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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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  • C12Q2600/00—Oligonucleotides characterized by their use
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  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/154—Methylation markers

Definitions

• the present invention relates to a method for detecting oxidative stress (OS) in a cell using epigenetic markers.
• the method is capable of identifying OS in cell by determining the methylation status of a CpG site in a test cell and comparing the resultant methylation status with a reference methylation status of a control cell without OS. Differential methylation of the CpG site in the test cell indicates that the test cell has OS.
• Oxidative stress refers to a serious imbalance between the levels of reactive oxygen species (ROS) in a cell and its antioxidant defense mechanism.
• ROS reactive oxygen species
• redox regulation to cope with the stress to maintain their homeostasis by regulating the redox state.
• This system functions to adapt to many external stress agents such as radiation, ultraviolet (UV) rays, environmental pollutants, high fever, low temperature, hypoxic condition, and infectious diseases as well as to oxidative stress from lifestyle- related diseases such as cancer, diabetes, arteriosclerosis, hypertension and obesity.
• OS oxidative stress
• the human skin is constantly exposed to oxidative stress and free radicals, such as to high quantities of ROS, derived not only from ordinary metabolic reactions but also continuous exposure to air, radiation and UV rays, environmental pollutants, as well as physical and/or chemical agents (e.g., cosmetics). Under some conditions, the production of ROS may become so great that is may contribute to the pathogenesis of, for example, psoriasis or skin cancer. Oxidative damage caused by free radicals such as ROS is also a main cause of physical ageing in general, and of the skin in particular. Accordingly, there is a need in the art for detection of OS in cells, for example skin cells to prevent further damage to the cells.
• the human skin is constantly exposed to oxidative stress and free radicals, such as to high quantities of ROS, derived not only from ordinary metabolic reactions but also continuous exposure to air, radiation and UV rays, environmental pollutants, as well as physical and/or chemical agents (e.g., cosmetics). Under some conditions, the production of ROS may become so great that it may contribute to the pathogenesis of, for example, psoriasis or skin cancer. Oxidative damage caused by free radicals such as ROS is also a main cause of physical ageing in general, and of the skin in particular. Accordingly, there is a need in the art for detection of OS in cells, for example skin cells to prevent further damage to the cells.
• the present invention attempts to solve the problems above by providing a method of detecting Oxidative Stress (OS) in a test cell by comparing the methylation status of at least one CpG site in the test cell and the corresponding CpG site in a control cell with no OS, wherein the presence of hypomethylation or hypermethylation at the CpG site in the test cell is indicative of the test cell having OS.
• OS Oxidative Stress
• CpG sites can be used as biomarkers for detecting OS in a cell.
• CpG sites in a cell with OS are differentially methylated (i.e. hypomethylated or hypermethylated) compared to the corresponding CpG sites in a cell without OS. Accordingly, these CpG sites may be effectively used to determine if a cell has OS.
• an epigenetic marker is a long-term biomarker, that is to say it is inheritable and can be used to detect OS in the next generation as well if need be.
• a method of identifying oxidative stress (OS) in a test cell comprising
• the term "cell” refers to an intact live cell, naturally occurring or modified.
• the cell may be isolated from other cells, mixed with other cells in a culture, or within a tissue (partial or intact), or an organism.
• the cell may be a eukaryote cell.
• the cell may be mammalian cell.
• mammalian cell refers to any cell derived from a mammalian subject.
• the cell may also be a cell derived from the culture and expansion of a cell obtained from a subject.
• the cell may also have been genetically modified to express a recombinant protein and/or nucleic acid.
• the mammalian cell may be from humans and other primates, including nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; rodents such as mice, rats, rabbits, hamsters, and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like.
• the subject is a mammal.
• the mammal is selected from the group consisting of a mouse, a rat, a guinea pig, a dog, a mini-pig, a human being, a cow, a sheep, a pig, a goat, a horse, a donkey, and a mule.
• the mammalian cell may be a skin cell, a stem cell or a cell derived therefrom. More in particular, the mammalian cell may be a skin cell.
• a “CpG site” or “methylation site” is a nucleotide within a nucleic acid (DNA or RNA) that is susceptible to methylation either by natural occurring events in vivo or by an event instituted to chemically methylate the nucleotide in vitro. Some of these sites may be hypermethylated and some may be hypomethylated in a cell with OS compared to a cell with no OS.
