*2.2. DNAm Age Clocks*

We used two recently developed epigenetic clocks, DNAmPhenoAge [44] and DNAmGrimAge [42], both developed by the Horvath group at UCLA. In short, DNAmPhenoAge is an age clock based on beta values of 513 CpG sites established via correlation with clinical markers, and DNAmGrimAge, the more recent and accurate clock, relies on 12 different sub-DNAm-estimators alongside actual age and gender. Both clocks aim to describe health and lifespan predictions through clinical and phenotypic measurements. Both clocks predict younger age of our groups (Figure 2), with DNAmGrimAge outperforming DNAmPhenoAge, and the ELLI estimations are the most juvenile (differences between actual age and clocks is largest). The differences between chronological age and DNAmGrimAge in the control and offspring groups were very slight (Tables A3 and A4), whereas the DNAmPhenoAge consistently underestimated the ages of control and offspring participants. This performance is consistent with the DNAmGrimAge performance in the validation data used by the developers, yet is the first to be reported in ELLI, whose ages were calculated to be younger by DNAmGrimAge.

**Figure 2.** Comparison of actual age and two age clocks. DNAmGrimAge and DNAmPhenoAge calculated by applying methylation beta values to DNAm online tool [37]. Ncontrol = 28, NELLI = 24, and Noffspring = 18.

Further, there is a high correlation between chronological age and both DNAm clocks (Figure 3), with DNAmGrimAge outperforming DNAmPhenoAge in actual age prediction. Though DNAmGrimAge is more closely related to chronological age (especially due to the use of actual age as a parameter of DNAmGrimAge), DNAmPhenoAge was originally designed to capture a phenotypic age (rather than chronological age). As depicted by our results, the phenotypic age prediction was lower than the chronological age especially for ELLI, indicating a juvenile phenotype of this group. With this high correlation in mind, we proceeded to examine correlation between age and DNAm clocks with cognitive state of IMECS participants. For this extent, we used the Mini-Mental State Exam (MMSE) questionnaire score as a measure for the cognitive impairment of participants. This assessment revealed no significant correlation between MMSE score and age in neither group (Figure 4).

**Figure 3.** Chronological age vs. DNAm epigenetic age clocks. Ncontrol = 28, NELLI = 24, and Noffspring = 18. (**A**) DNAmPhenoAge as a function of chronological age, R<sup>2</sup> = 0.716, *p* < 0.001. (**B**) DNAmGrimAge as a function of chronological age, R<sup>2</sup> = 0.919, *p* < 0.001. Linear regressions performed and plotted using JMP 14 (SAS Institute Inc., Cary, NC, USA).

**Figure 4.** Cognition vs. age. Mini-Mental State Exam scores used for measuring cognitive impairment. (**A**) MMSE score of controls as a function of chronological age, *N* = 28, *p* = 0.7021. (**B**) MMSE score of ELLI as a function of chronological age, *N* = 21, *p* = 0.0776. (**C**) MMSE score of offspring as a function of chronological age, *N* = 16, *p* = 0.2021.
