An epigenetic biomarker of aging for lifespan and healthspan

Results

Overview of the statistical model and analysis

Our development of the new epigenetic biomarker of aging proceeded along three main steps (Fig. 1). In step 1, a novel measure of ‘phenotypic age’ was developed using clinical data from the third National Health and Nutrition Examination Survey (NHANES). Details on the phenotypic age estimator can be found in Table 1 and in Supplement 1. In step 2, DNAm from whole blood was used to predict phenotypic age, such that:

$DNAm\mathrm{ }Pheno\mathrm{Age}=intercept{+CpG}_1\times {\beta }_1+{CpG}_2\times {\beta }_2+\dots {CpG}_{513}\times {\beta }_{513}$

Figure 1. Roadmap for developing DNAm PhenoAge. The roadmap depicts our analytical procedures. In step 1, we developed an estimate of ‘Phenotypic Age’ based on clinical measure. Phenotypic age was developed using the NHANES III as training data, in which we employed a proportional hazard penalized regression model to narrow 42 biomarkers to 9 biomarkers and chronological age. This measure was then validated in NHANES IV and shown to be a strong predictor of both morbidity and mortality risk. In step 2, we developed an epigenetic biomarker of phenotypic age, which we call DNAm PhenoAge, by regressing phenotypic age (from step 1) on blood DNA methylation data, using the InCHIANTI data. This produced an estimate of DNAm PhenoAge based on 513 CpGs. We then validated our new epigenetic biomarker of aging, DNAm PhenoAge, using multiple cohorts, aging-related outcomes, and tissues/cells. In step 3, we examined the underlying biology of the 513 CpGs and the composite DNAm PhenoAge measure, using a variety of complementary data (gene expression, blood cell counts) and various genome annotation tools including chromatin state analysis and gene ontology enrichment.

Table 1. Phenotypic aging measures and Gompertz coefficients.

Variable		Units	Weight
Albumin	Liver	g/L	-0.0336
Creatinine	Kidney	umol/L	0.0095
Glucose, serum	Metabolic	mmol/L	0.1953
C-reactive protein (log)	Inflammation	mg/dL	0.0954
Lymphocyte percent	Immune	%	-0.0120
Mean cell volume	Immune	fL	0.0268
Red cell distribution width	Immune	%	0.3306
Alkaline phosphatase	Liver	U/L	0.0019
White blood cell count	Immune	1000 cells/uL	0.0554
Age		Years	0.0804

The coefficient values of this model can be found in Supplement 2: Table S6. Predicted estimates from this model represent a person’s epigenetic age, which we refer to as ‘DNAm PhenoAge’. Using multiple independent datasets, we then tested whether DNAm PhenoAge was associated with a number of aging-related outcomes. We also tested whether it differed as a function of social, behavioral, and demographic characteristics, and whether it was applicable to tissues other than whole blood. Finally, in step 3, we examine the underlying biology of the 513 CpGs in the DNAm PhenoAge measure by examining differential expression, GO and pathway enrichment, chromosomal locations, and heritability.

Estimating phenotypic age from clinical biomarkers

For step 1, NHANES III was used to generate a measure of phenotypic age. NHANES III is a nationally-representative sample, with over twenty-three years of mortality follow-up, from which our analytical sample included 9,926 adults with complete biomarker data. A Cox penalized regression model—where the hazard of mortality was regressed on forty-two clinical markers and chronological age—was used to select variables for inclusion in our phenotypic age score. The forty-two biomarkers considered represent those that were available in both NHANES III and IV. Based on 10-fold cross-validation, ten variables (including chronological age) were selected for the phenotypic age predictor (Table 1, Table S1). These nine biomarkers and chronological age were then combined in a phenotypic age estimate (in units of years) as detailed in Methods.

