BACKGROUND
dna methylation  is one type of epigenetic modification that regulates gene expression heritably.
it is catalyzed by dna methyltransferase that adds and maintains a methyl group to the 5′ position of the cytosine ring to form 5′ methyl-cytosine  <cit> .
in mammalian genomes, this modification occurs almost exclusively on cytosine residues that precede guanine .
these cpg dinucleotides are generally about 60%–90% methylated  <cit> .
however, cpg islands , which are the genomic regions with the highest cpg density, exhibit the lowest levels of dnam  <cit> .
the potential role of aberrant dnam in human diseases such as cancer, both at a single-gene level and on a genome-wide scale  <cit> , is important.
recently, dnam has also been shown to be associated with aging in a wide array of organisms, ranging from yeast to humans  <cit> .
for example, horvath et al.
observed that a genome-wide decrease in dna methylation, preferentially hypermethylation at cgis, occurred during aging  <cit> .
some studies have investigated age-associated methylation of cpg loci dependent on sex, body mass index, specific tissue, or cell type .
these studies were performed using various linear-based methods, including conventional linear regression methods  <cit> , a weighted correlation network method  <cit> , and a multidimensional scaling method  <cit> .
however, most of these studies were restricted to specific tissue types, and included limited numbers of samples and/or limited ranges of sample ages.
more recently, several studies were performed to identify more reliable cpg sites associated with aging, which collected many samples with various tissue types from public data sets .
these studies also used linear-based regression methods of analysis.
although previous studies have reported that age-associated dnam can show both nonlinear and linear patterns  <cit> , there has been little identification of age-associated dnam signatures through systematic analysis of nonlinear dnam patterns.
more importantly, the characteristics of dnam age signatures that are applicable to multiple types of normal or cancer tissues are not well studied.
in this study, we identify for the first time tissue type-invariant dnam age signatures for healthy normal and cancer tissues using linear and nonlinear models.
for more reliable signatures, we collected diverse samples from a range of studies available in public resources that included multiple tissue types.
after identifying the dnam age signatures from normalized dnam levels of the samples, we extensively investigated the characteristics of the signatures and their biological meaning through a number of analyses, including analysis of changes in dnam pattern with age, gene ontology term analysis, and network and conservation analysis.
we also compared the signatures with the results of previous studies.
finally, we checked that the signatures could be used as an age predictor for multiple tissue types.
RESULTS
discovery of age-associated dna methylation signatures
to identify robust age-associated dnam signatures, we first searched and downloaded various dnam profiles from diverse studies available in the gene expression omnibus  database .
we then excluded studies without age information or with small numbers of samples .
we also excluded samples of diseased tissues other than cancer.
it is known that technical bias exists across different array platforms  <cit> , so we considered only the illumina infinium humanmethylation <dig> bead chip array, which was the most widely used among the downloaded profiles.
consequently, we collected dnam profiles of  <dig> samples  from eight studies available in the geo.
additionally, we downloaded  <dig> publicly available dnam profiles  of five cancer types  evaluated on the same platform from the cancer genome atlas  consortium .
in total, we gathered dnam profiles of  <dig> normal healthy and  <dig> cancer samples.
these samples included diverse tissue types and exhibited a wide range of ages from  <dig> to 91 years .
we next normalized dnam levels  using a single beta-score measure that indicates conceptually the normalized levels of dnam .
the normalized dnam levels were well correlated between normal or cancer samples, but had higher correlation scores between normal samples , and for both normal and cancer tissues, the dnam levels in cgi regions of individual samples were much lower than those in non-cgis .
moreover, the dnam levels of normal and cancer tissue showed different patterns depending on the genomic regions.
in cgis, for example, the average dnam levels were generally higher in cancer than those in normal tissue, except for ovarian cancer samples .figure  <dig>
study overview.
sources of dnam data with sample information.
eight studies from geo and five from open tcga data were included.
identifying an age-associated dnam signature.
linear and nonlinear regression models using single or combined studies were applied.
age prediction and characterization of identified age-associated signatures.
various analyses using dnam patterns and distributions, gene ontology, and protein networks in normal and cancer tissues were performed.
dna methylation correlation and age-associated cpg loci across individual studies.
heat map representing the pearson’s correlation coefficients between all samples of normal and cancer tissues used in this study.
a hierarchical clustering was used.
box plot representing the correlations between normal samples or between cancer samples.
p-value was calculated using a wilcoxon rank-sum test.
examples of cpg loci  hypermethylated with age in the gse <dig> study.
examples of cpg loci  hypomethylated with age in the gse <dig> study.
r: correlation coefficient.
the number of significant age-associated cpg loci in normal or cancer tissues from individual studies.
the number and color in each cell corresponds to the number of significant age-associated cpg loci and its significance in terms of a p-value, respectively.
p-value: z-test result using the random distribution of the  <dig> age-permutation tests.
na: not available.
next, using various regression methods, we identified an aging signature associated with the normalized dnam levels.
because some studies have reported that methylation levels can change most dramatically during childhood  <cit> , we applied nonlinear regression models as well as linear regression models.
after finding statistically significant age-associated dnam sites using single studies or combined multiple studies, we examined the characteristics and biological meaning of the sites through analysis of the associations of dnam patterns with age, gene ontology, sequence conservation, and protein networks.
we also compared the age-associated dna loci across different studies or tissue types.
to identify tissue type-invariant age-associated dnam signatures, we integrated data from all samples of normal tissues, cancers, or both, after removing noisy samples .
we compared the methylation patterns of the normal signature to those in cancer according to different genomic regions.
we finally checked the potential for age prediction using the methylation levels of the age-associated signature, regardless of tissue type.
cpg loci are widely associated with age in disease-free normal samples
the distribution of dnam levels showed a significant disparity between normal and cancer tissues depending on the genomic region.