• a “methylated nucleic acid molecule” refers to a nucleic acid molecule that contains one or more nucleotides that is/are methylated.
• a “CpG island” as used herein describes a segment of DNA sequence that comprises a functionally or structurally deviated CpG density.
• Yamada et al. have described a set of standards for determining a CpG island: it must be at least 400 nucleotides in length, has a greater than 50% GC content, and an OCF/ECF ratio greater than 0.6 (Yamada et al., 2004, Genome Research, 14, 247-266).
• Others have defined a CpG island less stringently as a sequence at least 200 nucleotides in length, having a greater than 50% GC content, and an OCF/ECF ratio greater than 0.6 (Takai et al., 2002, Proc. Natl.
• methylation profile “methylation pattern”, “methylation state” or “methylation status,” are used herein to describe the state, situation or condition of methylation of a genomic sequence, and such terms refer to the characteristics of a DNA segment at a particular genomic locus in relation to methylation. Such characteristics include, but are not limited to, whether any of the cytosine (C) residues within this DNA sequence are methylated, location of methylated C residue(s), percentage of methylated C at any particular stretch of residues, and allelic differences in methylation due to, e.g., difference in the origin of the alleles.
• C cytosine
• methylation status refers to the status of a specific methylation site (i.e. methylated vs. non-methylated) which means a residue or methylation site is methylated or not methylated. Then, based on the methylation status of one or more methylation sites, a methylation profile may be determined. Accordingly, the term “methylation profile” or also “methylation pattern” refers to the relative or absolute concentration of methylated C residues or unmethylated C residues at any particular stretch of residues in the genomic material of a biological sample.
• cytosine (C) residue(s) not typically methylated within a DNA sequence are methylated, it may be referred to as "hypermethylated”; whereas if cytosine (C) residue(s) typically methylated within a DNA sequence are not methylated, it may be referred to as "hypomethylated”.
• cytosine (C) residue(s) within a DNA sequence are methylated as compared to another sequence from a different region or from a different individual (e.g., relative to normal nucleic acid or to the standard nucleic acid of the reference sequence), that sequence is considered hypermethylated compared to the other sequence.
• the cytosine (C) residue(s) within a DNA sequence are not methylated as compared to another sequence from a different region or from a different individual, that sequence is considered hypomethylated compared to the other sequence.
• Measurement of the levels of differential methylation may be done by a variety of ways known to those skilled in the art.
• One method is to measure the methylation level of individual interrogated CpG sites determined by the bisulfite sequencing method, as a non-limiting example.
• a “methylated nucleotide” or a “methylated nucleotide base” refers to the presence of a methyl moiety on a nucleotide base, where the methyl moiety is usually not present in a recognized typical nucleotide base.
• cytosine in its usual form does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at position 5 of its pyrimidine ring. Therefore, cytosine in its usual form may not be considered a methylated nucleotide and 5-methylcytosine may be considered a methylated nucleotide.
• thymine may contain a methyl moiety at position 5 of its pyrimidine ring, however, for purposes herein, thymine may not be considered a methylated nucleotide when present in DNA.
• Typical nucleotide bases for DNA are thymine, adenine, cytosine and guanine.
• Typical bases for RNA are uracil, adenine, cytosine and guanine.
• a "methylation site" is the location in the target gene nucleic acid region where methylation has the possibility of occurring. For example, a location containing CpG is a methylation site wherein the cytosine may or may not be methylated.
• methylated nucleotide refers to nucleotides that carry a methyl group attached to a position of a nucleotide that is accessible for methylation. These methylated nucleotides are usually found in nature and to date, methylated cytosine that occurs mostly in the context of the dinucleotide CpG, but also in the context of CpNpG- and CpNpN-sequences may be considered the most common. In principle, other naturally occurring nucleotides may also be methylated but they will not be taken into consideration with regard to any aspect of the present invention.
• methylation profile In context of the present invention, the terms “methylation profile”, “methylation pattern”, “methylation state” or “methylation status,” are used herein to describe the state, situation or condition of methylation of a genomic sequence, and such terms refer to the characteristics of a DNA segment at a particular genomic locus in relation to methylation. Such characteristics include, but are not limited to, whether any of the cytosine (C) residues within this DNA sequence are methylated, location of methylated C residue(s), percentage of methylated C at any particular stretch of residues, and allelic differences in methylation due to, e.g., difference in the origin of the alleles.
