determined by Bisulfite Microarray Sequencing of RPE/Choroid That Showed a Significant Correlation (P < 0.1) Between HCS and NDRI Samples and Also Had a 5% Methylation Difference Between AMD and Control
Gene ID Chr Symbol CpG Loc TSS Coordinate Distance to TSS HCS NDRI
Control AMD AMD-Control NSD Control AMD AMD-Control P Value
117,194 11 MRGPRX2 cg22051636 19,038,166 19,038,804 638 0.271 0.804 0.534 19.988 0.691 0.802 0.112 0.073
3716 1 JAK1 cg15997411 65,124,972 65,124,574 −398 0.711 0.879 0.168 10.090 0.857 0.878 0.020 0.050
84,221 21 C21orf56 cg07747299 46,428,480 46,428,729 249 0.516 0.143 −0.373 8.264 0.218 0.128 −0.089 0.025
84,699 19 CREB3L3 cg23777956 4,104,671 4,104,629 42 0.487 0.267 −0.220 6.970 0.346 0.300 −0.046 0.054
9724 13 UTP14C cg24167928 51,496,903 51,496,828 75 0.856 0.918 0.062 6.303 0.871 0.894 0.023 0.041
51,179 1 HAO2 cg03762535 119,713,003 119,712,925 78 0.564 0.637 0.073 6.194 0.632 0.669 0.038 0.090
132,724 4 TMPRSS11B cg19510180 68,794,175 68,794,004 −171 0.661 0.736 0.074 6.161 0.681 0.709 0.027 0.076
4481 8 MSR1 cg01668126 16,095,111 16,094,595 −516 0.867 0.802 −0.065 6.126 0.859 0.836 −0.022 0.028
284,114 17 TMEM102 cg14782678 7,280,445 7,279,486 959 0.511 0.455 −0.056 6.011 0.488 0.469 −0.019 0.049
134,864 6 TAAR1 cg15582891 133,008,721 133,008,835 114 0.573 0.731 0.158 6.005 0.726 0.754 0.028 0.030
50,514 9 DEC1 cg26981881 116,943,245 116,943,918 −673 0.761 0.828 0.067 5.359 0.807 0.836 0.029 0.020
29,974 10 ACF cg03817621 52,315,405 52,315,441 36 0.727 0.844 0.117 4.866 0.789 0.815 0.025 0.061
55,856 6 THEM2 cg16381688 24,773,926 24,775,254 −1328 0.680 0.753 0.073 4.286 0.713 0.738 0.025 0.065
10,507 9 SEMA4D cg22496652 91,284,445 91,284,431 −14 0.707 0.625 −0.082 4.263 0.672 0.648 −0.024 0.070
6374 4 CXCL5 cg04559909 75,083,589 75,083,280 −309 0.438 0.581 0.143 4.173 0.554 0.593 0.039 0.092
2532 1 DARC cg23507131 157,440,780 157,441,134 −354 0.698 0.622 −0.076 4.088 0.677 0.652 −0.025 0.048
5478 7 PPIA cg17269548 44,802,815 44,802,777 38 0.179 0.255 0.076 3.998 0.163 0.207 0.045 0.011
1232 3 CCR3 cg11126313 46,259,266 46,258,692 574 0.785 0.846 0.061 3.991 0.800 0.845 0.044 0.003
83,876 18 MRO cg27318546 46,599,904 46,600,366 462 0.193 0.128 −0.064 3.829 0.191 0.165 −0.026 0.069
221,823 7 PRPS1L1 cg00911873 18,033,988 18,034,011 23 0.812 0.863 0.051 3.773 0.786 0.842 0.055 0.010
6613 17 SUMO2 cg19776090 70,690,552 70,690,693 141 0.378 0.430 0.053 3.752 0.352 0.376 0.025 0.039
337,977 21 KRTAP21-1 cg22373097 31,050,931 31,049,567 −1364 0.575 0.749 0.174 3.568 0.715 0.787 0.072 0.042
3694 2 ITGB6 cg21105318 160,764,766 160,764,836 70 0.825 0.895 0.070 3.547 0.857 0.881 0.024 0.049
84,218 17 TBC1D3 cg14532417 33,601,782 33,602,396 614 0.813 0.737 −0.076 3.492 0.783 0.751 −0.032 0.069
63,895 18 FAM38B cg21165219 10,688,044 10,687,814 −230 0.744 0.800 0.055 3.417 0.794 0.821 0.026 0.061