we first checked age-associated dnam sites separately in normal or cancer samples using a linear regression model.
for example, cg <dig>  and cg <dig>  sites showed linear hypermethylation patterns according to age in normal samples from the gse <dig> study  <cit>  .
in contrast, hypomethylation patterns were observed at cg <dig>  and cg <dig>  in normal samples from the gse <dig> study  <cit>  .
we checked the number of significantly age-associated cpg loci in each study .
the numbers of significant cpg loci were quite different between studies , mainly because of different ranges of age and/or different numbers of samples across studies .
although the numbers of age-associated loci in normal tissues varied between studies, the numbers were significant in all studies included .
in cancer samples, however, the numbers of age-associated loci were not significant in some studies, including gse <dig> and gse <dig>
individual studies included samples of various tissue types with different age ranges .
therefore, the average dnam levels per cpg site were quite different between studies in both disease-free normal samples  and cancer samples  .
similar results were also observed for the average dnam levels per sample unit .
however, most study pairs with normal samples showed significantly greater degrees of overlap of age-associated cpg loci than would be expected by chance .
moreover, the results of hierarchical clustering of the p-values of the degrees of overlap demonstrated that common age-associated cpg loci were independent of tissue or cell type.
in the case of cancer samples, the degrees of overlap of age-associated cpg loci between study pairs were also significant, but less so than for normal samples .figure  <dig>
comparison of age-associated cpg loci across different studies with different tissue types.
box plots of average methylation values  per cpg unit in normal and cancer tissues across individual studies .
box plots of average methylation values per sample unit in normal or cancer tissue across individual studies.
p-values were calculated by kruskal–wallis tests.
we checked the degree of overlap of age-associated cpg loci between studies by calculating the number of common cpg loci.
we performed  <dig> age-permutation tests with the samples of individual studies to verify the significance of the degree of overlap.
hierarchical clustering results using the degree of overlap of age-associated cpg loci between different studies with different tissue types in normal  and cancer .
p-value: a z-test result using the distribution of  <dig>  random selections.
age-associated dna methylation signatures in normal and cancer
to identify tissue type-invariant age-associated cpg loci, we integrated the normalized dnam levels of all the disease-free normal samples after removing noisy samples.
using this integrated data set, we first identified cpg loci with a linear relationship with age.
for example, the cg <dig> site was linearly hypomethylated  and cg <dig> was linearly hypermethylated  according to age, regardless of tissue type .
however, dnam levels of some loci showed nonlinear patterns according to age .
for these nonlinear relationships, we also observed more rapid changes in dnam levels at younger ages, which is consistent with previous studies  <cit> .
we also observed similar phenomena for gene-level dnam levels .
we thus identified tissue type-invariant age-associated dna methylation signatures using second- and third-degree nonlinear regression models in addition to the linear model.
for the threshold, we used three measures, including false discovery rate  , correlation coefficient , and residual error , to reduce tissue-type variations.
we identified  <dig> unique cpg loci in the combined normal samples, which we termed a “tissue type-invariant age-associated dnam signature”.
among these,  <dig> cpg loci had a linear relationship with age and the other  <dig> loci were identified using nonlinear models .
seventy-seven loci were hypermethylated and  <dig> were hypomethylated with age.
we also applied a similar approach to examine the dnam levels of the combined  <dig> cancer samples.
compared with normal samples, only  <dig> age-associated cpg loci were identified .
interestingly, there was no cpg locus common to the normal and cancer age-associated signatures .
these epigenetic phenomena were also observed with the gene-level dnam values .
in case of the combined normal and cancer samples, only  <dig> cpg loci were identified as age-associated.
we examined the positions on human chromosomes of the age-associated cpg loci of each of the signatures by separating hypomethylated  and hypermethylated  loci, in normal , cancer , or combined samples .
generally, the  <dig> age-associated loci in normal tissue were distributed throughout the human genome, except for chromosomes  <dig> and  <dig>  in contrast to a previous study using male pediatric samples  <cit> , the x chromosome had the largest number of age-associated loci.
this difference may be caused by differences in sex, age range, and tissue types.
we checked the significance of the numbers of loci by chromosome using hypergeometric tests .
chromosomes x ,  <dig> ,  <dig> ,  <dig> , and  <dig>  were preferentially enriched for hypermethylated loci with age, whereas chromosomes y , x ,  <dig> , and  <dig>  were enriched for hypomethylated loci.
thus, the sex chromosomes, especially x, were enriched for age-associated cpg loci in disease-free normal tissues.
in cancer samples, chromosomes  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig> ,  <dig> , and  <dig>  were enriched with age-associated hypomethylated loci .
interestingly, there were no age-associated loci on the x or y chromosomes in cancer samples.
with the normal and cancer samples matched in age distribution, we also observed similar trends such as no overlap in signature between the normal and cancer samples .figure  <dig>
age-associated dna methylation signature independent of tissue type.
examples of age-associated cpg loci with linear  or nonlinear  relationships identified in integrated normal samples.
venn diagrams showing the numbers of age-associated cpg loci with three regression models  in integrated normal, cancer, or all samples.
venn diagram showing the number of age-associated cpg loci among integrated normal, cancer, and all samples.
manhattan plot of age-associated cpg loci in integrated normal samples by chromosome.
hypermethylated cpg loci with age are shown with a –log  and hypomethylated loci are shown with a log .
the most significant p-values among linear and nonlinear models were chosen.
significant loci are marked as green  or blue  dots.
the numbers of significant age-associated cpg loci by chromosome.
bar plots of p-values with hypergeometric tests for the degrees of significance of the numbers of the loci.
manhattan plot of age-associated cpg loci in integrated cancer samples by chromosome.
it was previously suggested that a difference in methylation variation might exist with gender  <cit> .
we therefore identified age-associated signatures for males and females separately .
