The authors have declared that no competing interests exist.
Conceived and designed the experiments: LEW JTB FMKW PT CJS. Performed the experiments: LEW PT CJS. Analyzed the data: LEW. Contributed reagents/materials/analysis tools: PD TDS CJS. Wrote the paper: LEW.
Epigenetic regulation of gene expression has been shown to change over time and may be associated with environmental exposures in common complex traits. Age-related hearing impairment is a complex disorder, known to be heritable, with heritability estimates of 57–70%. Epigenetic regulation might explain the observed difference in age of onset and magnitude of hearing impairment with age. Epigenetic epidemiology studies using unrelated samples can be limited in their ability to detect small effects, and recent epigenetic findings in twins underscore the power of this well matched study design. We investigated the association between venous blood DNA methylation epigenome-wide and hearing ability. Pure-tone audiometry (PTA) and Illumina HumanMethylation array data were obtained from female twin volunteers enrolled in the TwinsUK register. Two study groups were explored: first, an epigenome-wide association scan (EWAS) was performed in a discovery sample (n = 115 subjects, age range: 47–83 years, Illumina 27 k array), then replication of the top ten associated probes from the discovery EWAS was attempted in a second unrelated sample (n = 203, age range: 41–86 years, Illumina 450 k array). Finally, a set of monozygotic (MZ) twin pairs (n = 21 pairs) within the discovery sample (Illumina 27 k array) was investigated in more detail in an MZ discordance analysis. Hearing ability was strongly associated with DNA methylation levels in the promoter regions of several genes, including
The term epigenetics
Epigenetic changes can be influenced by both environmental exposure
Age-related hearing impairment (ARHI) is a common complex trait affecting 46% of the population over the age of 48
The study was approved by the National Research Ethics service London-Westminster (REC reference number: 07/H0802/84). Fully informed written consent was obtained from all participants prior to study conduction. All research described was conducted according to the rules described in the Declaration of Helsinki.
Hearing data in form of air-conduction PTA was collected from participants of the TwinsUK cohort between 2009 and 2013. Hearing thresholds were determined at frequencies 0.125–8 kHz for each ear according to the recommendations of the British Society of Audiology
Whole blood samples for DNA methylation screening were profiled using two different DNA methylation assays, the Infinium HumanMethylation 27 k BeadChip (26,690 CpG sites) and the Infinium HumanMethylation 450 k BeadChip Kit. In both arrays, the DNA methylation level at a specific CpG site is expressed as the β value, which represents the ratio of the methylated probe signal over the methylated and unmethylated probe signals. The β score ranges from 0 to 1, where 0 indicates absence of methylation and 1 represents a fully methylated CpG site.
The Illumina Infinium HumanMethylation 27 k array measures methylation at 27,578 CpG sites, covering 14,495 genes. This array covers primarily CpG sites located in promoter regions of genes with on average two CpG sites per consensus coding sequence and three to twenty assays per cancer gene
To identify potential confounders of the Illumina Infinium HumanMethylation 27 k array, principal component analysis (PCA) was performed using the normalised DNA methylation values. The first five principal components resulting from this analysis were correlated with following covariates: chronological age, methylation chip and position of sample on the chip. Both methylation chip and position of sample on the chip were significantly correlated with the first two principal components from this analysis and therefore included as fixed effects in further analysis
The discovery EWAS of hearing was performed in 115 adult female subjects with available PTA data and Illumina Infinium HumanMethylation 27 k profiles