4719 2 NDUFS1 cg06868758 206,733,636 206,732,432 −1204 0.551 0.470 −0.081 3.409 0.456 0.434 −0.022 0.054
54,103 7 LOC54103 cg26594488 76,873,584 76,873,361 −223 0.789 0.858 0.069 3.255 0.797 0.839 0.042 0.023
2044 4 EPHA5 cg13701273 66,218,375 66,218,104 −271 0.136 0.191 0.055 3.120 0.146 0.195 0.049 0.057
260,436 4 C4orf7 cg25600236 71,125,801 71,126,404 −603 0.650 0.750 0.099 3.103 0.746 0.777 0.030 0.061
1184 X CLCN5 cg20062122 49,720,482 49,720,896 −414 0.842 0.892 0.050 3.063 0.855 0.880 0.025 0.081
390,212 11 GPR152 cg00587613 66,976,799 66,976,776 −23 0.877 0.827 −0.050 3.051 0.857 0.837 −0.020 0.069
79,861 10 TUBAL3 cg07803864 5,436,998 5,436,795 −203 0.748 0.805 0.057 3.039 0.767 0.794 0.028 0.100
10,148 19 EBI3 cg16592658 4,180,887 4,180,540 347 0.748 0.661 −0.087 3.018 0.745 0.712 −0.033 0.032
7531 17 YWHAE cg25299176 1,250,091 1,250,267 176 0.115 0.210 0.096 3.017 0.126 0.176 0.049 0.079
126,433 19 FBXO27 cg11402505 44,215,276 44,215,038 −238 0.183 0.124 −0.059 3.000 0.155 0.130 −0.025 0.033
1041 6 CDSN cg24735489 31,196,331 31,196,202 −129 0.729 0.638 −0.091 2.933 0.716 0.692 −0.024 0.038
351 21 APP cg00542846 26,465,416 26,465,003 −413 0.178 0.234 0.056 2.891 0.210 0.262 0.052 0.059
84,221 21 C21orf56 cg10296238 46,429,602 46,428,729 −873 0.515 0.298 −0.217 2.885 0.385 0.265 −0.120 0.053
3784 11 KCNQ1 cg16465939 2,510,986 2,439,259 71,727 0.149 0.200 0.051 2.847 0.209 0.228 0.019 0.056
339,500 1 ZNF678 cg26683023 225,817,515 225,817,867 −352 0.740 0.794 0.054 2.815 0.757 0.797 0.040 0.009
166,647 4 GPR125 cg26631477 22,126,293 22,126,770 477 0.173 0.245 0.072 2.771 0.168 0.197 0.029 0.054
196,472 12 FAM71C cg04282622 98,565,053 98,565,662 −609 0.742 0.801 0.059 2.726 0.790 0.823 0.033 0.042
81,493 1 SYNC1 cg05342835 32,933,378 32,933,460 82 0.585 0.452 −0.133 2.668 0.529 0.478 −0.050 0.033
8061 11 FOSL1 cg18818531 65,424,853 65,424,573 −280 0.697 0.595 −0.101 2.649 0.650 0.611 −0.039 0.065
388,818 21 KRTAP26-1 cg18822544 30,614,336 30,614,478 142 0.760 0.674 −0.086 2.611 0.727 0.697 −0.030 0.057
8369 6 HIST1H4G cg23540745 26,355,112 26,355,184 72 0.648 0.717 0.068 2.592 0.647 0.680 0.033 0.050
114,035 21 C21orf81 cg14384940 14,274,661 14,274,636 −25 0.339 0.459 0.121 2.439 0.399 0.497 0.098 0.021
120,065 11 OR5P2 cg13410437 7,774,741 7,775,065 324 0.828 0.886 0.058 2.412 0.882 0.907 0.025 0.024
127,943 1 FCRLM2 cg27495845 159,959,712 159,959,081 631 0.502 0.573 0.072 2.404 0.572 0.617 0.045 0.026
81,793 4 TLR10 cg23855121 38,461,333 38,460,984 −349 0.629 0.684 0.055 2.376 0.663 0.690 0.027 0.072
4848 12 CNOT2 cg10464585 68,922,758 68,923,489 −731 0.218 0.155 −0.064 2.329 0.202 0.161 −0.041 0.100
148,646 1 C1orf188 cg15731815 6,191,847 6,191,507 340 0.170 0.275 0.105 2.313 0.218 0.285 0.068 0.078