we found  <dig>  and  <dig>  cpg loci in the male and female samples, respectively.
even though the number of age-associated loci differed between male and female, their ratio distributions by chromosome were similar .
moreover, the number of hyper- and hypomethylated loci on the x chromosome were similar between males and females .
we built a feasible age-prediction model to see whether the normal 127-site signature could be used as a tissue-invariant age predictor.
we applied a multiple linear regression model after identifying a feasible subset of the signature using a genetic algorithm .
the selected age-prediction model was composed of  <dig> cpg loci of the signature .
the correlation between the actual ages of the combined normal samples and their predicted ages using the model was highly significant , which indicates that the dnam levels of the age-associated signature sites can be used to predict the age of tissues, regardless of tissue type.
we next compared the age-associated normal signature with those identified in previous studies .
most previous studies identified age-associated loci using a fdr threshold in a linear model.
thus, we compared the loci resulting from only linear regression  and found that  <dig> age-associated cpg loci were age-associated in the integrated normal samples.
for instance, a recent study using the illumina human 450 k platform and a linear regression model identified  <dig> cpg loci associated with age in blood cells of  <dig> healthy subjects aged from  <dig> to 94 years  <cit> .
of these  <dig> loci, the  <dig> cpg loci present on the illumina 27 k overlapped 73% with our  <dig> age-associated loci.
another study by day et al.
<cit>  found that  <dig> cpg loci correlated with age in four tissue types, including brain samples, using a linear regression method, and the degree of overlap with our loci was 47%.
notably, we observed higher degrees of overlap of cpgs with previous studies that used only normal samples than with other studies that included diseased samples .
sixteen of our  <dig> age-associated loci were not identified in the previous studies.
interestingly,  <dig> of these  <dig> loci were located on the x chromosome .figure  <dig>
characteristics of age-associated dna methylation signature.
age prediction using the age-associated normal dnam signature.
age was predicted with the normal signature using a multivariate linear regression after using a genetic algorithm to identify a feasible set of loci.
degrees of overlap with age-associated dnam signatures identified in previous studies.
overlap percentages were calculated by the common numbers divided by the smaller number of total loci in either study.
the studies with only normal samples are orange; other studies including disease samples are gray.
the fractions of hyper-  or hypomethylated  cpg loci in the age-associated signatures in normal  or cancer  according to genomic regions.
the number on each bar indicates the count of the corresponding loci.
p-value was calculated by a chi-square test.
the hyper-  or hypomethylation  patterns according to age group of normal age-associated dna loci in cgi or non-cgi.
blue or green dotted lines show the linear regressions of median values of individual age groups using only hypo-  or hypermethylated  loci, respectively.
numbers below are the counts of loci considered for the corresponding cases.
nucleotide compositions of the sequences surrounding the hypo-  or hypermethylated loci  of normal age-associated dnam signature.
–log  of the y axis was calculated by random selection tests representing overrepresentation for each base at each location of the surrounding cpg.
our collected dnam profiles included diverse tissue types in the normal samples.
we next identified tissue-type-specific age-associated signatures .
although the number of age-associated loci in normal samples varied from one  to  <dig>  loci across tissue types , most tissue-type pairs showed significant degrees of overlap of the age-associated cpg loci compared with random expectation, except for the prostate tissue samples .
in the cancer samples, we could not find significant tissue-type-specific age-associated loci with the threshold we used in most cases.
characteristics of tissue type-invariant age-associated dna methylation signature
we investigated the genomic locations of the loci in the age-associated dnam signatures in normal or cancer samples.
of the  <dig> loci in normal samples,  <dig> were located in cgi regions and the others in non-cgi regions ; whereas in cancer,  <dig> loci were located in cgis and four in non-cgis.
thus, while the normal signature was enriched in cgi regions, the cancer signature was even more enriched in cgi regions .
in the normal age-associated signature, hypermethylated loci were enriched in cgi regions , whereas hypomethylated loci were enriched in non-cgis  .
interestingly, the cancer signature had only age-hypomethylated loci in both cgi and non-cgi regions .
similar results were also detected for gene-level dnam patterns .
we next checked the changing rates of dna methylation in the normal signature within cgi or non-cgi regions.
age-hypermethylated loci increased more rapidly in cgi regions .
in contrast, hypomethylated loci decreased more rapidly in non-cgi regions .
the cancer signature of only hypomethylated loci showed much smaller changes with age than did the normal signature .
next, we investigated the nucleotide composition surrounding the  <dig> cpg loci in the normal signature .
the sequences surrounding the normal signature showed significant overrepresentation of thymine  residues at - <dig>  - <dig>  + <dig>  + <dig>  + <dig>  and + <dig> bases from the cpg loci , adenine  residues at + <dig> and + <dig> bases , and guanine  residues at - <dig> base .
interestingly, the sequence motifs surrounding age-associated cpg loci were quite different between hypermethylated and hypomethylated loci.
sequences surrounding age-hypomethylated loci presented at-rich sequences , whereas the sequences surrounding age-hypermethylated loci were enriched for gc-rich sequences ; these are also enriched in cgi regions  <cit> .
these phenomena were also observed in the cancer signature .
analysis of gene ontology descriptors for the age-associated dna methylation signature in normal samples indicated that the aging-related terms such as regulation of protein kinase activity , metabolic processes , immune system processes , and neuron differentiation  were significantly enriched in the cgi regions .
genes related to age-associated loci in non-cgi regions  also carried aging-related ontology terms including protein maturation , and negative regulation of cell proliferation  .
in cancer, the aging-related terms such as neuron apoptosis  and muscle organ development  were significantly enriched .
we compared the normal signature with bivalent chromatin domain regions.
previously, human aging-associated dna hypermethylation was found to occur preferentially at bivalent chromatin domains in es cells  <cit> .
