Introduction

Advanced maternal age during pregnancy has been associated with adverse birth outcomes [1–4] as well as health problems in children (childhood cancer [5], type 1 diabetes [6], and neurodevelopmental disorders [7,8]). The biologic mechanisms underlying most of these associations remain unknown. One mechanism by which maternal age could influence the health of offspring is through epigenetic modifications such as DNA methylation. DNA methylation refers to the addition of a methyl group to the 5’ position of a cytosine at cytosine-guanine dinucleotides (CpGs) and has been associated with a growing number of diseases.

During development, the parental genomes are completely demethylated between fertilization and implantation. New methylation marks start to be established around the time of blastocyst implantation, and continue throughout fetal growth [9]. Some of the only exceptions are a relatively small number of imprinted loci. Placement of the early epigenetic marks is of growing interest because of their potential for modulating gene transcription, development, and disease risk. In particular, DNA methylation has been proposed [10] as a mechanism by which parental or fetal exposure could determine subsequent risk of adult diseases (the “developmental origins of disease” hypothesis [11]). The mechanisms by which remethylation is controlled during development at various sites across the genome are still unknown; there are very few documented examples of parental exposures or direct fetal exposure in utero that lead to persistent epigenetic change in the offspring [12,13].

It is well established that as people age, there are changes in the levels of DNA methylation in blood and various other tissues (for example [14]). However, the epigenetic consequences of advanced maternal age in offspring have been little explored. Support for an epigenetic effect includes reports of age-related gene-expression changes in both mouse [15] and human oocytes [16]; some of these differentially expressed genes play a role in chromatin structure and DNA methylation. Age-related changes in DNA methylation have also been observed in mouse oocytes and mouse and human sperm [17–19]. To date, only one study has investigated the relationship between parental age and DNA methylation in offspring [20]. Although a number of maternal age-related DNA methylation changes were reported, the study was limited by a small sample size, the use of a low-density DNA methylation microarray, and the lack of an independent population for replication of their findings. Thus, the possible effects of maternal age on the newborn epigenome, and the persistence of such effects if they exist, remain mostly unexplored.

Results

In this epigenome-wide association study (EWAS), maternal age at delivery was associated with decreased methylation at four sites in the newborn epigenome. Using samples from a study of Norwegian newborns (NFCS), we identified four adjacent CpGs near exon 2 of KLHL35 (kelch-like family member 35) with P-values ranging from 3.3x10^-6 to 8.1x10^-7 (Figs 1 and 2; S2 Table: Model1; S2–S4 Figs). Although not significant after strict Bonferroni correction for 465,525 tests (genome-wide threshold p < 1.07x10^-7; S5 Fig), these CpGs showed surprisingly large effect sizes (Fig 1; S2 and S3 Tables): The mean methylation level (average β-values) for these four CpGs was 40%. A 20-year increase in maternal age would be estimated to decrease average absolute methylation at these sites by roughly 12.5% (Fig 3), representing a 35% relative decrease in methylation. We found no corresponding effect of paternal age on methylation levels at these sites (S4 Table).

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Fig 1. Epigenome-wide association results of maternal age in NFCS newborns.

Volcano plot showing the relationship between the effect magnitude (coefficient) and strength of association (p-value) for Model1. The red horizontal line denotes the strict threshold for epigenome-wide significance based on a conservative Bonferroni correction for 465525 tests (p < 1.07x10^-7). The four CpGs that are circled in purple are near the second exon of the gene, KLHL35.

https://doi.org/10.1371/journal.pone.0156361.g001

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Fig 2. KLHL35 methylation association results in NFCS.

All KLHL35 CpGs present on the Illumina 450K chip are shown as rectangles above the gene diagram. Each CpG is colored according to its mean absolute β-value adjusted for technical factors: red (70–100%), blue (30–70%), and orange (0–30%). The four CpGs that are circled in purple are near the second exon and show the strongest associations. The green rectangles below the gene diagram mark the CpG islands (UCSC, GC content ≥ 50%, length > 200 bp with islands < 300 bp shown in light green, Observed/Expected CpGs > 0.6). The red horizontal line denotes the strict threshold for epigenome-wide significance based on a conservative Bonferroni correction for 465525 tests (p < 1.07x10^-7).

https://doi.org/10.1371/journal.pone.0156361.g002

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Fig 3. Maternal age-related DNA methylation changes in newborns (NFCS and MoBa) and adults (Sister Study) per 20 years.

Methylation at each CpG (β-value) was tested for association with maternal age and the β coefficients from the regression analysis were converted into the percent difference in methylation per 20 years (maternal age). The covariates included in Model1 differed slightly across the NFCS, MoBa, and the Sister Study analyses (S1 Text).

https://doi.org/10.1371/journal.pone.0156361.g003

The Adkins study of maternal age and methylation (based on 168 newborns) does not allow us to attempt to replicate our findings, as the four CpGs identified in our study were not covered by the Illumina 27K array used by Adkins [20]. None of the associations reported by Adkins were replicated in our data.