284,424 19 C19orf30 cg03996793 4,720,537 4,720,152 385 0.157 0.245 0.088 2.284 0.210 0.252 0.042 0.072
3150 21 HMGN1 cg13791713 39,642,786 39,642,917 131 0.264 0.317 0.053 2.279 0.265 0.291 0.026 0.067
23,524 16 SRRM2 cg06736444 2,741,794 2,742,655 −861 0.317 0.481 0.164 2.262 0.362 0.399 0.037 0.090
3827 3 KNG1 cg12454167 187,917,754 187,917,814 −60 0.492 0.331 −0.162 2.235 0.352 0.305 −0.047 0.071
3624 7 INHBA cg16415646 41,709,526 41,709,231 −295 0.806 0.865 0.059 2.235 0.819 0.851 0.032 0.046
163,589 1 TDRD5 cg09656934 177,828,123 177,827,648 475 0.214 0.276 0.062 2.189 0.250 0.282 0.032 0.069
27,004 14 TCL6 cg05023540 95,186,723 95,187,268 −545 0.735 0.675 −0.060 2.181 0.734 0.702 −0.032 0.063
3212 17 HOXB2 cg09313705 43,977,490 43,977,391 −99 0.275 0.370 0.095 2.151 0.314 0.344 0.030 0.092
64,174 16 DPEP2 cg04774694 66,590,771 66,590,857 86 0.757 0.696 −0.061 2.124 0.728 0.708 −0.020 0.073
5369 5 PMCHL1 cg12530080 22,177,396 22,178,218 −822 0.683 0.741 0.057 2.119 0.725 0.750 0.024 0.066
9541 2 CIR cg14138171 174,969,892 174,968,689 −1203 0.719 0.781 0.062 2.092 0.676 0.753 0.077 0.017
22,901 17 ARSG cg15308737 63,814,923 63,815,191 −268 0.820 0.879 0.059 2.090 0.817 0.853 0.036 0.055
140,685 20 BTBD4 cg21291985 61,907,479 61,907,300 −179 0.592 0.660 0.068 2.065 0.582 0.616 0.033 0.093
359 12 AQP2 cg12650635 48,630,730 48,630,796 −66 0.804 0.737 −0.066 2.054 0.796 0.773 −0.023 0.046
10,974 10 C10orf116 cg12261786 88,717,810 88,718,168 −358 0.426 0.377 −0.050 2.033 0.404 0.377 −0.027 0.006
2949 1 GSTM5 cg04987894 110,056,139 110,056,388 −249 0.1119 0.1685 0.0566 0.5664 0.1496 0.2330 0.0834 0.0335
Table 4.
 
A Total of 63 Genes With Exon Microarray Absolute Expression Change (FC) >1.25 in Both HCS and NDRI, and Bisulfite Microarray Sequencing Methylation Difference >2%
Table 4.
 
A Total of 63 Genes With Exon Microarray Absolute Expression Change (FC) >1.25 in Both HCS and NDRI, and Bisulfite Microarray Sequencing Methylation Difference >2%
Gene ID Chr Symbol CpG CpG Location TSS Coordinate Distance to TSS Expression
HCS
Control AMD AMD-Control FC NSD
15 17 AANAT cg09382492 71,975,276 71,975,246 30 6.065 6.380 0.315 1.244 6.991
8038 10 ADAM12 cg13488201 128,067,313 128,067,055 −258 7.512 6.861 −0.652 −1.571 5.172
247 17 ALOX15B cg15799267 7,883,131 7,883,127 4 6.418 6.932 0.514 1.428 0.641
23,452 9 ANGPTL2 cg11213150 128,924,278 128,924,865 587 7.586 7.175 −0.412 −1.330 9.087
314 17 AOC2 cg19317715 38,250,104 38,250,135 −31 5.799 6.849 1.049 2.070 0.833
10,930 6 APOBEC2 cg22375610 41,129,139 41,128,991 148 4.749 6.450 1.701 3.250 3.154
22,901 17 ARSG cg15308737 63,814,923 63,815,191 −268 7.865 7.792 −0.072 −1.051 0.655
284,424 19 C19orf30 cg03996793 4,720,537 4,720,152 385 5.757 6.016 0.259 1.197 0.898
10,842 7 C7orf16 cg23216015 31,693,179 31,693,372 −193 5.561 5.717 0.156 1.115 1.139