• C cytosine
• hypomethylation refers to the average methylation state corresponding to an increased presence of 5-mCyt at one or a plurality of CpG dinucleotides within a DNA sequence of a test DNA sample, relative to the amount of 5-mCyt found at corresponding CpG dinucleotides within a normal control DNA sample.
• control refers to a cell with no indication of OS.
• hypomethylation refers to the average methylation state corresponding to a decreased presence of 5-mCyt at one or a plurality of CpG dinucleotides within a DNA sequence of a test DNA sample, relative to the amount of 5-mCyt found at corresponding CpG dinucleotides within a normal control DNA sample.
• control refers to a cell with no indication of OS.
• genomic material refers to nucleic acid molecules or fragments of the genome of the subject or group of subjects.
• nucleic acid molecules or fragments are DNA or RNA or hybrids thereof, and most preferably are molecules of the DNA genome of a subject or group of subjects.
• DNA sample refers to the DNA extracted from the cell according to any aspect of the present invention using known methods in the art.
• test cell when there is differential methylation detected in a test cell, that is to say that the cell displays hypermethylation or hypomethylation at, at least one CpG site in comparison to the control (i.e., a cell without indication of OS), then the test cell has OS. More in particular, when the CpG site displays hypomethylation in the test cell in comparison to the corresponding CpG site in the control cell, the test cell has OS. In another example, when the CpG site displays hypermethylation in the test cell in comparison to the corresponding CpG site in the control cell, the test cell has OS.
• step (a) in the method according to any aspect of the present invention, in step (a) the methylation status of at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 ,
• CpG sites are determined.
• a skilled person would be capable of determining the number of CpG sites that need to be used in step (a) according to any aspect of the present invention. Even more in particular, the methylation status of at least two CpG sites are determined in step (a) of the method according to any aspect of the present invention.
• step (a) the CpG site is selected from the list provided in Table 2:
• the method according to any aspect of the present invention further comprises the step of:
• ‘Bisulfite treatment’ of genomic DNA used interchangeably with the term ‘bisulfite modification’ refers to the treatment of the genomic DNA with a deaminating agent such as a bisulfite that may be used to treat all DNA, methylated or not.
• a deaminating agent such as a bisulfite that may be used to treat all DNA, methylated or not.
• bisulfite as used herein encompasses any suitable type of bisulfite, such as sodium bisulfite, or other chemical agents that are capable of chemically converting a cytosine (C) to an uracil (U) without chemically modifying a methylated cytosine and therefore can be used to differentially modify a DNA sequence based on the methylation status of the DNA, e.g., U.S. Pat. Pub. US 2010/0112595.
• a reagent that "differentially modifies" methylated or non-methylated DNA encompasses any reagent that modifies methylated and/or unmethylated DNA in a process through which distinguishable products result from methylated and non-methylated DNA, thereby allowing the identification of the DNA methylation status.
• processes may include, but are not limited to, chemical reactions (such as a C to U conversion by bisulfite) and enzymatic treatment (such as cleavage by a methylation-dependent endonuclease).
• an enzyme that preferentially cleaves or digests methylated DNA is one capable of cleaving or digesting a DNA molecule at a much higher efficiency when the DNA is methylated, whereas an enzyme that preferentially cleaves or digests unmethylated DNA exhibits a significantly higher efficiency when the DNA is not methylated.
• step (a) the genomic DNA contained/ obtained or extracted from the cell, is first bisulfite treated.
• TET-assisted pyridine borane sequencing may be used for detection of 5mC and 5hmC (Yibin Liu, et al., Nature Biotechnology, 37: 424-429 (2019).
• the cell used according to any aspect of the present invention is obtained from a biological sample selected from the group consisting of blood, brain, sperm and any other tissue or sample that provides genomic DNA to be used in the method according to any aspect of the present invention.
• the biological sample may comprise any biological material obtained from the subject that contains DNA, and may be liquid, solid or both, may be tissue or bone, or a body fluid such as blood, lymph, etc.