interestingly, we found that our normal age-associated hypermethylated loci overlapped significantly with the previously reported bivalent regions .
disruption of age-associated dna methylation signature in cancer
for several of the  <dig> normal age-associated dnam loci, multiple cpg sites were identified in a single gene.
for example, cg <dig> , cg <dig>  and cg <dig>  are located in the diras family, gtp-binding ras-like  <dig>  gene.
diras <dig> is known as a tumor-suppressor gene that is expressed in normal ovarian or breast epithelial cells, but is rarely expressed in tumors  <cit> .
these three loci are in cgi regions that show a positive correlation between dnam levels and age  in normal tissues .
in cancer, however, no correlation with age that observed and methylation levels were generally high regardless of age.
interestingly, the age-associated dnam increases or decreases of the normal signature were aberrantly accelerated in cancer samples, indicating that abnormal acceleration in age-associated dnam change might induce tumorigenesis.
in another example, cg <dig>  and cg <dig> , which are in non-cgi regions, are situated in mbd <dig>  methyl-cpg-binding domain protein  <dig>  which is associated with histone-modifying and chromatin-remodeling complexes  <cit> .
these two loci showed a negative correlation between dnam levels and age in normal tissues.
however, the correlation with age disappeared in cancer samples and the dnam levels of the loci were aberrantly lower .
similar phenomena were also observed in the multiple age-associated cpg loci in myf <dig>   and prr <dig> .figure  <dig>
disruption of age-associated dna methylation signature in cancer.
plots of dna methylation patterns of normal age-associated loci affiliated with diras <dig> , mbd <dig> , myf <dig> , and prr <dig>  in normal  and cancer  samples.
blue or red lines are the linear  regression results for normal or cancer samples, respectively.
plots of dna methylation patterns of tumor suppressor genes including sema3b, rrp <dig> and cdkn2b.
of the normal signature, eight genes were common with the tumor suppressor genes and pttg <dig> was common with the proto-oncogenes listed in the uniprot knowledgebase   <cit> .
the degree of overlap between the normal signature and the tumor suppressor genes was significant .
dnam levels of some tumor suppressor genes including rrp <dig> , cdkn2b , and diras <dig> increased with age; whereas those of sema3b  decreased .
those tumor suppressor genes also showed abnormal acceleration in methylation changes in cancer samples.
interaction network and sequence conservation analysis of the age-associated dna methylation signature
we examined the human protein interaction network of the  <dig> normal age-associated loci, mapped to  <dig> unique genes .
for this human protein interaction network analysis, we integrated protein interactions from a number of open databases .
we found that a protein interaction subnetwork that included the first neighbors of  <dig> gene products under the integrated network included  <dig> proteins and  <dig>  interactions between them.
analysis of the number of interacting neighbors revealed that the age-associated gene products in the normal signature had relatively more interacting partners than the average for all proteins in the prepared interaction network, and the overall distributions differed significantly between them .
furthermore, the  <dig> unique gene products tended not to interact directly with each other: only one interaction existed among the  <dig> gene products .
this indicates that the age-associated gene products cover a large portion of the human protein interaction network.
we also analyzed the dna sequence conservation scores of the  <dig> cpg positions using the average phylop  <cit>  .
the results showed that  <dig> cpg positions  had significantly higher conservation scores  and that this number was significantly higher than random expectations .
gene ontology term analysis of the highly conserved loci showed that aging-associated terms such as metabolic processes , muscle system processes , and cell proliferation  were enriched .
of highly conserved loci, myf <dig> and myf <dig> are associated with myogenic regulation, which is related with a decrease of muscle in mass, strength, and contraction in aging  <cit> .
the sequences surrounding myf <dig> and myf <dig> in cgi are enriched guanine and cytosine nucleotides .figure  <dig>
network characteristics and sequence conservation of the age-associated dna methylation signature.
protein interactions of age-associated dna methylation genes in normal tissue.
blue or green nodes indicate genes hypo- or hypermethylated with age; gray nodes indicate the interacting neighbors of the age-associated gene products.
red or yellow borders of nodes indicate whether the loci are located in cgis  or non-cgis .
edges between nodes indicate the protein–protein interactions of gene products.
node sizes are proportional to numbers of interacting proteins.
the distributions of numbers of interacting protein neighbors from the network shown in  , or from all protein interactions combined , respectively.
the number of direct protein interactions between gene products affiliated with the age-associated dna methylation signature is indicated by a red arrow.
the black curve indicates the distribution of  <dig> random selections of proteins with the same number of the age-associated genes.
the number of the age-associated dna loci with significant the average phylop scores  is indicated by a red arrow.
the black curve indicates the background distribution of the human genome.
surrounding sequences of age-associated loci in myf <dig> and myf <dig>
CONCLUSIONS
in this study, we extensively investigated the relation between dnam and age from a large number of collected samples from a range of diverse studies of normal and cancer tissues.
using these samples and both linear and nonlinear models, we identified tissue type-invariant dnam age-associated signatures for both healthy normal and cancer samples.
we observed that the characteristics of the genomic regions involved in the normal age signature were quite different from those of the cancer signature, and there was no common age-associated locus between them.
the normal age signature was particularly enriched on the x chromosome and satisfactorily predicted the chronological ages of samples of many different tissue types.
moreover, the dnam levels of the normal signature approached the corresponding cancer levels with age.
interaction network analysis showed that normal age-associated gene products had relatively more interacting partners and had a tendency not to interact directly with each other.
the genomic sequences of these age-associated loci were also well conserved.
the age-associated dnam increases or decreases of the normal signature were aberrantly accelerated in cancer samples.
although we could not completely address the biological significance of these characteristics, these findings can be used to address important questions in developmental biology and cancer research.