389,799 9 C9orf171 cg25344672 134,275,028 134,275,432 −404 5.898 6.162 0.264 1.200 1.342
27,091 17 CACNG5 cg06226384 62,303,813 62,303,913 −100 5.926 6.857 0.931 1.906 4.184
283,316 12 CD163L1 cg13986618 7,487,248 7,488,015 767 6.769 6.400 −0.369 −1.292 1.348
1184 X CLCN5 cg20062122 49,720,482 49,720,896 −414 7.999 7.677 −0.322 −1.250 0.638
119,587 10 CPXM2 cg09619146 125,641,024 125,641,490 466 9.596 9.303 −0.293 −1.225 0.628
1400 4 CRMP1 cg03544320 5,945,592 5,945,686 94 6.865 7.163 0.298 1.230 1.126
8451 13 CUL4A cg16155588 112,909,934 112,911,087 −1153 7.789 7.510 −0.280 −1.214 5.573
54,849 16 DEF8 cg25193494 88,543,505 88,542,652 853 8.086 8.420 0.333 1.260 1.425
126,433 19 FBXO27 cg11402505 44,215,276 44,215,038 −238 7.318 7.882 0.564 1.478 2.439
26,157 7 GIMAP2 cg20663831 150,014,087 150,013,727 360 5.760 5.930 0.169 1.125 1.459
51,659 16 GINS2 cg19890739 84,281,040 84,280,081 −959 5.896 6.010 0.114 1.082 0.607
55,105 1 GPATCH2 cg01727899 215,872,498 215,871,032 −1466 7.782 7.446 −0.336 −1.262 4.095
390,212 11 GPR152 cg00587613 66,976,799 66,976,776 −23 6.655 6.966 0.311 1.241 2.311
9402 22 GRAP2 cg03840259 38,626,982 38,627,032 −50 6.862 6.208 −0.654 −1.574 3.604
2949 1 GSTM5 cg04987894 110,056,139 110,056,388 −249 9.389 8.444 −0.946 −1.926 2.102
3149 X HMGB3 cg05935584 149,902,481 149,902,421 60 6.304 5.785 −0.519 −1.433 1.479
3624 7 INHBA cg16415646 41,709,526 41,709,231 −295 6.375 6.790 0.416 1.334 5.121
3664 1 IRF6 cg23283495 208,046,402 208,046,102 −300 5.971 6.498 0.527 1.441 2.399
55,600 1 ITLN1 cg08356693 159,121,824 159,121,584 −240 3.566 3.718 0.153 1.112 1.386
199,834 1 LCE4A cg17542385 150,948,603 150,948,176 427 5.804 6.322 0.518 1.432 3.224
84,856 10 LOC84856 cg00042156 42,290,848 42,290,967 −119 7.633 7.828 0.195 1.145 1.205
147,172 17 LRRC37B2 cg06488505 25,958,201 25,958,802 −601 6.213 6.374 0.161 1.118 1.452
2872 19 MKNK2 cg21030400 2,003,564 2,002,233 −1331 9.279 9.592 0.313 1.242 1.718
23,209 22 MLC1 cg05861567 48,865,813 48,866,041 228 6.762 7.026 0.264 1.201 1.667
4481 8 MSR1 cg01668126 16,095,111 16,094,595 −516 6.023 6.753 0.730 1.659 1.180
4481 8 MSR1 cg16303562 16,094,704 16,094,595 −109 6.023 6.753 0.730 1.659 1.180
4641 17 MYO1C cg00597076 1,342,630 1,342,745 115 9.737 9.593 −0.144 −1.105 1.087
55,264 21 NA cg13033054 32,870,432 32,870,062 −370 6.818 7.002 0.184 1.136 2.240
55,849 X NA cg19963797 110,811,123 110,811,069 54 4.242 4.080 −0.161 −1.118 1.175
4837 11 NNMT cg14209518 113,671,846 113,671,745 101 7.739 8.395 0.655 1.575 1.085
26,532 19 OR10H3 cg25843439 15,713,574 15,713,203 371 5.922 5.218 −0.705 −1.630 2.782
5016 1 OVGP1 cg22997415 111,772,543 111,771,922 −621 6.261 6.016 −0.245 −1.185 2.707
9796 8 PHYHIP cg05947740 22,145,723 22,145,549 −174 6.382 6.892 0.510 1.424 1.475
9271 12 PIWIL1 cg13861644 129,388,239 129,388,567 −328 5.519 5.138 −0.381 −1.302 2.035