Validation data for phenotypic age came from NHANES IV, and included up to 12 years of mortality follow-up for n=6,209 national representative US adults. In this population, phenotypic age is correlated with chronological age at r=0.94. Results from all-cause and cause-specific (competing risk) mortality predictions, adjusting for chronological age (Table 2), show that a one-year increase in phenotypic age is associated with a 9% increase in the risk of all-cause mortality (HR=1.09, p=3.8E-49), a 9% increase in the risk of mortality from aging-related diseases (HR=1.09, p=4.5E-34), a 10% increase in the risk of CVD mortality (HR=1.10, p=5.1E-17), a 7% increase in the risk of cancer mortality (HR=1.07, p=7.9E-10), a 20% increase in the risk of diabetes mortality (HR=1.20, p=1.9E-11), and a 9% increase in the risk of chronic lower respiratory disease mortality (HR=1.09, p=6.3E-4). Further, phenotypic age is highly associated with comorbidity count (p=3.9E-21) and physical functioning measures (p=2.1E-10, Supplement 1: Fig. S1).

Table 2. Mortality validations for phenotypic age.

Mortality Cause	Cases	HR	P-Value
All-Cause	1052	1.09	3.8E-49
Aging-Related	661	1.09	4.5E-34
CVD	272	1.10	5.1E-17
Cancer	265	1.07	7.9E-10
Alzheimer's	30	1.04	2.6E-1
Diabetes	41	1.20	1.9E-11
Chronic lower respiratory diseases	53	1.09	6.3E-4

An epigenetic biomarker of aging (DNAm PhenoAge)

For step 2 (Fig. 1), data from n=456 participants at two time-points in the Invecchiare in Chianti (InCHIANTI) study was used to relate blood DNAm levels to phenotypic age. InCHIANTI was used as training data for the new epigenetic biomarker because the study assessed all clinical measures needed to estimate phenotypic age, contained data on DNAm, and had a large age range population (21-100 years). A total of 20,169 CpGs were considered when generating the new DNAm measure. They represented those CpGs available on all three chips (27k, 450k, EPIC), so as to facilitate usability across platforms. Elastic net regression, with 10-fold cross-validation, produced a model in which phenotypic age is predicted by DNAm levels at 513 of the 20,169 CpGs. The linear combination of the weighted 513 CpGs yields a DNAm based estimator of phenotypic age that we refer to as ‘DNAm PhenoAge’ (mean=58.9, s.d.=18.2, range=9.1-106.1), in contrast to the previously published Hannum and Horvath ‘DNAm Age’ measures.

While our new clock was trained on cross-sectional data in InCHIANTI, we capitalized on the repeated time-points to test whether changes in DNAm PhenoAge are related to changes in phenotypic age. As expected, between 1998 and 2007, mean change in DNAm PhenoAge was 8.51 years, whereas mean change in clinical phenotypic age was 8.88 years. Moreover, participants’ clinical phenotypic age (adjusting for chronological age) at the two time-points was correlated at r=0.50, whereas participants’ DNAm PhenoAge (adjusting for chronological age) at the two time-points was correlated at r=0.68 (Supplement 1: Fig. S2). We also find that the change in phenotypic age between 1998 and 2007 is highly correlated with the change in DNAm PhenoAge between these two time-points (r=0.74, p=3.2E-80, Supplement 1: Fig. S2).

DNAm PhenoAge strongly relates to all-cause mortality

In step 2 (Fig. 1), the epigenetic biomarker, DNAm PhenoAge, was calculated in five independent large-scale samples—two samples from Women’s Health Initiative (WHI) (n=2,016; and n=2,191), the Framingham Heart Study (FHS) (n=2,553), the Normative Aging Study (NAS) (n=657), and the Jackson Heart Study (JHS) (n=1,747). The first four studies used the Illumina 450K array while the JHS employed the latest Illumina EPIC array platform. In these studies, DNAm PhenoAge correlated with chronological age at r=0.66 in WHI (Sample 1), r=0.69 in WHI (Sample 2), r=0.78 in FHS, r=0.62 in the NAS, and r=0.89 in JHS. The five validation samples were then used to assess the effects of DNAm PhenoAge on mortality in comparison to the Horvath and Hannum DNAm Age measures. DNAm PhenoAge was significantly associated with subsequent mortality risk in all studies (independent of chronological age), such that, a one-year increase in DNAm PhenoAge is associated with a 4.5% increase in the risk of all-cause mortality (Meta(FE)=1.045, Meta p=7.9E-47, Fig. 2). To better conceptualize what this increase represents, we compared the predicted life expectancy and mortality risk for person’s representing the top 5% (fastest agers), the average, and the bottom 5% (slowest agers). Results suggest that those in the top 5% of fastest agers have a mortality hazard of death that is about 1.62 times that of the average person, i.e. the hazard of death is 62% higher than that of an average person. Further, contrasting the 5% fastest agers with the 5% slowest agers, we find that the hazard of death of the fastest agers is 2.58 times higher than that of the bottom 5% slowest agers (HR=1.045^11.0/1.045^-10.5). Additionally, both observed and predicted Kaplan-Meier survival estimates showed that faster agers had much lower life expectancy and survival rates compared to average and/or slow agers (Fig. 2).