• the biological sample useful for the present invention may comprise biological cells or fragments thereof.
• test used in conjunction with the term cell herein refers to a cell that is subjected to the method according to any aspect of the present invention and is the basis for an analysis application of the present invention.
• a ‘test cell’ is therefore a cell or a group of cells being tested according to any aspect of the present invention or a profile being obtained or generated in this context.
• reference shall denote, mostly predetermined, entities which are used for a comparison with the test entity.
• a ‘test cell’ refers to a cell being tested for OS where the methylation status has to be determined and a ‘control’ refers to a cell without OS where the methylation status is already known and used as a reference.
• the OS according to any aspect of a the present invention may be caused or may be a result of UV light exposure, ageing, H2O2 exposure and a combination thereof (i.e. UV light and H2O2 exposure, UV light exposure and ageing, H2O2 exposure and ageing or UV light and H2O2 exposure and ageing.
• a method of detecting the incidence of oxidative stress (OS) in a cell comprising detecting an epigenetic change in at least one CpG site in the cell, wherein detection of the epigenetic change is indicative of the incidence of OS and wherein the epigenetic change is methylation.
• OS oxidative stress
• methylation is hypomethylation.
• DNA hypomethylation profiling may be very useful for stratifying cell cultures systems ranging from 1 D to 3D, stem cells to differentiated skin tissue models under stress.
• epigenetic change refers to a chemical (e.g., methylation) change or protein (e.g., histones) change that takes place to a gene body or a promoter thereof.
• chemical change e.g., methylation
• protein e.g., histones
• Figure 1 A is a scatter plot showing that a large number of probes have a different methylation status in cell with where artificial OS (high UV for 24hrs) was induced according to Example 1 . As can be seen, there is an equally large number of probes that are hypomethylated as there are probes hypermethylated.
• Figure 1 B is a box-plot confirming the results in Figure 1 A that a large number of probes have a different methylation status in cell with where artificial OS (high UV for 24hrs) was induced according to Example 1 .
• Figure 2A is a scatter plot showing that a large number of probes have a different methylation status in cell with where artificial OS (low UV for 72hrs) was induced according to Example 1 . As can be seen, there is an equally large number of probes that are hypomethylated as there are probes hypermethylated.
• Figure 2B is a box-plot confirming the results in Figure 2A that a large number of probes have a different methylation status in cell with where artificial OS (low UV for 72hrs) was induced according to Example 1 .
• Figure 3A is a scatter plot showing that a large number of probes have a different methylation status in cell with where artificial OS (low H2O2 for 24hrs) was induced according to Example 2. As can be seen, there is a large number of probes that are hypomethylated in cells with OS compared to cells without OS.
• Figure 3B is a box-plot confirming the results in Figure 3A that a large number of probes have a different methylation status in cell with where artificial OS (low H2O2 for 24hrs) was induced according to Example 2.
• Figure 4A is a scatter plot showing that a large number of probes have a different methylation status in cell with where artificial OS (high H2O2 for 24hrs) was induced according to Example 2. As can be seen, there is a large number of probes that are hypomethylated in cells with OS compared to cells without OS.
• Figure 4B is a box-plot confirming the results in Figure 4A that a large number of probes have a different methylation status in cell with where artificial OS (high H2O2 for 24hrs) was induced according to Example 2.
• Figure 5A is a scatter plot showing that a large number of probes have a different methylation status in cell with where artificial OS according to Example 3 with Medox® in cells was induced. As can be seen, there is a large number of probes that are hypomethylated in cells with OS compared to cells without OS.
• Figure 5B is a box-plot confirming the results in Figure 5A that a large number of probes have a different methylation status in cell with where artificial OS according to Example 3 with Medox® in cells was induced.
• T-Skin models were obtained from Episkin SA, France which is composed of reconstructed human skin.
• Each skin model consists of a dermal equivalent overlaid by a stratified, well-differentiated epidermis derived from normal human keratinocytes.
• UV radiation UVA 24 J/cm 2 + UVB 50mJ/cm 2
• high UV UV
• the genomic DNA (500ng) from tissue samples were subjected to bisulfite conversion using the EZ DNA Methylation-GoldTM Kit (Zymo Research).