5368 8 PNOC cg03642518 28,230,922 28,230,568 354 6.377 6.577 0.200 1.149 1.945
5446 7 PON3 cg24750391 94,864,147 94,863,598 −549 7.154 6.277 −0.877 −1.836 1.329
5478 7 PPIA cg17269548 44,802,815 44,802,777 38 6.990 6.808 −0.181 −1.134 0.996
5522 4 PPP2R2C cg07867360 6,526,057 6,524,911 −1146 6.081 6.476 0.396 1.315 1.343
5935 X RBM3 cg12251508 48,317,941 48,317,780 161 9.893 8.865 −1.028 −2.040 6.851
166,863 4 RBM46 cg22496683 155,922,060 155,921,950 110 5.157 4.750 −0.407 −1.326 1.006
27,316 X RBMX cg14642832 135,790,803 135,790,605 −198 7.708 7.907 0.199 1.148 1.090
55,511 X SAGE1 cg19856594 134,803,587 134,803,451 136 3.323 3.438 0.115 1.083 3.568
65,012 12 SLC26A10 cg12883767 56,299,376 56,299,960 −584 6.590 6.338 −0.251 −1.190 0.866
6817 16 SULT1A1 cg18530748 28,542,345 28,542,367 22 7.438 7.196 −0.242 −1.183 2.310
6855 X SYP cg10818284 48,943,549 48,943,605 56 7.245 7.044 −0.201 −1.150 1.093
6872 X TAF1 cg23986186 70,502,270 70,502,839 −569 8.135 8.003 −0.132 −1.096 6.151
84,218 17 TBC1D3F cg14532417 33,601,782 33,602,396 614 3.271 6.520 3.248 9.503 8.660
79,875 15 THSD4 cg04616566 69,807,614 69,807,942 −328 8.330 7.995 −0.335 −1.262 1.852
11,011 17 TLK2 cg23181434 57,909,890 57,910,136 −246 6.464 6.142 −0.323 −1.251 0.994
6399 X TRAPPC2 cg24352688 13,661,648 13,662,648 1000 4.961 4.689 −0.272 −1.208 1.076
10,346 11 TRIM22 cg12461141 5,667,230 5,667,664 −434 8.773 8.213 −0.560 −1.474 2.868
10,009 X ZBTB33 cg13128531 119,268,412 119,268,635 −223 7.937 8.092 0.154 1.113 0.965
64,429 10 ZDHHC6 cg17872476 114,195,644 114,196,662 1018 7.956 7.574 −0.383 −1.304 2.066
7542 11 ZFPL1 cg19507591 64,606,853 64,608,270 −1417 8.253 8.387 0.134 1.097 1.316
Table 4.
 
Extended
Table 4.
 
Extended
Expression Methylation
NDRI HCS NDRI
Control AMD AMD-Control FC P Value Control AMD AMD-Control NSD Control AMD AMD-Control P Value
6.265 6.561 0.296 1.228 0.044 0.513 0.567 0.054 1.099 0.519 0.546 0.027 0.234
6.763 6.400 −0.363 −1.286 0.091 0.190 0.240 0.049 1.149 0.178 0.207 0.028 0.131
6.348 6.553 0.205 1.153 0.090 0.492 0.361 −0.131 4.936 0.411 0.387 −0.024 0.208
7.211 7.048 −0.163 −1.119 0.245 0.451 0.401 −0.050 0.945 0.413 0.377 −0.036 0.135
6.646 7.177 0.531 1.444 0.290 0.534 0.463 −0.071 1.127 0.506 0.482 −0.024 0.152
6.245 6.665 0.420 1.338 0.028 0.297 0.373 0.076 1.966 0.338 0.358 0.020 0.230
8.595 8.364 −0.231 −1.173 0.257 0.820 0.879 0.059 2.090 0.817 0.853 0.036 0.055
5.838 6.018 0.180 1.133 0.095 0.157 0.245 0.088 2.284 0.210 0.252 0.042 0.072
5.477 5.616 0.139 1.101 0.035 0.657 0.569 −0.088 0.535 0.649 0.613 −0.036 0.023
6.030 6.178 0.149 1.108 0.100 0.587 0.498 −0.089 5.956 0.549 0.522 −0.027 0.108
7.007 7.596 0.589 1.504 0.127 0.582 0.517 −0.065 1.466 0.577 0.554 −0.023 0.076
6.596 6.280 −0.316 −1.245 0.158 0.657 0.711 0.054 1.024 0.665 0.710 0.045 0.021
