Cohort characteristics

Details of LBC1936 participant characteristics at Waves 1 and 2 are presented in Supplementary File 1. Briefly, 47.6% of participants in this study were female. At Wave 1 (relating to the mortality and longitudinal analyses), mean chronological age for both males and females was 69.6 years (SD 0.8), whereas the mean DNAm GrimAge was 67.4 years (SD 5.2). At Wave 2 (relating to cross-sectional analyses), mean chronological age for both males and females was 72.5 years (SD 0.7), whereas the mean DNAm GrimAge was 70.0 years (SD 4.9). The lower mean measure of epigenetic age when compared to chronological age may reflect overall good health of the cohort. However, the variance associated with DNAm GrimAge is much higher than that of chronological age. When calculated across all four available waves of the LBC1936 study, DNAm GrimAge exhibits an intra-class correlation coefficient of 0.85. Mean age 11 IQ scores were 100.69 (SD: 15.37). Notably, lower IQ scores at age 11 (β = −0.11, P = 0.02) were associated with an accelerated DNAm GrimAge. Associations between age 11 IQ and tested phenotypes are presented in Supplementary File 2.

DNAm GrimAge predicts mortality and associates with frailty factors in the LBC1936

Mortality in LBC1936 participants was assessed in relation to an accelerated DNAm GrimAge as well as age-adjusted DNAm-based surrogate markers for plasma protein levels and smoking pack years. DNAm GrimAge was derived for 906 participants with methylation data (at Wave 1: age 70 years). There were 226 deaths (24.9%) over 9 years of follow-up.

A higher DNAm GrimAge was significantly associated with risk of all-cause mortality (Hazard Ratio (HR) = 1.81 per SD increase in DNAm GrimAge, 95% confidence interval (CI) = [1.58, 2.07], P < 2.0 × 10−16). Furthermore, higher levels of age-adjusted DNAm Pack Years were associated with all-cause mortality in the LBC1936 (HR = 1.64 per SD, 95% CI [1.46, 1.86], P = 2.0 × 10−16). In relation to methylation-based surrogates for plasma protein levels, six of the seven DNAm protein surrogates (DNAm ADM, B2M, Cystatin C, GDF15, PAI1 and TIMP1) were significantly associated with all-cause mortality (see Supplementary File 3; Fig. 1a). Following adjustment for age 11 IQ, there was very little change in the HRs and all of the predictors remained significant. Indeed, HRs from all survival models ranged from an attenuation of 2.4% to an increase of 1.8% following adjustment for childhood intelligence.

Fig. 1 DNAm GrimAge and its component surrogate markers predict mortality in the LBC1936. a Forest plot showing hazard ratios and 95% confidence intervals (horizontal lines) from Cox proportional hazard models for DNAm GrimAge and its constituent DNAm surrogate markers in the LBC1936 (n = 906, no. of deaths = 226 following nine years of follow-up). All associations with the exceptions of DNAm Leptin were significant. b Kaplan–Meier survival curve exhibiting the survival probabilities for the top (highest DNAm GrimAge) and bottom quartiles (lowest DNAm GrimAge) for DNAm GrimAge in the LBC1936 following 9 years of follow-up Full size image

A Kaplan–Meier survival plot for an accelerated DNAm GrimAge, split into the highest and the lowest quartiles, is presented in Fig. 1b illustrating the higher mortality risk for those with a higher DNAm GrimAge. Kaplan–Meier survival plots for methylation-based surrogates for smoking pack years and plasma protein levels are presented in Supplementary Fig. 2.

For the remainder of the results, only those associations with an FDR-corrected significant P value ( < 0.05) are presented herein and in Fig. 2. Full results are presented in Supplementary File 4. In relation to major mortality- and frailty-associated physical traits in the LBC1936, an accelerated DNAm GrimAge was associated with increased levels of interleukin-6 (β = 0.37, P = 2.3 × 10-18), C-reactive protein (β = 0.25, P = 2.8 × 10−8), creatinine (β = 0.16, P = 1.1 × 10−4), an increased body mass index (β = 0.16, P = 2.9 × 10−4), triglyceride concentration (β = 0.13, P = 5.0 × 10−3) and body weight (β = 0.09, P = 0.04) (Fig. 2). The relationship between accelerated DNAm GrimAge and triglycerides was no longer significant after controlling for childhood cognitive ability with the effect size decreasing from 0.13 to 0.09 (32.5% attenuation) (Supplementary File 4).