 dna methylation is one type of epigenetic modification that regulates gene expression heritably. it is catalyzed by dna methyltransferase that adds and maintains a methyl group to the 5′ position of the cytosine ring to form 5′ methyl-cytosine. in mammalian genomes, this modification occurs almost exclusively on cytosine residues that precede guanine. these cpg dinucleotides are generally about 60%–90% methylated. however, cpg islands, which are the genomic regions with the highest cpg density, exhibit the lowest levels of dnam. the potential role of aberrant dnam in human diseases such as cancer, both at a single-gene level and on a genome-wide scale, is important. recently, dnam has also been shown to be associated with aging in a wide array of organisms, ranging from yeast to humans. for example, horvath et al. observed that a genome-wide decrease in dna methylation, preferentially hypermethylation at cgis, occurred during aging. some studies have investigated age-associated methylation of cpg loci dependent on sex, body mass index, specific tissue, or cell type. these studies were performed using various linear-based methods, including conventional linear regression methods, a weighted correlation network method, and a multidimensional scaling method. however, most of these studies were restricted to specific tissue types, and included limited numbers of samples and/or limited ranges of sample ages. more recently, several studies were performed to identify more reliable cpg sites associated with aging, which collected many samples with various tissue types from public data sets. these studies also used linear-based regression methods of analysis. although previous studies have reported that age-associated dnam can show both nonlinear and linear patterns, there has been little identification of age-associated dnam signatures through systematic analysis of nonlinear dnam patterns. more importantly, the characteristics of dnam age signatures that are applicable to multiple types of normal or cancer tissues are not well studied. in this study, we identify for the first time tissue type-invariant dnam age signatures for healthy normal and cancer tissues using linear and nonlinear models. for more reliable signatures, we collected diverse samples from a range of studies available in public resources that included multiple tissue types. after identifying the dnam age signatures from normalized dnam levels of the samples, we extensively investigated the characteristics of the signatures and their biological meaning through a number of analyses, including analysis of changes in dnam pattern with age, gene ontology term analysis, and network and conservation analysis. we also compared the signatures with the results of previous studies. finally, we checked that the signatures could be used as an age predictor for multiple tissue types.  discovery of age-associated dna methylation signatures to identify robust age-associated dnam signatures, we first searched and downloaded various dnam profiles from diverse studies available in the gene expression omnibus database. we then excluded studies without age information or with small numbers of samples. we also excluded samples of diseased tissues other than cancer. it is known that technical bias exists across different array platforms, so we considered only the illumina infinium humanmethylation bead chip array, which was the most widely used among the downloaded profiles. consequently, we collected dnam profiles of samples from eight studies available in the geo. additionally, we downloaded publicly available dnam profiles of five cancer types evaluated on the same platform from the cancer genome atlas consortium. in total, we gathered dnam profiles of normal healthy and cancer samples. these samples included diverse tissue types and exhibited a wide range of ages from to 91 years. we next normalized dnam levels using a single beta-score measure that indicates conceptually the normalized levels of dnam. the normalized dnam levels were well correlated between normal or cancer samples, but had higher correlation scores between normal samples, and for both normal and cancer tissues, the dnam levels in cgi regions of individual samples were much lower than those in non-cgis. moreover, the dnam levels of normal and cancer tissue showed different patterns depending on the genomic regions. in cgis, for example, the average dnam levels were generally higher in cancer than those in normal tissue, except for ovarian cancer samples.figure study overview. sources of dnam data with sample information. eight studies from geo and five from open tcga data were included. identifying an age-associated dnam signature. linear and nonlinear regression models using single or combined studies were applied. age prediction and characterization of identified age-associated signatures. various analyses using dnam patterns and distributions, gene ontology, and protein networks in normal and cancer tissues were performed. dna methylation correlation and age-associated cpg loci across individual studies. heat map representing the pearson’s correlation coefficients between all samples of normal and cancer tissues used in this study. a hierarchical clustering was used. box plot representing the correlations between normal samples or between cancer samples. p-value was calculated using a wilcoxon rank-sum test. examples of cpg loci hypermethylated with age in the gse study. examples of cpg loci hypomethylated with age in the gse study. r: correlation coefficient. the number of significant age-associated cpg loci in normal or cancer tissues from individual studies. the number and color in each cell corresponds to the number of significant age-associated cpg loci and its significance in terms of a p-value, respectively. p-value: z-test result using the random distribution of the age-permutation tests. na: not available. next, using various regression methods, we identified an aging signature associated with the normalized dnam levels. because some studies have reported that methylation levels can change most dramatically during childhood, we applied nonlinear regression models as well as linear regression models. after finding statistically significant age-associated dnam sites using single studies or combined multiple studies, we examined the characteristics and biological meaning of the sites through analysis of the associations of dnam patterns with age, gene ontology, sequence conservation, and protein networks. we also compared the age-associated dna loci across different studies or tissue types. to identify tissue type-invariant age-associated dnam signatures, we integrated data from all samples of normal tissues, cancers, or both, after removing noisy samples. we compared the methylation patterns of the normal signature to those in cancer according to different genomic regions. we finally checked the potential for age prediction using the methylation levels of the age-associated signature, regardless of tissue type. cpg loci are widely associated with age in disease-free normal samples the distribution of dnam levels showed a significant disparity between normal and cancer tissues depending on the genomic region. we first checked age-associated dnam sites separately in normal or cancer samples using a linear regression model. for example, cg and cg sites showed linear hypermethylation patterns according to age in normal samples from the gse study. in contrast, hypomethylation patterns were observed at cg and cg in normal samples from the gse study. we checked the number of significantly age-associated cpg loci in each study. the numbers of significant cpg loci were quite different between studies, mainly because of different ranges of age and/or