Figure 2. Mortality Prediction by DNAm PhenoAge. (A) Using five samples from large epidemiological cohorts—two samples from the Women’s health Initiative, the Framingham Heart Study, the Normative Aging Study, and the Jackson Heart Study—we tested whether DNAm PhenoAge was predictive of all-cause mortality. The Fig. displays a forest plot for fixed-effect meta-analysis, based on Cox proportional hazard models, and adjusting for chronological age. Results suggest that DNAm PhenoAge is predictive of mortality in all samples, and that overall, a one-year increase in DNAm PhenoAge is associated with a 4.5% increase in the risk of death (p=9.9E-47). This is contrasted against the first generation of epigenetic biomarkers of aging by Hannum and Horvath, which exhibit less significant associations with lifespan (p=1.7E-21 and p=4.5E-5, respectively). (B and C) Using the WHI sample 1, we plotted Kaplan-Meier survival estimates using actual data from the fastest versus the slowest agers (panel B). We also applied the equation from the proportional hazard model to predict remaining life expectancy and plotted predicted survival assuming a chronological age of 50 and a DNAm PhenoAge of either 40 (slow ager), 50 (average ager), or 60 (fast ager) (panel C). Median life expectancy at age 50 was predicted to be approximately 81 years for the fastest agers, 83.5 years for average agers, and 86 years for the slowest agers.

As shown in Fig. 2, the DNAm age based measures from Hannum and Horvath also related to all-cause mortality, consistent with what has been reported previously [15,19,23,33,34]. To directly compare the three epigenetic measures, we contrasted their accuracy in predicting 10-year and 20-year mortality risk, using receiver operating characteristics (ROC)curves. DNAm PhenoAge (adjusted for age) predicts both 10-year mortality and 20-year mortality significantly better than Horvath and Hannum DNAmAge measures (Supplement 1: Table S2). When examining a model that includes all three measures (Supplement 1: Table S3), we find that only DNAm PhenoAge is positively associated with mortality (HR=1.04, p=1.33E-8), whereas Horvath DNAm Age is now negatively associated (HR=0.98, p=2.72E-2), and Hannum DNAm Age has no association (HR=1.01, p=4.66E-1).

DNAm PhenoAge strongly relates to aging-related morbidity

Given that aging is believed to also influence disease incidence/prevalence, we examined whether DNAm PhenoAge relates to diverse age-related morbidity outcomes. We observe strong associations between DNAm PhenoAge and a variety of other aging outcomes using the same five validation samples (Table 3). For instance, independent of chronological age, higher DNAm PhenoAge is associated with an increase in a person’s number of coexisting morbidities (β=0.008 to 0.031; Meta P-value=1.95E-20), a decrease in likelihood of being disease-free (β=-0.002 to -0.039; Meta P-value=2.10E-10), an increase in physical functioning problems (β=-0.016 to -0.473; Meta P-value=2.05E-13), an increase in the risk of coronary heart disease (CHD) risk (β=0.016 to 0.073; Meta P-value=3.35E-11).

Table 3. Morbidity validation for DNAm PhenoAge.