Fig. 2 Cross-sectional association between age-adjusted DNAm GrimAge and cognitive, neuroimaging and physical traits in the LBC1936. Cognitive: An accelerated DNAm GrimAge was negatively associated with the general factor of cognitive ability, digit symbol coding, symbol search and matrix reasoning tasks. DNAm GrimAge was also associated with an increased mean four choice reaction time. Neuroimaging: Age-adjusted DNAm GrimAge was negatively associated with the ratios of white matter volume, brain volume and grey matter volume to intracranial volume, and positively associated with the ratio of volume of white matter hyperintensities to intracranial volume. Physical: An accelerated DNAm GrimAge was negatively associated with four measures of lung function: forced expiratory volume in 1 s, forced vital capacity, forced expiratory ratio and peak expiratory flow, as well as levels of iron, low-density lipoprotein cholesterol and total cholesterol. Age-adjusted DNAm GrimAge was positively associated with weight, levels of creatinine, body mass index as well as levels of C-reactive protein and interleukin-6. Horizontal lines indiciate 95% confidence intervals. BMI body mass index, CRP C-reactive protein, FCRT four choice reaction time, FER forced expiratory ratio, FEV forced expiratory volume, FVC forced vital capacity, GM grey matter, ICV intracranial volume, IL6 interleukin-6, LDL low-density lipoprotein, PEF peak expiratory flow, WM white matter, WHM white matter hyperintensities Full size image

An accelerated DNAm GrimAge was negatively associated with all four measures of lung function (β = [−0.16 to −0.27], P = [9.4 × 10−7 to 1.7 × 10−16]), iron levels (β = −0.24, P = 7.2 × 10−7), low-density lipoprotein cholesterol levels (β = −0.17, P = 1.1 × 10−4), total cholesterol levels (β = −0.13, P = 1.1 × 10−4) and height (β = −0.08, P = 0.01) (Fig. 2). Only the relationship between accelerated DNAm GrimAge and height was non-significant after controlling for childhood intelligence, with the effect size attenuating from −0.08 to −0.06 (% attenuation: 24.5%) (Supplementary File 4). On average, associations were attenuated by 2.5% after controlling for age 11 IQ [ranged from: 19.1% increase (total cholesterol) to 32.5% attenuation (triglycerides)]. All associations between blood and physical traits and an accelerated DNAm GrimAge in this study are presented in Supplementary Fig. 3. Relationships between all phenotypes tested in this study and age-adjusted DNAm Pack Years as well as age-adjusted plasma protein levels are presented in Supplementary File 5. Significant relationships are further detailed in Supplementary Note 2.

DNAm GrimAge associates with lower cognitive ability in the LBC1936

An accelerated DNAm GrimAge was significantly associated with lower measures of general cognitive ability (g: β = −0.18, P = 8.0 × 10−6; n = 709). Furthermore, an accelerated DNAm GrimAge was negatively associated with all six component tests for fluid intelligence from which g was derived (see Section “Phenotypic data”; β = [−0.11 to −0.16], P = [0.02 to 2.4 × 10−4]). In addition, an accelerated DNAm GrimAge was associated with an increased four choice reaction time mean (β = 0.16, P = 2.9 × 10−4). Lower IQ scores at age 70 (which correlated 0.70 with age 11 IQ scores) were associated with age-adjusted DNAm GrimAge (β = −0.11, P = 0.02). An accelerated DNAm GrimAge was also negatively associated with the following measures of crystallised intelligence: the Wechsler Test of Adult Reading (β = −0.13, P = 4.0 × 10−3) and the National Adult Reading Test (β = −0.10, P = 0.03).

Following adjustment for age 11 IQ, an accelerated DNAm GrimAge remained significantly associated with general cognitive ability (g: β = −0.12, P = 2.0 × 10−3; 33.9% attenuation). Three out of the six tests which constitute the general intelligence factor remained significant after adjustment for age 11 IQ (digit-symbol coding, symbol search, and matrix reasoning). Furthermore, the association between an accelerated DNAm GrimAge and an increased mean four choice reaction time remained significant following adjustment for age 11 IQ (Fig. 2). On average, associations between cognitive tasks and an accelerated DNAm GrimAge were attenuated by 41.1% following controlling for age 11 IQ (ranging from 21.7% attenuation [four choice reaction time] to 77.4% attenuation [National Adult Reading Test]). All associations between cognitive traits and an accelerated DNAm GrimAge in this study are presented in Supplementary Fig. 4. Finally, an accelerated DNAm GrimAge was not associated with APOE ε4 carrier status—the strongest genetic risk factor for Alzheimer’s disease (odds ratio = 0.96, 95% CI = [0.93, 1.00], P = 0.06).

Accelerated DNAm GrimAge showed a borderline significant association with faster cognitive decline (interaction term between an accelerated DNAm GrimAge at Wave 1 and age: β = −0.018, P = 0.05; n = 906). This association was attenuated following adjustment for age 11 IQ (β = −0.015, P = 0.11, % attenuation: 16.7%). Secondly, restricting the set of individuals to just those incorporated into our cross-sectional design (n = 709), accelerated DNAm GrimAge at Wave 1 was significantly associated with decline in general cognitive ability across the eighth decade (β = −0.020, P = 0.03; n = 709). After adjusting for childhood cognitive ability, this association was attenuated to non-significance (β = −0.017, P = 0.07, % attenuation: 15%).