different numbers of samples across studies. although the numbers of age-associated loci in normal tissues varied between studies, the numbers were significant in all studies included. in cancer samples, however, the numbers of age-associated loci were not significant in some studies, including gse and gse individual studies included samples of various tissue types with different age ranges. therefore, the average dnam levels per cpg site were quite different between studies in both disease-free normal samples and cancer samples. similar results were also observed for the average dnam levels per sample unit. however, most study pairs with normal samples showed significantly greater degrees of overlap of age-associated cpg loci than would be expected by chance. moreover, the results of hierarchical clustering of the p-values of the degrees of overlap demonstrated that common age-associated cpg loci were independent of tissue or cell type. in the case of cancer samples, the degrees of overlap of age-associated cpg loci between study pairs were also significant, but less so than for normal samples.figure comparison of age-associated cpg loci across different studies with different tissue types. box plots of average methylation values per cpg unit in normal and cancer tissues across individual studies. box plots of average methylation values per sample unit in normal or cancer tissue across individual studies. p-values were calculated by kruskal–wallis tests. we checked the degree of overlap of age-associated cpg loci between studies by calculating the number of common cpg loci. we performed age-permutation tests with the samples of individual studies to verify the significance of the degree of overlap. hierarchical clustering results using the degree of overlap of age-associated cpg loci between different studies with different tissue types in normal and cancer. p-value: a z-test result using the distribution of random selections. age-associated dna methylation signatures in normal and cancer to identify tissue type-invariant age-associated cpg loci, we integrated the normalized dnam levels of all the disease-free normal samples after removing noisy samples. using this integrated data set, we first identified cpg loci with a linear relationship with age. for example, the cg site was linearly hypomethylated and cg was linearly hypermethylated according to age, regardless of tissue type. however, dnam levels of some loci showed nonlinear patterns according to age. for these nonlinear relationships, we also observed more rapid changes in dnam levels at younger ages, which is consistent with previous studies. we also observed similar phenomena for gene-level dnam levels. we thus identified tissue type-invariant age-associated dna methylation signatures using second- and third-degree nonlinear regression models in addition to the linear model. for the threshold, we used three measures, including false discovery rate, correlation coefficient, and residual error, to reduce tissue-type variations. we identified unique cpg loci in the combined normal samples, which we termed a “tissue type-invariant age-associated dnam signature”. among these, cpg loci had a linear relationship with age and the other loci were identified using nonlinear models. seventy-seven loci were hypermethylated and were hypomethylated with age. we also applied a similar approach to examine the dnam levels of the combined cancer samples. compared with normal samples, only age-associated cpg loci were identified. interestingly, there was no cpg locus common to the normal and cancer age-associated signatures. these epigenetic phenomena were also observed with the gene-level dnam values. in case of the combined normal and cancer samples, only cpg loci were identified as age-associated. we examined the positions on human chromosomes of the age-associated cpg loci of each of the signatures by separating hypomethylated and hypermethylated loci, in normal, cancer, or combined samples. generally, the age-associated loci in normal tissue were distributed throughout the human genome, except for chromosomes and in contrast to a previous study using male pediatric samples, the x chromosome had the largest number of age-associated loci. this difference may be caused by differences in sex, age range, and tissue types. we checked the significance of the numbers of loci by chromosome using hypergeometric tests. chromosomes x,,,, and were preferentially enriched for hypermethylated loci with age, whereas chromosomes y, x,, and were enriched for hypomethylated loci. thus, the sex chromosomes, especially x, were enriched for age-associated cpg loci in disease-free normal tissues. in cancer samples, chromosomes,,,,,, and were enriched with age-associated hypomethylated loci. interestingly, there were no age-associated loci on the x or y chromosomes in cancer samples. with the normal and cancer samples matched in age distribution, we also observed similar trends such as no overlap in signature between the normal and cancer samples.figure age-associated dna methylation signature independent of tissue type. examples of age-associated cpg loci with linear or nonlinear relationships identified in integrated normal samples. venn diagrams showing the numbers of age-associated cpg loci with three regression models in integrated normal, cancer, or all samples. venn diagram showing the number of age-associated cpg loci among integrated normal, cancer, and all samples. manhattan plot of age-associated cpg loci in integrated normal samples by chromosome. hypermethylated cpg loci with age are shown with a –log and hypomethylated loci are shown with a log. the most significant p-values among linear and nonlinear models were chosen. significant loci are marked as green or blue dots. the numbers of significant age-associated cpg loci by chromosome. bar plots of p-values with hypergeometric tests for the degrees of significance of the numbers of the loci. manhattan plot of age-associated cpg loci in integrated cancer samples by chromosome. it was previously suggested that a difference in methylation variation might exist with gender. we therefore identified age-associated signatures for males and females separately. we found and cpg loci in the male and female samples, respectively. even though the number of age-associated loci differed between male and female, their ratio distributions by chromosome were similar. moreover, the number of hyper- and hypomethylated loci on the x chromosome were similar between males and females. we built a feasible age-prediction model to see whether the normal 127-site signature could be used as a tissue-invariant age predictor. we applied a multiple linear regression model after identifying a feasible subset of the signature using a genetic algorithm. the selected age-prediction model was composed of cpg loci of the signature. the correlation between the actual ages of the combined normal samples and their predicted ages using the model was highly significant, which indicates that the dnam levels of the age-associated signature sites can be used to predict the age of tissues, regardless of tissue type. we next compared the age-associated normal signature with those identified in previous studies. most previous studies identified age-associated loci using a fdr threshold in a linear model. thus, we compared the loci resulting from only linear regression and found that age-associated cpg loci were age-associated in the integrated normal samples. for instance, a recent study using the illumina human 450 k platform and a linear regression model identified cpg loci associated with age in blood cells of healthy subjects aged from to 94 years. of these loci, the cpg loci present on the illumina 27 k overlapped 