		Comorbidity		Disease Free		CHD Risk		Physical Functioning
Sample		Coefficient	P-value	Coefficient	P-value	Coefficient	P-value	Coefficient	P-value
DNAm PhenoAge
	WHI BA23 White	0.008	2.38E-01	-0.002	3.82E-01	0.016	5.36E-02	-0.396	1.04E-04
	WHI BA23 Black	0.013	6.15E-02	-0.006	2.40E-02	0.021	2.02E-02	-0.423	4.50E-04
	WHI BA23 Hispanic	0.024	1.64E-02	-0.004	3.67E-01	0.033	5.07E-02	-0.329	7.37E-02
	WHI EMPC White	0.031	2.95E-07	-0.026	1.63E-02	0.023	1.89E-01	-0.361	3.81E-05
	WHI EMPC Black	0.014	7.67E-02	-0.023	6.98E-02	0.048	2.27E-02	-0.473	3.75E-04
	WHI EMPC Hispanic	0.003	7.83E-01	0.002	9.28E-01	0.073	1.98E-01	-0.377	6.54E-02
	FHS	0.022	3.93E-07	-0.034	1.59E-03	0.028	5.47E-06	-0.016	4.60E-01
	NAS	0.023	7.59E-06	-0.062	2.00E-04	0.030	2.27E-02	NA	NA
	JHS	0.018	1.86E-08	-0.039	5.92E-05	0.033	4.73E-02	NA	NA
	Meta P-value (Stouffer)		1.95E-20		2.14E-10		3.35E-11		2.05E-13
DNAmAge Hannum
	WHI BA23 White	0.007	3.90E-01	-0.003	3.48E-01	0.013	2.36E-01	-0.399	2.90E-03
	WHI BA23 Black	0.022	2.72E-02	-0.007	6.03E-02	0.015	2.67E-01	-0.345	4.29E-02
	WHI BA23 Hispanic	0.010	4.33E-01	-0.010	6.24E-02	0.011	6.10E-01	-0.599	1.16E-02
	WHI EMPC White	0.025	1.53E-03	-0.020	1.55E-01	0.022	3.30E-01	-0.284	1.43E-02
	WHI EMPC Black	0.022	6.34E-02	-0.008	6.62E-01	0.055	6.12E-02	-0.323	9.56E-02
	WHI EMPC Hispanic	-0.012	4.17E-01	0.035	2.09E-01	-0.012	8.85E-01	-0.345	2.54E-01
	FHS	0.019	5.94E-04	-0.030	2.55E-02	0.022	1.55E-02	0.040	1.32E-01
	NAS	0.009	2.19E-01	-0.026	2.26E-01	0.025	1.83E-01	NA	NA
	JHS	0.020	2.09E-05	-0.036	9.91E-03	0.086	1.64E-04	NA	NA
	Meta P-value (Stouffer)		1.50E-08		1.64E-04		1.40E-05		2.03E-05
DNAmAge Horvath
	WHI BA23 White	0.007	3.49E-01	-0.004	1.69E-01	0.001	9.12E-01	-0.440	5.10E-04
	WHI BA23 Black	0.018	3.96E-02	-0.006	6.25E-02	0.009	4.07E-01	-0.305	4.52E-02
	WHI BA23 Hispanic	0.012	3.65E-01	-0.007	1.86E-01	-0.001	9.78E-01	-0.204	4.12E-01
	WHI EMPC White	0.031	1.99E-04	-0.043	5.56E-03	0.000	9.88E-01	-0.288	1.74E-02
	WHI EMPC Black	0.016	1.93E-01	-0.003	8.56E-01	0.033	2.87E-01	-0.144	4.68E-01
	WHI EMPC Hispanic	-0.025	8.99E-02	-0.016	5.70E-01	-0.064	4.63E-01	-0.012	9.70E-01
	FHS	0.011	5.82E-02	-0.021	8.34E-02	0.007	5.19E-01	0.027	3.16E-01
	NAS	0.011	7.90E-02	-0.039	4.53E-02	0.006	7.14E-01	NA	NA
	JHS	0.014	2.03E-03	-0.040	1.78E-03	0.049	3.93E-02	NA	NA
	Meta P-value (Stouffer)		3.26E-06		6.36E-07		1.49E-01		1.43E-03