DNAm GrimAge is associated with gross neurostructural differences in the LBC1936

An accelerated DNAm GrimAge was associated with lower white matter volume (β = −0.28, P = 1.7 × 10−8), total brain volume (β = −0.25, P = 1.4 × 10−7) and grey matter volume (β = −0.22, P = 1.3 × 10−5). Furthermore, an accelerated DNAm GrimAge was associated with an increased volume of white matter hyperintensities (β = 0.17, P = 1.0 × 10−3) (Fig. 2). All associations remained significant following adjustment for age 11 IQ (Supplementary File 4). On average, these associations were attenuated by 6.98% after adjusting for age 11 IQ. All associations between neuroimaging traits and an accelerated DNAm GrimAge in this study are presented in Supplementary Fig. 5.

An accelerated DNAm GrimAge was not significantly associated with general factors of white matter microstructural metrics i.e. fractional anisotropy (β = −0.009, P = 0.89) or mean diffusivity (β = −0.001, P = 0.98), hence additional regional analyses were not performed. However, given that DNAm GrimAge was associated with grey matter volume, we further tested whether there was regional cortical heterogeneity in relation to the DNAm GrimAge-grey matter association. The negative association between accelerated DNAm GrimAge and cortical volume showed a degree of regional heterogeneity across the cortical surface (Fig. 3). The strongest magnitudes were evident in lateral and medial frontal and temporal regions, extending into motor and somatosensory cortex as well as into the posterior cingulate and precuneal areas. In contrast, associations in occipital and inferior lateral and medial frontal regions were non-significant. When the associations were additionally corrected for age 11 IQ, the magnitude of the effect sizes at the FDR-significant loci were weakly attenuated (mean t-value attenuation = 3.36%; Supplementary Fig. 6).

Fig. 3 Cross-sectional association between age-adjusted DNAm GrimAge and regional cortical volume in the LBC1936. Left panel: t values indicate the magnitude of the negative association (values have been flipped for visualisation purposes). An accelerated DNAm GrimAge was negatively associated with cortical volume. Right panel: Corresponding FDR-corrected P values indicate the spatial distribution of significant associations. FDR false discovery rate Full size image

Association of DNAm GrimAge with neurological protein biomarkers

Forty of the 92 neurology-related Olink® proteins were significantly associated with an accelerated DNAm GrimAge at FDR-corrected P < 0.05 (n = 709). These proteins explained between 0.73% (β = −0.09, NC-Dase) to 7.19% (β = 0.30, SKR3) of inter-individual variation in an accelerated DNAm GrimAge (in a model which was not adjusted for age and sex; Supplementary File 6). Following adjustment for age 11 IQ, 36/40 associations (90%) remained significant. After adjusting for age 11 IQ, associations were, on average, attenuated by 3.03%.

Correlation between DNAm GrimAge and DNAm Pack Years

We observed that DNAm GrimAge and DNAm Pack Years were highly correlated (correlation coefficient: 0.82) and were cross-sectionally associated with many of the same variables in our phenotypic analyses (Supplementary File 7). Therefore, we carried out a follow-up analysis to determine the difference in magnitude between the effect sizes for DNAm GrimAge or DNAm Pack Years in relation to phenotypes associated with both predictors. Prior to adjusting for age 11 IQ, the effect sizes had a correlation coefficient of 0.88. However, they were, on average, 16.5% greater for DNAm GrimAge when compared to DNAm Pack Years. Following adjustment for age 11 IQ, the correlation coefficient was 0.84, and the effect sizes were, on average, 23.1% greater for DNAm GrimAge upon comparison to DNAm Pack Years. A plot demonstrating the correlation between effect sizes for DNAm GrimAge and DNAm Pack Years from our cross-sectional phenotypic analyses is presented in Supplementary Fig. 7.

Sex-specific differences in associations with DNAm GrimAge

As a sensitivity analysis, we accounted for an interaction between age-adjusted DNAm GrimAge and sex. Prior to adjusting for age 11 IQ, there was evidence for a sex-specific difference only in the relationships between an accelerated DNAm GrimAge and all four measures of lung function (FVC: β GrimAge × males = 0.50, P GrimAge × males = 5.6 × 10−39; FEV: β GrimAge × males = 0.39, P GrimAge × males = 1.3 × 10−23; PEF: β GrimAge × males = 0.17, P GrimAge × males = 1.2 × 10−4; FER: β GrimAge × males = −0.14, P GrimAge × males = 0.049) (Supplementary File 8). The same interaction model was also rerun accounting for age 11 IQ. Three of the lung function tests (all but FER), as well as reading ability and general cognitive ability, exhibited significant interactions between sex and DNAm GrimAge (Supplementary File 9).

Adjustment for educational attainment

In a further sensitivity analysis, we found that an accelerated DNAm GrimAge was significantly associated with years of education (β = −0.12, P = 1.7 × 10−3). Models adjusted for age 11 IQ were rerun with an additional adjustment for years of education. Of the 57 relationships which remained significant after adjusting for age 11 IQ, eight were attenuated to non-significance when adjusting for education. These included associations with symbol search and weight (β = −0.10 to −0.08, % attenuation = 22.3%; β = 0.09 to 0.05, % attenuation = 44.4%, respectively) and with six proteins (THY 1, RGMA, CDH3, TNFRSF21, NEP and TMPRSS5, mean attenuation: 8.8%) (Supplementary File 10).