73% with our age-associated loci. another study by day et al. found that cpg loci correlated with age in four tissue types, including brain samples, using a linear regression method, and the degree of overlap with our loci was 47%. notably, we observed higher degrees of overlap of cpgs with previous studies that used only normal samples than with other studies that included diseased samples. sixteen of our age-associated loci were not identified in the previous studies. interestingly, of these loci were located on the x chromosome.figure characteristics of age-associated dna methylation signature. age prediction using the age-associated normal dnam signature. age was predicted with the normal signature using a multivariate linear regression after using a genetic algorithm to identify a feasible set of loci. degrees of overlap with age-associated dnam signatures identified in previous studies. overlap percentages were calculated by the common numbers divided by the smaller number of total loci in either study. the studies with only normal samples are orange; other studies including disease samples are gray. the fractions of hyper- or hypomethylated cpg loci in the age-associated signatures in normal or cancer according to genomic regions. the number on each bar indicates the count of the corresponding loci. p-value was calculated by a chi-square test. the hyper- or hypomethylation patterns according to age group of normal age-associated dna loci in cgi or non-cgi. blue or green dotted lines show the linear regressions of median values of individual age groups using only hypo- or hypermethylated loci, respectively. numbers below are the counts of loci considered for the corresponding cases. nucleotide compositions of the sequences surrounding the hypo- or hypermethylated loci of normal age-associated dnam signature. –log of the y axis was calculated by random selection tests representing overrepresentation for each base at each location of the surrounding cpg. our collected dnam profiles included diverse tissue types in the normal samples. we next identified tissue-type-specific age-associated signatures. although the number of age-associated loci in normal samples varied from one to loci across tissue types, most tissue-type pairs showed significant degrees of overlap of the age-associated cpg loci compared with random expectation, except for the prostate tissue samples. in the cancer samples, we could not find significant tissue-type-specific age-associated loci with the threshold we used in most cases. characteristics of tissue type-invariant age-associated dna methylation signature we investigated the genomic locations of the loci in the age-associated dnam signatures in normal or cancer samples. of the loci in normal samples, were located in cgi regions and the others in non-cgi regions; whereas in cancer, loci were located in cgis and four in non-cgis. thus, while the normal signature was enriched in cgi regions, the cancer signature was even more enriched in cgi regions. in the normal age-associated signature, hypermethylated loci were enriched in cgi regions, whereas hypomethylated loci were enriched in non-cgis. interestingly, the cancer signature had only age-hypomethylated loci in both cgi and non-cgi regions. similar results were also detected for gene-level dnam patterns. we next checked the changing rates of dna methylation in the normal signature within cgi or non-cgi regions. age-hypermethylated loci increased more rapidly in cgi regions. in contrast, hypomethylated loci decreased more rapidly in non-cgi regions. the cancer signature of only hypomethylated loci showed much smaller changes with age than did the normal signature. next, we investigated the nucleotide composition surrounding the cpg loci in the normal signature. the sequences surrounding the normal signature showed significant overrepresentation of thymine residues at - - + + + and + bases from the cpg loci, adenine residues at + and + bases, and guanine residues at - base. interestingly, the sequence motifs surrounding age-associated cpg loci were quite different between hypermethylated and hypomethylated loci. sequences surrounding age-hypomethylated loci presented at-rich sequences, whereas the sequences surrounding age-hypermethylated loci were enriched for gc-rich sequences; these are also enriched in cgi regions. these phenomena were also observed in the cancer signature. analysis of gene ontology descriptors for the age-associated dna methylation signature in normal samples indicated that the aging-related terms such as regulation of protein kinase activity, metabolic processes, immune system processes, and neuron differentiation were significantly enriched in the cgi regions. genes related to age-associated loci in non-cgi regions also carried aging-related ontology terms including protein maturation, and negative regulation of cell proliferation. in cancer, the aging-related terms such as neuron apoptosis and muscle organ development were significantly enriched. we compared the normal signature with bivalent chromatin domain regions. previously, human aging-associated dna hypermethylation was found to occur preferentially at bivalent chromatin domains in es cells. interestingly, we found that our normal age-associated hypermethylated loci overlapped significantly with the previously reported bivalent regions. disruption of age-associated dna methylation signature in cancer for several of the normal age-associated dnam loci, multiple cpg sites were identified in a single gene. for example, cg, cg and cg are located in the diras family, gtp-binding ras-like gene. diras is known as a tumor-suppressor gene that is expressed in normal ovarian or breast epithelial cells, but is rarely expressed in tumors. these three loci are in cgi regions that show a positive correlation between dnam levels and age in normal tissues. in cancer, however, no correlation with age that observed and methylation levels were generally high regardless of age. interestingly, the age-associated dnam increases or decreases of the normal signature were aberrantly accelerated in cancer samples, indicating that abnormal acceleration in age-associated dnam change might induce tumorigenesis. in another example, cg and cg, which are in non-cgi regions, are situated in mbd methyl-cpg-binding domain protein which is associated with histone-modifying and chromatin-remodeling complexes. these two loci showed a negative correlation between dnam levels and age in normal tissues. however, the correlation with age disappeared in cancer samples and the dnam levels of the loci were aberrantly lower. similar phenomena were also observed in the multiple age-associated cpg loci in myf and prr.figure disruption of age-associated dna methylation signature in cancer. plots of dna methylation patterns of normal age-associated loci affiliated with diras, mbd, myf, and prr in normal and cancer samples. blue or red lines are the linear regression results for normal or cancer samples, respectively. plots of dna methylation patterns of tumor suppressor genes including sema3b, rrp and cdkn2b. of the normal signature, eight genes were common with the tumor suppressor genes and pttg was common with the proto-oncogenes listed in the uniprot knowledgebase. the degree of overlap between the normal signature and the tumor suppressor genes was significant. dnam levels of some tumor suppressor genes including rrp, cdkn2b, and diras increased with age; whereas those of sema3b decreased. those tumor suppressor genes also showed abnormal acceleration in methylation changes in cancer samples. interaction network and sequence conservation analysis of the age-associated dna methylation signature we examined the human protein interaction network of