• the methylation levels were quantified using Infinium MethylationEPIC v2.0 Kit (Illumina) which can analyze over 850,000 methylation sites quantitatively across the genome at single-nucleotide resolution.
• Methylation EPIC array data processing was performed in R version 4.1 .2 (2021-11-01) using the minfi version 1 .40.0.
• the raw intensity data (IDAT) were imported into the R (4.1 .2), processed using the minfi (1 .4.0) Bioconductor packaged 8.
• Quality check on samples was performed to keep probes that have a detection P-value ⁇ 0.01 in one or more samples or have a mean detection P- value ⁇ 0.05 in all samples. Then samples were normalized using functional normalization (implemented by preprocessFunnorm function in minfi) for type-bias correction and background correction.
• the probes with non-specific binding, cross reactive probes, probes affected by common SNPs, and probes annotated to the X,Y chromosomes were also filtered out.
• Beta-value and M-value of normalized and filtered samples were calculated using getBeta and getM function respectively, the samples were then subjected to further downstream analysis.
• Differential methylation analysis Differential methylation analysis was performed using packages limma version 3.50.1 and DMRcate version 2.8.5. Contrast matrix was set up by comparing each corresponding treatment and control group and empirical Bayesian algorithm was used to fit the M-values based on the design and contrast model. Probes with adjusted P-value lower than 0.05 were considered as differentially methylation positions (DMPs). Annotation was performed using HluminaHumanMethylationEPICkanno.ilmn12.hg19 and annotatr package (1 .20.0).
• Example 2 Same method of quality control and data processing as that disclosed in Example 1 was carried out on the samples here. Further, the same differential methylation analysis as disclosed in Example 1 was carried out on the data obtained from Example 2.
• MSCs Mesenchymal Stem Cells
• Medox Evonik, Batch:H-080719
• Bone marrow derived MSCs were cultured for 1 week in Mesencult ACF Plus Medium with two doses of Medox (4x replicates): 25 pg/ml (low) and 100 pg/ml (high). The media with Medox was replaced every second day for 1 week.
• MSCs were cultured for 1 week in Mesencult ACF Plus Medium without any Medox® treatment. Medox® treatment is expected to produce the opposite reaction to OS. This was followed by collection of cell pellet and genomic DNA was purified from the cell pellet using the DNeasy® Blood & Tissue Kit (Qiagen). The genomic DNA was quantified using the PicroGreen® or NanoDropTM 2000.
• the genomic DNA (500ng) from the cell pellet was subjected to bisulfite conversion using the EZ DNA Methylation-GoldTM Kit (Zymo Research).
• the methylation levels were quantified using Infinium MethylationEPIC v2.0 Kit (Illumina) which can analyze over 850,000 methylation sites quantitatively across the genome at single-nucleotide resolution.
• DNA methylation profiling has been proven to be a powerful analytical tool to accurately identify the origin of tissue and the effect of environmental factors. It has several advantages as a biomarker classifier as it is a stable marker, and it can facilitate quantitative analysis at single-nucleotide resolution.
• Example 3 Same method of quality control and data processing as that disclosed in Example 1 was carried out on the samples here. Further, the same differential methylation analysis as disclosed in Example 1 was carried out on the data obtained from Example 3.
• T-Skin models were obtained from Episkin SA, France which is composed of reconstructed human skin.
• Each skin model consists of a dermal equivalent overlaid by a stratified, well-differentiated epidermis derived from normal human keratinocytes.
• the genomic DNA (500ng) from tissue samples were subjected to bisulfite conversion using the EZ DNA Methylation-GoldTM Kit (Zymo Research).
• the methylation levels were quantified using Infinium MethylationEPIC v2.0 Kit (Illumina) which can analyze over 850,000 methylation sites quantitatively across the genome at single-nucleotide resolution.
• the genomic DNA (500ng) from tissue samples were subjected to bisulfite conversion using the EZ DNA Methylation-GoldTM Kit (Zymo Research).
• the methylation levels were quantified using Infinium MethylationEPIC v2.0 Kit (Illumina) which can analyze over 850,000 methylation sites quantitatively across the genome at single-nucleotide resolution.
• PM2.5 Particulate Matter 2.5
• the skin models (5x replicates) were treated with two different concentrations of PM2.5 [15 pg/cm 2 (low) and 30 pg/cm 2 (high)] and were maintained for 24hrs.