DNAm PhenoAge and smoking

Cigarette exposure has been shown to have an epigenetic fingerprint [35–37], which has been reflected in previous DNAm risk predictors [38]. Similarly, we find that DNAm PhenoAge significantly differs between never (n=1,097), current (n=209), and former smokers (n=710) (p=0.0033) (Supplement 1: Fig. S3A); however, conversely, we do not find a robust association between pack-years and DNAm PhenoAge (Supplement 1, Fig. S3B-D). Given the association between DNAm PhenoAge and smoking, we re-evaluated the morbidity and mortality associations (fully-adjusted) in our four samples, stratifying by smoking status (Supplement 1: Fig. S4 and Table S4). We find that DNAm PhenoAge is associated with mortality among both smokers (adjusted for pack-years) (Meta(FE)=1.050, Meta p=7.9E-31), and non-smokers (Meta(FE)=1.033, Meta p=1.2E-10). DNAm PhenoAge relates to the number of coexisting morbidities, physical functioning status, disease free status, and CHD for both smokers and non-smokers (Supplement 1: Table S4). In previous work we showed that Horvath DNAm age of blood predicts lung cancer risk in the first WHI sample [20]. Using the same data, we find that a one-year increase in DNAm PhenoAge (adjusting for chronological age, race/ethnicity, pack-years, and smoking status) is associated with a 5% increase in the risk of lung cancer incidence and/or mortality (HR=1.05, p=0.031). Further, when restricting the model to current smokers only, we find that the effect of DNAm PhenoAge on future lung cancer incidence and/or mortality is even stronger (HR=1.10, p=0.014).

DNAm PhenoAge in other tissues

One advantage of developing biological aging estimates based on molecular markers (like DNAm), rather than clinical risk measures (e.g. those in the phenotypic age variable), is that they may lend themselves to measuring tissue/cell specific aging. Although DNAm PhenoAge was developed using samples from whole blood, our empirical results show that it strongly correlates with chronological age in a host of different tissues and cell types (Fig. 3). For instance, when examining all tissues concurrently, the correlation between DNAm PhenoAge and chronological age was 0.71. Age correlations in brain tissue ranged from 0.54 to 0.92, while correlations were also found in breast (r=0.47), buccal cells (r=0.88), dermal fibroblasts (r=0.87), epidermis (r=0.84), colon (r=0.88), heart (r=0.66), kidney (r=0.64), liver (r=0.80), lung (r=055), and saliva (r=0.81).

Figure 3. Chronological age versus DNAm PhenoAge in a variety of tissues and cells. Although DNAm PhenoAge was developed using methylation data from whole blood, it also tracks chronological age in a wide variety of tissues and cells. (A) The correlation across all tissues/cells we examined is r=0.71. (B-ZJ) report results in different sources of DNA as indicated in panel headings. The numbers correspond to the data sets from (Horvath 2013). Overall, correlations range from r=0.35 (breast, panel O) to r=0.92 (temporal cortex in brain, panel L).

Alzheimer's disease and brain samples

Based on the accuracy of the age prediction in other tissues/cells, we examined whether aging in a given tissue was associated with tissue-associated outcomes. For instance, using data from approximately 700 post-mortem samples from the Religious Order Study (ROS) and the Memory and Aging Project (MAP) [39,40] we tested the association between pathologically diagnosed Alzheimer’s disease and DNAm PhenoAge in dorsolateral prefrontal cortex (DLPFX). Results suggest (Fig. 4) that those who are diagnosed with Alzheimer’s disease (AD), based on postmortem autopsy, have DLPFX that appear more than one-year older than same aged individuals who are not diagnosed with AD postmortem (p=4.6E-4). Further, age adjusted DNAm PhenoAge was found to be positively associated with neuropathological hallmarks of Alzheimer’s disease, such as amyloid load (r=0.094, p=0.012), neuritic plaques (r=0.11, p=0.0032), and neurofibrillary tangles (r=0.10, p=0.0073).

Figure 4. DNAm PhenoAge measured in dorsolateral prefrontal cortex relates to Alzheimer’s disease and related neuropathologies. Using postmortem data from the Religious Order Study (ROS) and the Memory and Aging Project (MAP), we find a moderate/high correlation between chronological age and DNAm PhenoAge (panel A). We also estimate the Pproportion of neurons via the CETS algorithm and show that it correlates with DNAm PhenoAge (B). Further furtherthe correlation between chronological agen and DNAm PhenoAge is increased after adjusting for the estimated proportion on neurons in each sample (panel C). We also find that DNAm PhenoAge is significantly higher (p=0.00046) among those with Alzheimer’s disease versus controls (panel D), and that it positively correlates with amyloid load (p=0.012, panel E), neuritic plaques (p=0.0032, panel F), diffuse plaques (p=0.036, panel G), and neurofibrillary tangles (p=0.0073, panel H).