the normal age-associated loci, mapped to unique genes. for this human protein interaction network analysis, we integrated protein interactions from a number of open databases. we found that a protein interaction subnetwork that included the first neighbors of gene products under the integrated network included proteins and interactions between them. analysis of the number of interacting neighbors revealed that the age-associated gene products in the normal signature had relatively more interacting partners than the average for all proteins in the prepared interaction network, and the overall distributions differed significantly between them. furthermore, the unique gene products tended not to interact directly with each other: only one interaction existed among the gene products. this indicates that the age-associated gene products cover a large portion of the human protein interaction network. we also analyzed the dna sequence conservation scores of the cpg positions using the average phylop. the results showed that cpg positions had significantly higher conservation scores and that this number was significantly higher than random expectations. gene ontology term analysis of the highly conserved loci showed that aging-associated terms such as metabolic processes, muscle system processes, and cell proliferation were enriched. of highly conserved loci, myf and myf are associated with myogenic regulation, which is related with a decrease of muscle in mass, strength, and contraction in aging. the sequences surrounding myf and myf in cgi are enriched guanine and cytosine nucleotides.figure network characteristics and sequence conservation of the age-associated dna methylation signature. protein interactions of age-associated dna methylation genes in normal tissue. blue or green nodes indicate genes hypo- or hypermethylated with age; gray nodes indicate the interacting neighbors of the age-associated gene products. red or yellow borders of nodes indicate whether the loci are located in cgis or non-cgis. edges between nodes indicate the protein–protein interactions of gene products. node sizes are proportional to numbers of interacting proteins. the distributions of numbers of interacting protein neighbors from the network shown in, or from all protein interactions combined, respectively. the number of direct protein interactions between gene products affiliated with the age-associated dna methylation signature is indicated by a red arrow. the black curve indicates the distribution of random selections of proteins with the same number of the age-associated genes. the number of the age-associated dna loci with significant the average phylop scores is indicated by a red arrow. the black curve indicates the background distribution of the human genome. surrounding sequences of age-associated loci in myf and myf  in this study, we extensively investigated the relation between dnam and age from a large number of collected samples from a range of diverse studies of normal and cancer tissues. using these samples and both linear and nonlinear models, we identified tissue type-invariant dnam age-associated signatures for both healthy normal and cancer samples. we observed that the characteristics of the genomic regions involved in the normal age signature were quite different from those of the cancer signature, and there was no common age-associated locus between them. the normal age signature was particularly enriched on the x chromosome and satisfactorily predicted the chronological ages of samples of many different tissue types. moreover, the dnam levels of the normal signature approached the corresponding cancer levels with age. interaction network analysis showed that normal age-associated gene products had relatively more interacting partners and had a tendency not to interact directly with each other. the genomic sequences of these age-associated loci were also well conserved. the age-associated dnam increases or decreases of the normal signature were aberrantly accelerated in cancer samples. although we could not completely address the biological significance of these characteristics, these findings can be used to address important questions in developmental biology and cancer research. 
we studied approximately normal and cancer samples with multiple tissue types from diverse studies, and using linear and nonlinear regression models identified reliable tissue type-invariant dnam age signatures. these tissue type-invariant dnam age signatures in normal and cancer can be used to address important questions in developmental biology and cancer research. dna methylation levels can be used to predict the chronological age of tissues; however, the characteristics of dnam age signatures in normal and cancer tissues are not well studied using multiple studies. however, the cancer signature comprised only age-hypomethylated loci, none on the x chromosome, and with no overlap with the normal signature. genes related to the normal signature were enriched for aging-related gene ontology terms including metabolic processes, immune system processes, and cell proliferation. age-hypermethylated loci were enriched for guanine–and-cytosine-rich regions in cpg islands, whereas age-hypomethylated loci were enriched for adenine–and-thymine-rich regions in non-cgis. the related gene products of the normal signature had more than the average number of interacting partners in a protein interaction network and had a tendency not to interact directly with each other. the genomic sequences of the normal signature were well conserved and the age-associated dnam levels could satisfactorily predict the chronological ages of tissues regardless of tissue type. a normal signature comprising cpg loci was highly enriched on the x chromosome. interestingly, the age-associated dnam increases or decreases of the normal signature were aberrantly accelerated in cancer samples.
 dna methylation levels can be used to predict the chronological age of tissues; however, the characteristics of dnam age signatures in normal and cancer tissues are not well studied using multiple studies.  we studied approximately normal and cancer samples with multiple tissue types from diverse studies, and using linear and nonlinear regression models identified reliable tissue type-invariant dnam age signatures. a normal signature comprising cpg loci was highly enriched on the x chromosome. age-hypermethylated loci were enriched for guanine–and-cytosine-rich regions in cpg islands, whereas age-hypomethylated loci were enriched for adenine–and-thymine-rich regions in non-cgis. however, the cancer signature comprised only age-hypomethylated loci, none on the x chromosome, and with no overlap with the normal signature. genes related to the normal signature were enriched for aging-related gene ontology terms including metabolic processes, immune system processes, and cell proliferation. the related gene products of the normal signature had more than the average number of interacting partners in a protein interaction network and had a tendency not to interact directly with each other. the genomic sequences of the normal signature were well conserved and the age-associated dnam levels could satisfactorily predict the chronological ages of tissues regardless of tissue type. interestingly, the age-associated dnam increases or decreases of the normal signature were aberrantly accelerated in cancer samples.  these tissue type-invariant dnam age signatures in normal and cancer can be used to address important questions in developmental biology and cancer research. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords dna methylationepigeneticsage signatureagingmeta-analysissystems biologycancerissue-copyright-statement© the author 2014 