Human longevity is always a biological hotspot and so much effort has been devoted to identifying genes and genetic variations associated with longer lives. Most of the demographic studies have highlighted that females have a longer life span than males. The reasons for this are not entirely clear. In this study, we carried out a pool-based, epigenome-wide investigation of DNA methylation profiles in male and female nonagenarians/centenarians using the Illumina 450 K Methylation Beadchip assays. Although no significant difference was detected for the average methylation levels of examined CpGs (or probes) between male and female samples, a significant number of differentially methylated probes (DMPs) were identified, which appeared to be enriched in certain chromosome regions and certain parts of genes. Further analysis of DMP-containing genes (named DMGs) revealed that almost all of them are solely hypermethylated or hypomethylated. Functional enrichment analysis of these DMGs indicated that DNA hypermethylation and hypomethylation may regulate genes involved in different biological processes, such as hormone regulation, neuron projection, and disease-related pathways. This is the first effort to explore the gender-based methylome difference in nonagenarians/centenarians, which may provide new insights into the complex mechanism of longevity gender gap of human beings.
Over the last 100 years, humans experienced a huge increase of life expectancy. These advances were largely driven by extrinsic improvements of their living environment (such as diet and disease prevalence) as well as genetic variations (such as polymorphism and DNA methylation). Since human aging and longevity is a very complex trait where environmental, genetic, and stochastic factors are involved, it has largely aroused the attention of scientists around the world. A great number of studies have been carried out to investigate the mechanisms and key factors that may influence human mortality, aging, and lifespan [
As specific cohorts, nonagenarians and centenarians are always considered as the most valuable models to study the mechanisms involved in human aging and longevity [
Currently, the majority of genome-based studies focused on the association between longevity and sequence variations including single nucleotide polymorphism or copy number variation [
A significant trend observed in most parts of the world is that females have a longer life span than males. In particular, when nonagenarians and centenarians are considered, the male/female ratio has been reported to range between 1 : 4 and 1 : 7 [
China has the largest population of adults aged 60+ years in the world [
This project is an extension of the “Longevity and Health of Aging Population in Guangxi China” project conducted in 2008 and 2010 [
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood for genomic DNA extraction using the Qiagen mini kit (Qiagen, Germany) following the manufacturer’s protocol. DNA concentrations were determined by NanoDrop microvolume quantitation assay and 1% agarose electrophoresis. After validation of quality and integrity of individual genomic DNA, we equally pooled each sample into male and female groups, respectively.
The prepared genomic DNA (0.5
As described above, the measurement of whole genome DNA methylation used a pool-based approach, in which the
Gene ontology (GO) and KEGG (Kyoto encyclopedia of genes and genomes) pathway enrichment analyses were conducted using the
It has been suggested that DNA pooling allows accurate assessment of average DNA methylation in large groups of individual genomes [
A general view of whole genome methylation profiles in autosomes of male and female nonagenarians/centenarians from Yongfu County in China was shown in Figure
General view of DNA methylation level for autosomes of Chinese nonagenarians/centenarians. The average genome-wide DNA methylation levels in male and female samples are represented using Circos. The inner and outer tracks indicate the average methylation levels for female and male samples, respectively. All autosomes are represented via 10 Mbp-wide windows. The average methylation level in each region represents the average
The discrepancies of DNA methylomes between male and female nonagenarians/centenarians prompted us to search for particular DMPs. In this study, DMPs were predicted based on a Gaussian model with the same mean and variation of
Distribution of DMPs between male and female samples. (a) Circos representation of the total number of DMPs in each region. The number is calculated using 10 Mbp-wide windows for each autosome. (b) Distribution of DMPs according to different regions of genes.
We further examined the location of DMPs based on different parts of genes: 1500 bp above transcription start site (TSS1500), 200 bp above TSS (TSS200), 5′ untranslated region (5′-UTR), the 1st exon, gene body (other exons except the 1st exon), and 3′-UTR (Figure
We also analyzed the trend of methylation changes of DMPs. The majority of DMPs (54.5%) were hypermethylated in female compared to those in male samples (Figure
Distribution of hypermethylated and hypomethylated DMPs and DMGs. (a) Distribution of hypermethylated and hypomethylated DMPs. (b) Distribution of hypermethylated and hypomethylated DMPs in different parts of genes. (c) Distribution of hypermethylated, hypomethylated, and mixed DMGs.
To investigate the potential relationship between DMPs and genes, all DMPs were mapped to 564 genes (named differential methylated genes or DMGs; see Table S1 in Supplementary Material available online at
It is known that epigenetic changes may affect the aging process and may be one of the central mechanisms of many age-related diseases [
To extrapolate the biological processes of DMGs, a
GO analysis of hypomethylated and hypermethylated DMGs.