Lifestyle and demographic variables

In evaluating the relationship between DNAm PhenoAge in blood and additional characteristics we observe significant differences between racial/ethnic groups (p=5.1E-5), with non-Hispanic blacks having the highest DNAm PhenoAge on average, and non-Hispanic whites having the lowest (Supplement 1: Fig. S5). We also find evidence of social gradients in DNAm PhenoAge, such that those with higher education (p=6E-9) and higher income (p=9E-5) appear younger (Figure 5). DNAm PhenoAge relates to exercise and dietary habits, such that increased exercise (p=7E-5) and markers of fruit/vegetable consumption (such as carotenoids, p=2E-27) are associated with lower DNAm PhenoAge (Figure 5, Supplement 1: Fig. S6). Cross sectional studies in the WHI also revealed that DNAmPhenoAge acceleration is positively correlated with C-reactive protein (r=0.18, p=5E-22, Figure 5), insulin (r=0.15, p=2E-20), glucose (r=0.10, p=2E-10), triglycerides (r=0.09, p=5E-9), waist to hip ratio (r=0.15, p=5E-22) but it is negatively correlated with HDL cholesterol (r=-0.09, p=7E-9).

Figure 5. Lifestyle factors versus DNAm PhenoAge acceleration in blood in the WHI. In this cross- sectional analysis, the correlation test analysis (bicor, biweight midcorrelation) between select variables and DNAm PhenoAgeAccel reveals that education, income, exercise, proxies of fruit/vegetable consumption, and HDL cholesterol are negatively associated (blue) with DNAm PhenoAge, i.e. younger epigenetic age. Conversely, CRP, insulin, glucose, triglycerides, BMI, waist-to-hip ratio, systolic blood pressure, and smoking have a positive association (red) with DNAm PhenoAge. All results have been adjusted for ethnicity and batch/data set. Similar results based on multivariate regression models can be found in Supplement 1: Fig. S6B.

DNAm PhenoAge and immunosenescence

To test the hypothesis that DNAm PhenoAge captures aspects of age-related decline of the immune system, we correlated DNAm PhenoAge with estimated blood cell count (Supplement 1: Fig. S7). After adjusting for age, we find that DNAm PhenoAgeAccel is negatively correlated with naïve CD8+ T cells (r=-0.35, p=9.2E-65), naïve CD4+ T cells (r=-0.29, p=4.2E-42), CD4+ helper T cells (r=-0.34, p=3.6E-58), and B cells (r=-0.18, p=8.4E-17). Further, DNAm PhenoAgeAccel is positively correlated with the proportion of granulocytes (r=0.32, p=2.3E-51), exhausted CD8+ (defined as CD28-CD45RA-) T cells (r=0.20, p=1.9E-20), and plasma blast cells (r=0.26, p=6.7E-34). These results are consistent with age related changes in blood cells [41] and suggest that DNAm PhenoAge may capture aspects of immunosenescence in blood. However, three lines of evidence suggest that DNAm PhenoAge is not simply a measure of immunosenescence. First, another measure of immunosenescence, leukocyte telomere length, is only weakly correlated with DNAm PhenoAgeAccel (r=-0.13 p=0.00019 in the WHI; r=-0.087, P=7.6E-3 in Framingham Heart study; JHS p=7.83E-7, Supplement 1: Fig. S8). Second, the strong association between DNAm PhenoAge and mortality does not simply reflect changes in blood cell composition, as can be seen from the fact that in Supplement 1: Fig. S9 the robust association remains even after adjusting for estimates of seven blood cell count measures (Meta(FE)=1.036, Meta p=5.6E-21). Third, DNAmPhenoAge correlates with chronological age in non-blood tissue.

DNA sequence characteristics of the 513 CpGs in DNAm PhenoAge

Of the 513 CpGs in DNAm PhenoAge, we find that, 41 CpGs were also in the Horvath DNAm age measure (Supplement 2: Table S6). This represents a 4.88-fold increase over what would be expected by chance (p=8.97E-15). Of the 41 overlapping CpGs, the average absolute value for their age correlations was r=0.40, and 31 had age correlations with absolute values in the top 20% of what is found among the 513 CpGs in the DNAm PhenoAge score. We also observed 6 CpGs that overlapped between the Hannum DNAm Age score and the DNAm PhenoAge score—five of which were also found in the Horvath DNAm Age measure. All six CpGs had extremely high age correlations (half positive, half negative), with absolute values between r=0.49 and r=0.76. The five CpGs that are found in all three epigenetic aging measures were: cg05442902 (P2RXL1), cg06493994 (SCGN), cg09809672 (EDARADD), cg19722847 (IPO8), and cg22736354 (NHLRC1). Several additional DNAm biomarkers have been described in the literature [12,13]. A direct comparison of 6 DNAm biomarkers (including DNAm PhenoAge) reveals that DNAm PhenoAge stands out in terms of its predictive accuracy for lifespan, its relationship with smoking status, its relationship with leukocyte telomere length, naïve CD8+ T cells and CD4+ T cells (Supplement 1: Table S5).