We sought replication of the four KLHL35 CpGs associated with maternal age in an independent study of Norwegian newborns (MoBa). Although effect sizes were somewhat smaller, all four CpGs near exon 2 showed statistically significant decreases in methylation (p<0.05, with 3 of the 4 meeting an appropriate Bonferroni correction; Fig 3 and S5 Table).

To assess the long-term persistence of these maternal-age effects, we conducted a second replication of the candidate KLHL35 CpGs in a smaller sample of adult women from the Sister Study [21]. All four CpGs near exon 2 showed reduced methylation with advanced maternal age (p<0.05, although none met an appropriate Bonferroni correction). Moreover, the reduction in methylation levels were remarkably similar to those found in the original analysis of NFCS newborns (Fig 3 and S5 Table: Model1).

Discussion

We have identified a distinctive pattern of reduced DNA methylation in the blood of newborn infants that is linked to the mother’s age at delivery. Importantly, this pattern at four CpG sites in the KLHL35 gene was replicated in an independent sample of Norwegian newborns, and again in a sample of adult US women. Although we were unable to obtain serial biologic samples from the same individuals, the identification of the same maternal age-related DNA methylation changes in both newborns and adults suggests that these changes likely persist from birth for more than 40 years. To date, there are very few examples of parental exposures before conception, or even direct fetal exposure in utero, leading to a persistent epigenetic change throughout life [12,13]. While the reported maternal age association could be due to unmeasured or uncontrolled confounding, we have adjusted for several factors in our statistical model, including infant’s birth weight, maternal alcohol use, maternal smoking, maternal education, and parity. Additional factors such as gestational age, maternal body mass index, multivitamin use, dietary folate, and folic acid supplement use during pregnancy were explored but not included in the model as potential confounders as they were not associated with maternal age at delivery (p<0.05).

A mechanism by which maternal age might lead to persistent epigenetic differences in the offspring is unclear. These changes could be due to epigenetic inheritance of an altered maternal chromatin state. Perhaps more likely is that an altered in utero environment affects the re-establishment of DNA methylation marks within KLHL35 after the initial wave of demethylation. In the absence of experimental data, it is unclear whether the altered DNA methylation state is biologically significant, influencing gene expression or chromatin structure. While CpGs at only one gene were found to show this methylation pattern with maternal age, there are vast numbers of CpG sites not captured by the 450K panel. If these sites are affected by maternal age, it seems plausible that other sites could also be affected. Further, our study only examined one form of epigenetic mechanism, thus there remains the possibility that other forms of epigenetic modifications, such as histone modifications or miRNAs, could also be influenced by maternal age.

Not much is known about KLHL35. This gene is part of the kelch-like gene family [22] and has been associated with cancer. In particular, KLHL35 is hypermethylated in hepatocellular carcinoma [23], renal cell carcinoma [24], and various other cancers, based on data from The Cancer Genome Atlas [14]. In addition, RNAi knockdown of KLHL35 in human embryonic kidney cells results in an anchorage-independent growth advantage [24]. Taken together, epigenetic dysregulation of KLHL35 appears to be a feature shared across multiple cancers.

An unusual feature of the implicated CpG sites in the KLHL35 gene is that they exhibit intermediate levels of absolute DNA methylation (Adjusted β-values in NFCS range from: 0.30 to 0.54). CpG methylation levels are more typically near 100% or zero percent at given sites. One way a CpG site might have intermediate levels of methylation is if one parental allele is imprinted (with the different methylation levels in the maternal and paternal alleles averaging out to an apparent intermediate level of methylation at the site). However, the KLHL35 gene does not seem to be one of the imprinted genes. Known examples of maternally imprinted loci escape the wave of demethylation that occurs following fertilization [9]. In contrast, the exon 2 region of KLHL35 is likely susceptible to epigenetic reprogramming after fertilization, since no DNA methylation is observed in single blastocysts for the single CpG site in this region (cg05353869) with data in GSE51239 (see S1 Text, for details) [25]. If there is differential methylation of parental alleles in this region, traditional imprinting is unlikely to explain the observed relationship.

In sum, we find a novel and robust pattern of gene methylation associated with mother’s age. This epigenetic change is apparently persistent (detectable > 40 years after birth), and one of only a few examples of epigenetic modifications associated with the prenatal environment. The relationship between epigenetic changes in the newborn and subsequent health outcomes remains to be seen.

Material and Methods

Supporting Information

Acknowledgments

We would like to thank all individuals for participating in the Norway Facial Clefts Study, Norwegian Mother and Child Cohort Study, and the Sister Study. We are grateful to all the participating families in Norway who take part in this ongoing cohort study.