The replication sample consisted of 203 females from the TwinsUK registry. For the replication study only the 10 probes most highly associated in the discovery EWAS were investigated, while the remaining 485567 probes from the 450 k array were neglected. The 10 selected probes were examined for replication in the second sample using a linear mixed effect model. DNA methylation was transformed to standard normal per probe using a quantile normalisation. DNA methylation at each CpG site was regressed against hearing ability (PC1) with adjustment for age, methylation chip, order of samples on the chip, bisulfite conversion levels and twin relatedness. To exclude association with DNA methylation due to covariates, the full model was compared to a null model, in which hearing was excluded as a predictor variable. The null and the full models were compared for model fit in an analysis of variance. For each of the 10 probes, the effect size (beta), standard error of effect (se) and the p-value from the analysis of variance comparing full and null model were reported. Replication of association was considered if association was in the same direction and nominally significant (p≤0.05). To confirm that replicating probes were not age-dependent DMRs, models including and excluding age as a fixed effect were compared. To determine the significance and effect of joint association signals in the discovery (27 k) and replication (450 k) samples, a meta-analysis was conducted for the ten most highly associated probes using METAL
To further validate the findings from the EWAS (27 k) and replication study (450 k) using an alternative technique, the top ranked DMR was also explored using methylated DNA immunoprecipitation followed by high throughput sequencing (MeDIPseq) data. The MeDIPseq validation sample consisted of 46 unrelated healthy females with PTA scores and previously published MeDIPseq profiles
Previous studies have reported that association with DNA methylation measured in whole blood samples can be driven by blood cell subtype heterogeneity
Monozygotic twin pairs with PTA and Illumina HumanMethylation 27 k data were selected for the MZ discordance analysis (n = 21 pairs). Intra-pair DNA methylation difference per probe was calculated as the difference in DNA methylation residuals (adjusted for chip and position on the chip) between co-twins. DNA methylation residuals were calculated from quantile normalised β values per probe. Differences in DNA methylation were compared to differences in PC1 were using Spearman rank correlation.
To investigate the influence of DNA methylation on gene expression, expression levels in skin tissue collected as part of the Multiple Tissue Human Expression Resource (MuTHER) (
Two independent samples with hearing data and DNA methylation profiles were selected from the TwinsUK registry to perform the discovery EWAS (n = 115) using Illumina HumanMethylation 27 k profiles, and the replication EWAS (n = 203) using Illumina HumanMethylation 450 k profiles. Subjects included in the discovery EWAS had a mean age of 56.7 years (±7.9 years of standard deviation from the mean, age range 33–80 years) and included 25 dizygotic twin (DZs) pairs, 21 monozygotic twin (MZs) pairs and 23 singletons. The replication sample included 203 females, comprising 61 MZ twin pairs, 22 DZ twin pairs and 37 unpaired twins (singletons), with a mean age of 63.21 (± 8.87 years of standard deviation from the mean, age range 41–82 years). The discovery and replication samples are summarised in
sample | zygosity | n | age at DNA extraction | age at hearing test | PC1 ±sd | ||
mean ±sd | range | mean ±sd | range | ||||
discovery (27 k) | MZ | 42 | 55.43±6.93 | 45–68 | 62.00±6.60 | 50–72 | −0.28±1.47 |
DZ | 50 | 57.68±8.88 | 33–80 | 64.32±7.68 | 47–83 | 0.72±1.53 | |
singleton | 23 | 56.91±7.35 | 43–70 | 64.83±6.12 | 50–75 | 0.29±1.78 | |
Total | 115 | 56.70±7.91 | 33–80 | 63.57±7.05 | 47–83 | 0.27±1.61 | |
replication (450 k) | MZ | 122 | 55.64±8.83 | 37–73 | 63.82±8.79 | 46–82 | 0.60±2.13 |
DZ | 44 | 52.88±10.59 | 33–78 | 60.86±10.58 | 41–86 | 0.21±2.48 | |
singleton | 37 | 55.87±6.20 | 42–66 | 63.97±6.28 | 49–75 | 0.21±1.74 | |
Total | 203 | 55.09±8.87 | 33–78 | 63.21±8.87 | 41–86 | 0.45±2.15 | |
validation (MeDIPseq) | singleton | 46 | 60.02±7.85 | 41–83 | 62.28±7.86 | 43–86 | −0.10±2.08 |
Demographic characteristics of the discovery (27 k DNA methylation bead chip), replication (450 k DNA methylation bead chip) and validation (methylated DNA immunoprecipitation and high throughput sequencing (MeDIPseq) samples are listed. Samples zygosity is shown (monozygotic (MZ) and dizygotic (DZ) twins) as well as unpaired twins (singletons). Demographic measures include number of subjects (n), mean chronological age at DNA extraction in years and age range, as well as mean chronological age at hearing assessment in years. Mean and standard deviation (sd) of hearing principal component 1 (PC1), representing the overall threshold shift in the audiogram, are given.