Finally, we conducted a functional enrichment analysis of the chromosomal locations of the 513 CpGs, we found that 149 CpGs whose age correlation exceeded 0.2 tended to be located in CpG islands (p=0.0045, Supplement 1: Fig. S10) and were significantly enriched with polycomb group protein targets (p=8.7E-5, Supplement 1: Fig. S10), which echoes results of epigenome wide studies of aging effects [4,5,42].

Transcriptional and genetic studies of DNAm PhenoAge

Using the genome-wide data from FHS and WHI, we estimated the heritability of DNAm PhenoAge. The heritability estimated by the SOLAR polygenic model for those of European ancestry in the FHS was $h^2$=0.33, while the heritability estimated for those of European ancestry in WHI, using GCTA-GREML analysis [43] was $h^2$=0.51.

Using the monocyte data mentioned above, as well as PBMC expression data on 2,188 persons from the FHS, we conducted a transcriptional analysis to identify differential expression associated with DNAm PhenoAgeAccel (Supplement 3: Table S7). Overall, we find that genes show similar associations with chronological age and DNAm PhenoAgeAccel. DNAm PhenoAgeAccel represents aging differences among same-aged individuals and is adjusted so as to exhibit a correlation of r=0.0 with chronological age. Thus, this observation suggests that genes whose transcription increases with age are upregulated among epigenetically older compared to epigenetically younger persons of the same chronological age (Supplement 1: Fig. S11) the same applies for genes that show decreases with chronological age being downregulated in epigenetically older versus younger persons of the same age.

Using the transcriptional data from monocytes described above (adjusting for array, sex, race/ethnicity, age, and imputed cell counts), we tested for GO enrichment among genes that are positively associated with DNAm PhenoAge and those that are negatively associated with DNAm PhenoAge (Supplement 4: Table S8). Among those with positive aging associations (overexpression among epigenetically older individuals), we observed enrichment for a number of pro-inflammatory signaling pathways. These pathways included, but are not limited to: multiple toll-like receptor signaling pathways (7,9,3,2), regulation of inflammatory response, JAK-STAT cascade, response to lipopolysaccharide, tumor necrosis factor-mediated signaling pathway, and positive regulation of NF-kappaB transcription factor activity. Additionally, positively associated genes were also enriched for a number anti-viral response pathways—type I interferon signaling, defense response to virus, interferon-gamma-mediated signaling pathway, cellular response to interferon-alpha, etc. Other interesting GO terms enriched among positively associated genes included: response to nutrient, JAK-STAT cascade involved in growth hormone signaling pathway, multicellular organism growth, and regulation of DNA methylation.

When testing for enrichment among genes that were negatively associated with DNAm PhenoAgeAccel (decreased expression among epigenetically older persons) we observed that many were implicated in processes involving transcriptional and translational machinery, as well as damage recognition and repair. These included: translational initiation; regulation of translational initiation; ribosomal large subunit assembly; ribosomal small subunit assembly; translational elongation; transcription initiation from RNA polymerase I promoter; transcription-coupled nucleotide-excision repair; nucleotide-excision repair, DNA incision, 5'-to lesion; nucleotide-excision repair, DNA damage recognition; DNA damage response, detection of DNA damage; and regulation of DNA damage checkpoint.

Author Contributions

ML and SH developed the DNAmPhenoAge estimator and wrote the article. SH, ML, LF conceived of the study. ML, SH, AL, AQ carried out the statistical analysis. The remaining authors contributed data and participated in the interpretation of the results.

Acknowledgements

We would like to acknowledge The WHI Investigators listed below:

Program Office: (National Heart, Lung, and Blood Institute, Bethesda, Maryland) Jacques Rossouw, Shari Ludlam, Dale Burwen, Joan McGowan, Leslie Ford, and Nancy Geller.