7.205 6.895 −0.310 −1.239 0.151 0.842 0.892 0.050 3.063 0.855 0.880 0.025 0.081
8.668 8.193 −0.476 −1.391 0.164 0.172 0.237 0.065 1.590 0.229 0.251 0.022 0.024
7.930 8.297 0.366 1.289 0.257 0.193 0.314 0.121 1.340 0.164 0.298 0.135 0.050
7.618 7.403 −0.215 −1.161 0.003 0.355 0.399 0.045 0.875 0.373 0.401 0.028 0.155
7.921 8.088 0.167 1.123 0.056 0.475 0.588 0.113 1.453 0.539 0.576 0.037 0.203
7.464 7.598 0.134 1.098 0.204 0.183 0.124 −0.059 3.000 0.155 0.130 −0.025 0.033
5.465 5.831 0.366 1.289 0.184 0.292 0.240 −0.052 1.139 0.362 0.332 −0.030 0.269
6.047 6.189 0.143 1.104 0.279 0.531 0.615 0.084 1.515 0.630 0.659 0.029 0.147
7.795 7.550 −0.244 −1.184 0.075 0.749 0.796 0.047 2.008 0.794 0.837 0.043 0.012
6.777 7.019 0.243 1.183 0.074 0.877 0.827 −0.050 3.051 0.857 0.837 −0.020 0.069
6.169 5.984 −0.185 −1.137 0.206 0.648 0.571 −0.077 1.078 0.621 0.602 −0.019 0.235
9.117 8.622 −0.495 −1.409 0.037 0.112 0.169 0.057 0.566 0.150 0.233 0.083 0.017
5.614 5.386 −0.228 −1.171 0.144 0.562 0.332 −0.231 0.707 0.284 0.251 −0.033 0.426
6.775 7.008 0.233 1.175 0.126 0.806 0.865 0.059 2.235 0.819 0.851 0.032 0.046
6.321 6.462 0.141 1.103 0.140 0.064 0.118 0.054 1.254 0.149 0.169 0.020 0.095
3.559 3.754 0.196 1.145 0.003 0.710 0.763 0.053 1.032 0.751 0.783 0.033 0.127
5.935 6.071 0.135 1.098 0.072 0.704 0.655 −0.049 1.222 0.662 0.637 −0.025 0.224
7.253 7.414 0.161 1.118 0.166 0.716 0.649 −0.066 1.345 0.647 0.597 −0.050 0.035
6.684 6.845 0.161 1.118 0.210 0.714 0.765 0.051 1.881 0.729 0.753 0.024 0.216
9.194 9.322 0.128 1.093 0.118 0.730 0.669 −0.060 0.673 0.741 0.682 −0.059 0.004
6.719 7.101 0.382 1.303 0.046 0.476 0.407 −0.070 1.094 0.508 0.490 −0.018 0.242
6.306 6.881 0.575 1.490 0.135 0.867 0.802 −0.065 6.126 0.859 0.836 −0.022 0.028
6.306 6.881 0.575 1.490 0.135 0.713 0.659 −0.053 0.752 0.714 0.696 −0.018 0.102
9.390 9.071 −0.319 −1.247 0.134 0.564 0.492 −0.073 2.492 0.596 0.578 −0.018 0.256
6.883 7.030 0.148 1.108 0.077 0.639 0.486 −0.154 2.903 0.538 0.509 −0.029 0.281
4.381 4.228 −0.153 −1.112 0.136 0.371 0.169 −0.201 1.317 0.168 0.131 −0.038 0.328
7.846 8.138 0.292 1.224 0.281 0.587 0.532 −0.055 1.457 0.582 0.557 −0.025 0.171
5.096 4.559 −0.537 −1.451 0.004 0.374 0.422 0.048 0.750 0.411 0.448 0.036 0.164
6.048 5.905 −0.143 −1.104 0.143 0.775 0.820 0.046 2.560 0.771 0.794 0.023 0.087
6.554 6.749 0.196 1.145 0.166 0.241 0.300 0.059 3.392 0.266 0.286 0.021 0.119
5.339 5.002 −0.337 −1.263 0.076 0.554 0.671 0.117 1.169 0.713 0.789 0.076 0.179
6.299 6.498 0.199 1.148 0.191 0.562 0.476 −0.086 1.840 0.534 0.504 −0.029 0.146
6.062 5.889 −0.174 −1.128 0.289 0.156 0.210 0.054 1.269 0.195 0.255 0.060 0.001
7.078 6.865 −0.213 −1.159 0.164 0.179 0.255 0.076 3.998 0.163 0.207 0.045 0.011
6.562 6.823 0.261 1.198 0.146 0.769 0.724 −0.045 1.292 0.778 0.744 −0.034 0.070
9.693 8.963 −0.730 −1.659 0.100 0.344 0.202 −0.142 0.840 0.174 0.154 −0.020 0.407