Genome-wide DNA methylation levels were obtained in the set of 115 female twins using the Illumina 27 k array. The majority of autosomal CpG sites included in this analysis were unmethylated (β<0.3, 68.9% of probes), few probes were hemi-methylated (β: 0.3–0.7, 11.2% of probes) or fully methylated (β>0.7, 19.9% of probes).
Genome-wide DNA methylation levels at 24,461 autosomal probes were previously obtained in the set of 115 discovery female twins using the Illumina 27 k array
The manhattan plot depicts the significance of association with PC1 as the negative logarithm of the p-value (-log(p-value)) versus the chromosomal location (chromosomes) for each of the 24,641 tested DNA-methylation probes. The red line defines a Bonferroni adjusted genome-wide suggestive significance threshold of p = 6.9×10−6. The ten most highly associated probes are located above the horizontal blue line corresponding to p<6.985×10−5.
The most highly associated probe was cg01161216 which maps to the promoter region of transcription factor 25 (
probe | gene | 27 k (n = 115) | 450 k (n = 203) | meta-analysis (n = 318) | |||||||||
beta | se | p-value | p-value (no age) | beta | se | p-value | p-value (no age) | dir | beta | se | p-value | ||
cg25383093 | PGM3 | −0.26056 | 0.05631 | 4.46E-05 | 7.46E-05 | −0.00816 | 0.03367 | 8.08E-01 | 7.66E-02 | −0.0746 | 0.0289 | 9.80E-03 | |
cg07644368 | CDO1 | −0.23819 | 0.05639 | 4.67E-05 | 2.06E-03 | 0.02340 | 0.03877 | 5.50E-01 | 4.40E-01 | −0.0606 | 0.0319 | 5.80E-02 | |
cg19923810 | NOC2L | 0.04784 | 5.38E-05 | 3.24E-05 | 0.03111 | 4.26E-01 | 8.16E-01 | 0.0261 | 3.05E-03 | ||||
cg21370143 | MYBPC3 | 0.04546 | 5.44E-05 | 1.03E-05 | 0.03407 | 1.30E-01 | 1.62E-01 | 0.0273 | 1.95E-04 | ||||
cg15791248 | FGFR1 | 0.05799 | 5.73E-05 | 5.01E-05 | 0.03846 | 6.96E-01 | 5.13E-01 | 0.0321 | 8.46E-03 | ||||
cg05934874 | VPS4B | 0.19644 | 0.04751 | 6.55E-05 | 8.98E-04 | 0.02993 | 3.67E-03 | 1.88E-02 | + |
0.0253 | 7.80E-01 | ||
cg12241297 | HNRNPA0 | 0.13623 | 0.03269 | 6.90E-05 | 4.52E-04 | 0.02882 | 2.65E-02 | 3.18E-02 | + |
0.0234 | 0.0216 | 2.80E-01 | |
cg25017250 | APOC4 | 0.05495 | 6.98E-05 | 1.06E-03 | 0.03585 | 2.71E-01 | 1.71E-02 | 0.0300 | 1.25E-03 |
The ten most highly associated differentially methylated regions in the discovery EWAS (27 k) are shown. Probes are characterised by the nearest gene, the association effect (beta), standard error of the effect (se) and significance of model fit (p-value). Significance of model fit excluding age as a model parameter (p-value (no age)) is reported for both the discovery (27 k) and replication (450 k) sample. The ten most highly associated probes were taken forward for replication (450 k) with effect (beta) standard error of the effect and significance of model fit (p-value) listed. Results of the meta-analysis are presented as direction of effect (dir, discovery and replication direction shown), combined effect (beta), standard error of the combined effect (se) and significance of the combined association (p-value). Association of DNA methylation and hearing PC1 was replicated for probes cg01161216 and cg18877514 (highlighted in bold).