Clinical Coordinating Center: (Fred Hutchinson Cancer Research Center, Seattle, WA) Garnet Anderson, Ross Prentice, Andrea LaCroix, and Charles Kooperberg.

Investigators and Academic Centers: (Brigham and Women's Hospital, Harvard Medical School, Boston, MA) JoAnn E. Manson; (MedStar Health Research Institute/Howard University, Washington, DC) Barbara V. Howard; (Stanford Prevention Research Center, Stanford, CA).

Marcia L. Stefanick; (The Ohio State University, Columbus, OH) Rebecca Jackson; (University of Arizona, Tucson/Phoenix, AZ) Cynthia A. Thomson; (University at Buffalo, Buffalo, NY).

Jean Wactawski-Wende; (University of Florida, Gainesville/Jacksonville, FL) Marian Limacher; (University of Iowa, Iowa City/Davenport, IA) Robert Wallace; (University of Pittsburgh, Pittsburgh, PA) Lewis Kuller; (Wake Forest University School of Medicine, Winston-Salem, NC) Sally Shumaker.

The Framingham Heart Study is funded by National Institutes of Health contract N01-HC-25195 and HHSN268201500001I. The laboratory work for this investigation was funded by the Division of Intramural Research, National Heart, Lung, and Blood Institute, National Institutes of Health. The analytical component of this project was funded by the Division of Intramural Research, National Heart, Lung, and Blood Institute, and the Center for Information Technology, National Institutes of Health, Bethesda, MD. The Framingham Heart Study is conducted and supported by the National Heart, Lung, and Blood Institute (NHLBI) in collaboration with Boston University. This manuscript was not prepared in collaboration with investigators of the Framingham Heart Study and does not necessarily reflect the opinions or views of the Framingham Heart Study, Boston University, or the NHLBI.

The United States Department of Veterans Affairs (VA) Normative Aging Study (NAS) is supported by the Cooperative Studies Program/ERIC and is a research component of the Massachusetts Veterans Epidemiology Research and Information Center (MAVERIC), Boston Massachusetts.

The MESA Epigenomics and Transcriptomics Studies were funded by R01HL101250, R01 DK103531-01, R01 DK103531, R01 AG054474, and R01 HL135009-01 to Wake Forest University Health Sciences.

We thank the Jackson Heart Study (JHS) participants and staff for their contributions to this work. The JHS is supported by contracts HHSN268201300046C, HHSN268201300047C, HHSN268201300048C, HHSN268201300049C, HHSN268201300050C from the National Heart, Lung, and Blood Institute and the National Institute on Minority Health and Health Disparities. Dr. Wilson is supported by U54GM115428 from the National Institute of General Medical Sciences.

Conflicts of Interest

The Regents of the University of California is the sole owner of a provisional patent application directed at this invention for which MEL, SH are named inventors.

Funding

This study was supported by NIH/NIA U34AG051425-01 (Horvath) and NIH/NIA K99AG052604 (Levine). The WHI epigenetic studies were supported by NIH/NHLBI 60442456 BAA23 (Assimes, Absher, Horvath) and by the National Institute of Environmental Health Sciences R01-ES020836 WHI-EMPC (Whitsel, Baccarelli, Hou). The WHI program is funded by the National Heart, Lung, and Blood Institute, National Institutes of Health, U.S. Department of Health and Human Services through contracts HHSN268201100046C, HHSN268201100001C, HHSN268201100002C, HHSN268201100003C, HHSN268201100004C, and HHSN271201100004C. The authors thank the WHI investigators and staff for their dedication, and the study participants for making the program possible. A full listing of WHI investigators can be found at: www.whi.org/researchers/Documents%20%20Write%20a%20Paper/WHI%20Investigator%20Short%20List.pdf.

The InCHIANTI study baseline (1998-2000) was supported as a "targeted project" (ICS110.1/RF97.71) by the Italian Ministry of Health and in part by the U.S. National Institute on Aging (Contracts: 263 MD 9164 and 263 MD 821336).

Funding for the DNA methylation in JHS provided by NHLBI R01HL116446.

The funding bodies played no role in the design, the collection, analysis, or interpretation of the data.

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