4.725 4.078 −0.647 −1.566 0.014 0.586 0.632 0.046 0.571 0.595 0.641 0.045 0.174
7.454 7.660 0.206 1.153 0.087 0.451 0.243 −0.208 0.895 0.209 0.185 −0.024 0.414
3.361 3.505 0.144 1.105 0.008 0.642 0.687 0.045 0.580 0.677 0.708 0.031 0.182
6.451 6.237 −0.214 −1.160 0.102 0.736 0.691 −0.045 0.987 0.767 0.745 −0.022 0.095
6.811 6.580 −0.231 −1.173 0.019 0.132 0.081 −0.051 3.120 0.117 0.092 −0.025 0.122
9.124 8.774 −0.350 −1.275 0.297 0.539 0.294 −0.245 0.811 0.255 0.237 −0.019 0.455
8.060 7.921 −0.139 −1.101 0.025 0.364 0.241 −0.124 2.505 0.258 0.229 −0.030 0.262
7.072 7.206 0.134 1.098 0.374 0.813 0.737 −0.076 3.492 0.783 0.751 −0.032 0.069
8.196 7.948 −0.247 −1.187 0.244 0.688 0.734 0.047 1.616 0.645 0.708 0.063 0.189
6.790 6.574 −0.216 −1.161 0.118 0.198 0.246 0.048 1.748 0.217 0.246 0.029 0.065
5.141 4.975 −0.166 −1.122 0.236 0.666 0.606 −0.061 0.673 0.596 0.576 −0.019 0.344
8.399 8.191 −0.208 −1.155 0.248 0.379 0.451 0.072 1.406 0.465 0.487 0.022 0.179
8.224 8.452 0.228 1.171 0.099 0.326 0.201 −0.125 0.995 0.190 0.167 −0.023 0.360
7.725 7.461 −0.263 −1.200 0.020 0.449 0.603 0.154 3.004 0.479 0.520 0.040 0.262
8.184 8.326 0.142 1.104 0.068 0.654 0.783 0.130 3.533 0.684 0.704 0.020 0.200
Table 5.
 
Comparison of Percent Methylation Data Between AMD and Controls: Comparison of Bisulfite Pyrosequencing Percent Methylation Average, Median, Maximum, and Minimum for GSTM1 and GSTM5 Promoter for NSR and RPE
Table 5.
 
Comparison of Percent Methylation Data Between AMD and Controls: Comparison of Bisulfite Pyrosequencing Percent Methylation Average, Median, Maximum, and Minimum for GSTM1 and GSTM5 Promoter for NSR and RPE
AMD Cases (n = 10) Controls (n = 11) P Value*
n Mean (SD) n Mean (SD)
GSTM1 NSR
 CPG average 5 37.7 (5.07) 9 32.9 (7.91) 0.25
 CPG median 5 35.4 (10.1) 9 28.0 (10.0) 0.21
 CPG maximum 5 68.3 (7.07) 9 59.5 (15.7) 0.26
 CPG minimum 5 8.22 (4.84) 9 10.1 (5.48) 0.53
GSTM1 RPE
 CPG average 10 22.9 (2.37) 10 16.2 (7.47) 0.04
 CPG median 10 19.2 (5.88) 10 12.6 (7.67) 0.04
 CPG maximum 10 38.6 (7.51) 10 31.1 (13.4) 0.14
 CPG minimum 10 7.83 (3.72) 10 5.59 (3.79) 0.20
GSTM5 NSR
 CPG average 5 51.7 (3.11) 9 52.5 (3.02) 0.62
 CPG median 5 53.2 (6.32) 9 54.3 (3.01) 0.72
 CPG maximum 5 86.5 (4.44) 9 87.9 (6.09) 0.66
 CPG minimum 5 16.4 (9.42) 9 18.0 (6.26) 0.70
GSTM5 RPE
 CPG average 10 22.4 (6.33) 10 25.7 (5.64) 0.24
 CPG median 10 20.9 (6.38) 10 24.4 (5.75) 0.21
 CPG maximum 10 48.8 (11.8) 10 50.8 (9.45) 0.68
 CPG minimum 10 8.81 (2.96) 10 9.88 (4.58) 0.54
Table 6.
 
The Correlation Between RPE GSTM1 Promoter Methylation and GSTM1 and GSTM5 Gene Expression: Pearson and Spearman Correlation of Percent Methylation (Individual CpG Dinucleotides, Average, Media, Maximum, and Minimum) and GSTM1 and GSTM5 Relative Gene Expression Determined by qPCR for RPE/Choroid
Table 6.