After exclusion of chronological age as a fixed effect, association of DNA methylation with hearing PC1 remained significant for all of the ten most highly associated probes (
The ten most highly associated CpG probes from the discovery sample were examined in the replication sample (
A, B Hearing PC1 values were plotted versus raw DNA methylation betas for both the discovery (27 k, red dots) and the replication (450 k, blue dots) samples. Linear regression lines were fitted for both datasets (27 k:red line, 450 k:blue line).
After exclusion of chronological age as a fixed effect, association of DNA methylation with PC1 remained significant at
To assess the behaviour of additional probes mapping to the
. Hearing PC1 association with all available Illumina 450 k DNA methylation probes annotated to
Although DNA methylation was not found significantly associated at 8 out of 10 probes in the replication sample, DNA methylation at five further probes (cg25383093, cg19923810, cg21370143, cg15791248 and cg25017250) showed the same direction of effect as in the discovery sample (
To validate the peak EWAS DMR using a different technology, TCF25 DNA methylation levels based on MeDIPseq data were also explored for association with hearing in 46 unrelated females from TwinsUK
To account for potential effects of blood cell heterogeneity, the peak DMRs were also explored for association with proportion of eosinophils, lymphocytes, neutrophils and monocytes in a subset of 106 subjects from the discovery sample. The ten most highly associated probes in the discovery EWAS remained significantly associated (p>0.005) with PC1 after adjustment for blood cell heterogeneity.
MZ discordance analyses were performed in 21 female MZ twin pairs (n = 42) selected from the discovery sample with a mean age of 55.43 years (±6.93 years of standard deviation, age range: 45–68 years) (
MZ pair difference analysis (n = 42) | |||
probe | gene | rho | p-value |
cg01377755 | ACP6 | −0.753 | 1.24E-04 |
cg08156349 | MEF2D | −0.749 | 1.41E-04 |
cg07550362 | TAC1 | −0.722 | 3.25E-04 |
cg27383362 | ATAD3C | −0.703 | 5.49E-04 |
cg21208104 | PRSS12 | 0.697 | 6.26E-04 |
cg23566335 | ADAM18 | 0.696 | 6.47E-04 |
cg22892904 | CBX2 | −0.688 | 7.82E-04 |
cg04283938 | SEPT3 | −0.684 | 8.59E-04 |
cg23886551 | TMEM121 | −0.684 | 8.59E-04 |
cg14299800 | TOR1B | −0.682 | 9.13E-04 |
This table shows the results for the MZ discordance analysis. Results are listed for the ten most highly correlated probes with corresponding gene, Spearman rank correlation coefficient (rho) and significance of correlation (p-value).
To investigate the influence of DNA methylation on gene expression, expression profiles in skin were explored because skin originates from the same embryonic tissues as the inner ear, and expression profiles were not available for the cochlea. For 172 individuals with 27 k array data, DNA methylation at the two replicating probes (cg01161216 and cg18877514) was examined for association with gene expression (
. DNA methylation residuals showed a weak negative correlation (r = −0.02) with expression residuals of TCF25 in skin samples. Both quantile normalised DNA methylation betas and quantile normalised gene expression values were adjusted for experimental batch effects (chip and position on the chip for methylation betas and experimental batch and RNA concentration for gene expression profiles) previous to analysis. The regression line (blue line) depicts the linear association between DNA methylation residuals and gene expression residuals.
