*2.3. Telomere length*

Finally, we turned to telomere length measurement using qPCR and the DNAm estimator of telomere length, DNAmTL. Our qPCR results did not demonstrate different T/S ratios between the three groups (Figure 5 and Table A5). However, the DNAmTL estimator found the telomeres of ELLI to be approximately 500 bp shorter compared to the control and offspring groups (Figure 6 and Table A6). When comparing T/S ratio and DNAmTL (Figure A1), there is no correlation between the two TL measures. Interestingly, when T/S ratio is tested between ELLI and controls with adjustment by DNAmGrimAge, it approaches significant correlation (*p* = 0.0508), hinting at a masking effect of the physiological age (representing juvenile methylation levels of centenarians) on the T/S ratio obtained with qPCR.

**Figure 5.** Average T/S ratio as measured by qPCR. T/S ratio obtained by dividing concentration of telomeric reaction by concentration of SCG (Single Copy Gene) reaction, as calculated using standard curve reactions. NELLI = 12, Ncontrol = 17, and Noffspring = 12. All pair comparison (Dunn Joint Ranking) non-significant.

**Figure 6.** DNAmTL calculated using the online tool. DNAmTL was calculated by applying methylation beta values of 140 CpGs to online tool [37]. Ncontrol = 28, NELLI = 24, and Noffspring = 18. \* Significant differences, *p* <0.05.

#### **3. Discussion**

Many studies are aimed at biomarker discovery and improvement for aging [38,41,42,44,47–52]. The need for such characterization is of upmost importance in light of efforts to achieve longer health and lifespans across the world. Such biomarker detection would enable tracking and even reversal [45] of aging processes and allow for drug targeting and development to benefit the already graying population. Molecular and genomic biomarkers for aging are still sparse and inaccurate with the exception of the very recent development of DNAmGrimAge [42]. This DNA methylation biomarker outperforms all previously reported methylation age estimators and serves as a very accurate estimate of chronological age. Although this is expected due to the use of chronological as a surrogate for the age prediction, DNAmGrimAge, as DNAmPhenoAge, also serve as an evaluation of health status, indicative of the rate of epigenetic aging. Use of such biomarkers as indication of rate of age acceleration

could promote better understanding of the processes underlying progression of aging and replace use of chronological age in clinical assessments relating to those conditions.

That said, the centenarian DNAm still remains elusive, even to the most accurate DNAmGrimAge. We show here that although accurate in offspring of ELLI and unrelated controls, DNAmGrimAge, along with DNAmPhenoAge, underestimates the chronological age of our IMECS ELLI participants, predicting a younger epigenetic age. We believe that this represents a slower rate of aging processes occurring in ELLI, and enabling them to reach such exceptional chronological age. This is in agreement with the methylation profile of semi-supercentenarians and their offspring, described by Horvath et al. [36], and replicates their results in our independent cohort.

The juvenile DNAm profile demonstrated in our cohort together with mostly intact cognition add support to the idea that ELLI age at a slower rate. Even though there was a small decline in the MMSE scores of the ELLI, this decline was not statistically significant, indicating intact cognition in the majority of the ELLI participants.

Further, DNAmTL estimated telomere length compared to T/S ratio of qPCR measurement showed no correlation with each other, until adjusted by DNAmGrimAge, at which the correlation approached significance. This masking effect of the physiological age (measured by DNAmGrimAge) adds support to the slower rate of aging. Telomere length has long been argued for and against use as an age indicator, but it is well-established to be decreased with age. Our qPCR measurements are consistent with previous observations of longer telomeres in ELLI [2]. While T/S ratio of the ELLI was expected to shorten in respect to offspring and controls because of their relatively advanced age, it remained unchanged, indicating a similar telomere length despite almost 30 years average age difference between group participants, demonstrating once again, a decreased aging rate. Taken together with the juvenile methylation rates in ELLI, we suggest that ELLI age slower than the general population through a beneficial methylation profile that may affect telomere length and other aspects of the hallmarks of aging.

To further draw conclusions, there is a need for bigger sample size and thorough molecular validation. We acknowledge that these are limitations in our current study and are already planning to pursue various directions for validation of our results. In addition, since the work presented here is part of an ongoing study, new IMECS participants are recruited and new recruitment centers should be established to increase ease and rate of recruitment. We believe that with adequate sample size and further validation in primary cells from participants we will be able to obtain more information on the juvenile epigenetic profile of ELLI and their offspring.