• a control set of skin models (5x replicates) were maintained for 24hrs without any treatment with PM2.5.
• skin models were collected, and genomic DNA was purified from the tissue samples using the DNeasy® Blood & Tissue Kit (Qiagen). The genomic DNA was quantified using the PicroGreen® or NanoDropTM 2000.
• the genomic DNA (500ng) from tissue samples were subjected to bisulfite conversion using the EZ DNA Methylation-GoldTM Kit (Zymo Research).
• the methylation levels were quantified using Infinium MethylationEPIC v2.0 Kit (Illumina) which can analyze over 850,000 methylation sites quantitatively across the genome at single-nucleotide resolution.
• Glyoxal treatment Another way to induce oxidative stress on a human tissue system is through Glyoxal treatment which provokes oxidative stress by increasing the level of ROS within the cells by producing advanced glycation end-products.
• the skin models (5x replicates) were treated with two different concentrations of glyoxal [0.5 mM (low) and 1 mM (high)] and were maintained for 24hrs.
• a control set of skin models (5x replicates) were maintained for 24hrs without any treatment with glyoxal.
• skin models were collected, and genomic DNA was purified from the tissue samples using the DNeasy® Blood & Tissue Kit (Qiagen). The genomic DNA was quantified using the PicroGreen® or NanoDropTM 2000.
• the genomic DNA (500ng) from tissue samples were subjected to bisulfite conversion using the EZ DNA Methylation-GoldTM Kit (Zymo Research).
• the methylation levels were quantified using Infinium MethylationEPIC v2.0 Kit (Illumina) which can analyze over 850,000 methylation sites quantitatively across the genome at single-nucleotide resolution.
• the skin models were maintained in the deep well plate with media 14 days (6x replicates) with media being renewed after 7days to induce ageing in the skin tissue.
• Skin models were collected after 14 days, and genomic DNA was purified from the tissue samples using the DNeasy® Blood & Tissue Kit (Qiagen). The genomic DNA was quantified using the PicroGreen® or NanoDropTM 2000.
• the genomic DNA (500ng) from tissue samples were subjected to bisulfite conversion using the EZ DNA Methylation-GoldTM Kit (Zymo Research).
• the methylation levels were quantified using Infinium MethylationEPIC v2.0 Kit (Illumina) which can analyze over 850,000 methylation sites quantitatively across the genome at single-nucleotide resolution.
• Methylation EPIC array data processing was performed in R version 4.2.2 (2021-11-10 r83330) using the minfi version 1 .42.0.
• the raw intensity data (IDAT) were imported into the R (4.2.2), processed using the minfi (1 .42.0) Bioconductor package. Quality check on samples were performed to keep probes that had a detection P-value ⁇ 0.01 in one or more samples or had a mean detection P-value ⁇ 0.05 in all samples.
• the samples were then normalized using functional normalization (implemented by preprocesssFunnorm function in minfi) for type-bias correction and background correction.
• the probes with non-specific binding, cross reactive probes, probes affected by common SNPs, and probes annotated to the X,Y chromosomes were also filtered out.
• Beta-value and M-value of normalized and filtered samples were calculated using getBeta and getM function respectively, the samples were then subjected to further downstream analysis.
• Table 1 Sample information for batch 1 Differential methylation analysis
• Pair-wise differential methylation analysis (total of 16 pairs) was performed using the limma package version 3.52.4 .
• the batch 1 samples were analyzed together. Contrast matrix was set up by comparing each corresponding treatment and control group and empirical Bayesian algorithm was used to fit the M-values based on the design and contrast model. Probes with adjusted P- value lower than 0.05 were considered as differentially methylation positions (DMPs). After which, the DMPS within the batch 1 comparisons, batch 1 High concentration comparisons, and batch 1 Low concentration comparisons were compared to identify common DMPs that are present in all comparisons within each group and have the same methylation status throughout.
• Batch 1 comparisons had a total of 96 common DMPs
• Batch 1 High comparisons had a total of 616 common DMPs
• Batch 1 Low comparisons had a total of 238 common DMPs.
• Tables 2,3 and 4 show the list of common DMPs within each group for Batch 1 comparisons, Batch 1 High comparisons, and Batch 1 Low comparisons respectively.

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