Changes in DNA methylation have been associated with increasing age and age-related disorders
Nominally significant associations (p<0.05) with PC1, which represents the overall threshold shift in the pure-tone audiogram and hence impaired hearing ability, were identified at 2,519 CpG sites. The ten most highly associated probes remained nominally significant after exclusion of chronological age as a fixed effect in the model, showing that none of these associated probes are age-related differentially methylated probes. Furthermore association remained significant after adjustment for blood cell heterogeneity, indicating that blood cell subtypes were not driving these association signals. Two of the ten signals were replicated in a second independent sample. Changes in DNA methylation in the promoter region of
As both the discovery and replication data used the same array design from Illumina based on DNA hybridisation an alternative technique, MeDIPseq, was used to validate our findings. MeDIPseq in venous blood from 46 unrelated samples confirmed the association between hearing PC1 and DNA methylation levels at
We also identified a differentially methylated DNA methylation probe mapping to the promoter of the
Among the top associations in the discovery EWAS was a DMR in the promoter of
DNA methylation in the promoter of genes has been associated with repression of gene expression. At our peak DMR in
Monozygotic twin pairs are a preferred study sample for epigenetic studies as they are assumed to be genetically identical. In addition, both dizygotic and monozygotic twin pairs show an increased proportion of shared environment due to the nature of their time shared in uterus and upbringing. The MZ discordance analysis was performed to best utilise the unique study sample presented here and for completeness. Nevertheless, the relatively low sample size and restricted discordance within the twin pairs limited the statistical power to detect strong epigenetic effects. Association was examined between intra-pair discordance for PC1 and intra-pair differences in DNA methylation at CpG sites genome-wide. The most highly associated probes were found in the promoters of
Our study has several strengths and limitations. DNA methylation is likely to play an important role in gene expression contributing to important phenotypic differences between tissues, between individuals and with age. Methods of analysis of methylation data are in their infancy: there are many important covariates to be considered. We elected to remove one of these, gender, by confining our studies to females, which predominate in the TwinsUK database. Thus our results pertain to women and may not extrapolate to men. Strengths included ability to exclude age and blood cell heterogeneity as potential confounders. The high proportion of related individuals in this sample reduced both the genetic and environmental variance compared to a population sample of unrelated individuals. Although the discovery and replication datasets were well matched for gender, ethnicity, age and hearing ability, the replication sample included by chance a higher proportion of monozygotic twin pairs (450 k sample: 60% MZs) compared to the discovery sample (27 k sample: 37% MZs), which might have resulted in the reduced significance of associations obtained in the replication sample. Association in the EWAS did not reach epigenome-wide significance by Bonferroni corrected significance levels (considering 24,641 independent tests: p≤2.03×10−6). However, DNA methylation of neighbouring CpG sites is unlikely to be independent thus a Bonferroni correction may be considered overly stringent. Taking co-methylation into account by correcting for the number of genes, the peak DMR in
In conclusion, this is the first study investigating the association between hearing ability with age and DNA methylation genome-wide in humans. Strong associations with DNA methylation in the promoters of 10 genes were identified, of which two (
Replication study dataset (450 k). The replication study dataset shows DNA methylation betas at the 10 probes selected for replication from the Illumina HumanMethylation 450 k Beadchip for all subjects of the replication study (n = 203). Each subject has been allocated an anonymous identification number (PUBLIC.ID) and is presented by a family-identification number to identify twin siblings (family_zygosity), the age at hearing test (Age_pta), their hearing PC1 value (PC1_unadjusted), gender (SEX), age at DNA extraction (DNA_age), bisulfite conversion values (BSCng_ul), DNA methylation chip and order on the chip (chip and chipo, respectively).
(TXT)
Association of hearing PC1 values and DNA methylation residuals at
(TIF)
This work was funded by Action on Hearing Loss and AgeUK.
The authors would like to thank all volunteers from the TwinsUK register, who contributed to this study and acknowledge the work of all researchers who helped in the completion of this work. The study was funded by the Wellcome Trust; European Community's Seventh Framework Programme (FP7/2007–2013). The study also receives support from the National Institute for Health Research (NIHR) BioResource Clinical Research Facility and Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust and King's College London. Tim Spector is holder of an ERC Advanced Principal Investigator award.