GO ID | Description |
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FDR |
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Hypomethylated genes | |||
Biological process | |||
GO:0016043 | Cellular component organization |
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GO:0071840 | Cellular response to vitamin A |
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GO:0071299 | Cell surface receptor linked signaling pathway |
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GO:0007166 | Regulation of hormone levels |
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GO:0010817 | Cell projection organization |
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GO:0030030 | Hormone secretion |
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GO:0046879 | Cellular component organization at cellular level |
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GO:0071842 | Cellular response to vitamin |
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GO:0071295 | Regulation of transcription from RNA polymerase II promoter |
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GO:0006357 | Epithelial cell development |
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GO:0002064 | Hormone transport |
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GO:0009914 | Production of molecular mediator involved in inflammatory response |
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GO:0002532 | Cell morphogenesis involved in differentiation |
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GO:0000904 | Transmembrane receptor protein tyrosine kinase signaling pathway |
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GO:0007169 | Wnt receptor signaling pathway |
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Cellular component | |||
GO:0015629 | Actin cytoskeleton |
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Hypermethylated genes | |||
Biological process | |||
GO:0000902 | Cell morphogenesis |
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GO:0021955 | Central nervous system neuron axonogenesis |
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GO:0048869 | cellular developmental process |
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GO:0032989 | cellular component morphogenesis |
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GO:0048858 | cell projection morphogenesis |
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GO:0032990 | cell part morphogenesis |
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GO:0051179 | localization |
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GO:0048667 | cell morphogenesis involved in neuron differentiation |
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GO:0030154 | cell differentiation |
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Cellular component | |||
GO:0044459 | plasma membrane part |
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GO:0016020 | membrane |
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GO:0044425 | membrane part |
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GO:0005911 | cell-cell junction |
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GO:0030054 | cell junction |
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Molecular function | |||
GO:0015108 | chloride transmembrane transporter activity |
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GO:0015103 | inorganic anion transmembrane transporter activity |
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GO:0015296 | anion:cationsymporter activity |
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GO:0004714 | glycoprotein binding |
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Hypomethylated DMGs were mainly enriched in cellular component organization, cell surface receptor signaling, hormone regulation, and some disease-related pathways (such as Wnt receptor signaling pathway). It has been known for a long time that Wnt signaling pathway may lead to tumor development [
On the other hand, hypermethylated DMGs were found to be enriched in cell morphogenesis, cell-cell junction, and cell projection. Some of the enriched biological processes are known to be involved in neuron projection and central nervous system development [
KEGG analysis showed that very few pathways could be significantly enriched for either hypomethylated or hypermethylated DMGs (Table
KEGG pathway enrichment analysis of hypomethylated and hypermethylated DMGs.
KEGG ID | Description |
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FDR |
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Hypomethylated genes | |||
KEGG:04512 | ECM-receptor interaction |
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Hypermethylated genes | |||
KEGG:04360 | Axon guidance |
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KEGG:04514 | Cell adhesion molecules (CAMs) |
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Although the
We are collecting samples for examining the DNA methylomes of male and female adults from Yongfu County to identify epigenetic patterns that may be related to the mechanisms of longevity in the Han Chinese population. Based on our preliminary data, less DMPs in autosomes and more DMPs in sex chromosomes were observed in adults than in nonagenarians/centenarians (unpublished data). The average methylation level was significantly higher in both male and female adult samples compared to those in nonagenarians/centenarians, implying that hypomethylation in certain genomic regions may be related to longer life span. Hormone regulation and cell morphogenesis regulation appeared to be important for gender-specific longevity. These findings may help us figure out the difference of aging process between male and female. However, as longevity is a very complex trait, it should be understood that reliance on a single aspect of genetics has its limitations. In the future, by increasing the sample size, generating different levels of data, and developing more reliable methods, these defects may be rectified, providing scientists with more opportunities to explore in detail the role of DNA methylation and other factors involved in gender-specific longevity.
In summary, we are unique in reporting a comprehensive comparison of DNA methylome between male and female nonagenarians/centenarians in a Han Chinese population. The average methylation level in female samples was similar to that in male samples in spite of the fact that a significant number of DMPs were identified. These DMPs prefer to be enriched in certain chromosome regions. Further analysis of DMPs in different parts of genes revealed that most of them are located in gene body regions. Almost all of the DMGs are solely hypermethylated or hypomethylated. Functional enrichment analysis of these genes revealed that DNA hypermethylation and hypomethylation may regulate genes involved in different processes or pathways, some of which may contribute to the gender gap of life span. In addition, identification of
The authors have declared that no competing interests exist.
Liang Sun and Jie Lin contributed equally to the work.
This work was supported by Beijing Nova program (Z121107002512058), National Natural Science Foundation of China (no. 81370445, 31171233), and a grant from Chinese Academy of Sciences (CAS) (2012OHTP10).