 
The Correlation Between RPE GSTM1 Promoter Methylation and GSTM1 and GSTM5 Gene Expression: Pearson and Spearman Correlation of Percent Methylation (Individual CpG Dinucleotides, Average, Media, Maximum, and Minimum) and GSTM1 and GSTM5 Relative Gene Expression Determined by qPCR for RPE/Choroid
GSTM1 Methylation Location Correlation Coefficient and P ValueWith GSTM1 Gene Expression Correlation Coefficient and P Value With GSTM5 Gene Expression
Pearson Correlation Spearman Correlation Pearson Correlation Spearman Correlation
CpG__1 −0.01669 −0.05904 −0.11474 −0.23509
0.9443 0.8047 0.64 0.3326
CpG__2 −0.47005 −0.45366 −0.34243 −0.04386
0.0365 0.0445 0.1513 0.8585
CpG__3 0.25108 0.21285 0.04744 −0.14035
0.2856 0.3676 0.8471 0.5666
CpG__4 −0.38924 −0.53911 −0.1975 −0.06842
0.0898 0.0142 0.4177 0.7808
CpG__5 −0.6437 −0.29363 −0.54658 −0.22807
0.0022 0.2089 0.0155 0.3477
CpG__6 −0.58249 −0.2245 −0.53872 −0.29825
0.007 0.3413 0.0173 0.2149
CpG__7 −0.22386 −0.07224 −0.33567 −0.56491
0.3427 0.7621 0.16 0.0117
CpG__8 −0.53213 −0.42958 −0.59881 −0.63509
0.0157 0.0587 0.0067 0.0035
CpG__9 −0.51406 −0.26256 −0.57479 −0.43333
0.0204 0.2634 0.01 0.0638
CpG_average −0.57789 −0.55464 −0.59848 −0.54386
0.0076 0.0111 0.0068 0.0161
CpG_media −0.40782 −0.47852 −0.45723 −0.58596
0.0743 0.0328 0.049 0.0084
CpG_maximum −0.5434 −0.49716 −0.44711 −0.08947
0.0133 0.0257 0.0549 0.7157
CpG_minimum −0.10095 −0.11186 −0.34811 −0.60351
0.6719 0.6387 0.1442 0.0062
Table 7.
 
The Correlation Between NSR GSTM1 Methylation and GSTM1 and GSTM5 Gene Expression: Pearson and Spearman Correlation of Percent Methylation (Individual Cpg Dinucleotides, Average, Media, Maximum, and Minimum) and GSTM1 and GSTM5 Relative Gene Expression Determined by qPCR for NSR
Table 7.
 
The Correlation Between NSR GSTM1 Methylation and GSTM1 and GSTM5 Gene Expression: Pearson and Spearman Correlation of Percent Methylation (Individual Cpg Dinucleotides, Average, Media, Maximum, and Minimum) and GSTM1 and GSTM5 Relative Gene Expression Determined by qPCR for NSR
GSTM1 Methylation Location Correlation Coefficient and P Value With GSTM1 Gene Expression Correlation Coefficient and P Value With GSTM5 Gene Expression
Pearson Correlation Spearman Correlation Pearson Correlation Spearman Correlation
CpG__1 0.08844 −0.56037 0.34053 0.13986
0.796 0.073 0.2788 0.6646
CpG__2 −0.75036 −0.73349 −0.57685 −0.66434
0.0078 0.0102 0.0496 0.0185
CpG__3 0.17865 −0.15034 0.30995 0.2662
0.5992 0.659 0.3269 0.403
CpG__4 −0.47443 −0.60046 −0.25531 −0.59895
0.1404 0.0508 0.4232 0.0396
CpG__5 −0.27526 −0.26879 −0.22223 −0.12587
0.4127 0.4242 0.4876 0.6967
CpG__6 −0.24482 0.07289 −0.21525 0.04895
0.4681 0.8313 0.5017 0.8799
CpG__7 0.38965 0.55125 0.26116 0.44755
0.2362 0.0788 0.4123 0.1446
CpG__8 0.20985 0.1139 0.20004 0.23077
0.5357 0.7388 0.5331 0.4705
CpG__9 −0.26344 −0.21868 −0.18274 −0.06993
0.4338 0.5183 0.5697 0.829
CpG_average −0.38165 −0.4328 −0.16966 −0.16783
0.2468 0.1836 0.5981 0.6021
CpG_media −0.30005 −0.48747 −0.04052 −0.00699
0.37 0.1283 0.9005 0.9828
CpG_maximum −0.5863 −0.6287 −0.49273 −0.59441
0.058 0.0383 0.1036 0.0415
CpG_minimum 0.57967 0.48747 0.48633 0.51138
0.0616 0.1283 0.1089 0.0893
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