Human longevity, which is coupled to compression of age-related disease, is a heritable trait. However, only few common genetic variants have been linked to longevity, suggesting that rare, family-specific variants may also play a role. We therefore investigated whole-genome sequencing data of long-lived individuals from the Leiden Longevity Study and identified family-specific variants residing in genes involved in the mitogen-activated protein kinase (MAPK) cascade, a lifespan-associated and evolutionarily conserved pathway emerging from studies in model organisms. We subsequently generated and functionally characterised mouse embryonic stem cells (mESCs) harbouring these variants. Two variants, located in NF1 (Phe1112Leu) and RAF1 (Asp633Tyr), reduce MAPK/extracellular signal-regulated kinase (ERK) signalling pathway activity in mESCs. At the proteomic and transcriptomic level, we observed prominent changes that were shared (e.g. upregulation of ribosomal proteins and Foxo3 expression) and opposing between the variants (e.g. downregulation of mTORC1 signalling-related proteins and Ets2 expression in the RAF1^Asp633Tyr variant cell line versus upregulation in the NF1^Phe1112Leu variant cell lines). These changes were accompanied by opposing effects on proliferation. Moreover, the RAF1^Asp633Tyr variant improved resistance to replication stress, while this was not the case for the NF1^Phe1112Leu variant. In conclusion, we identified two rare genetic variants in long-lived families that influence MAPK/ERK signalling in a manner that has previously been linked to increased lifespan in model organisms. Our findings suggest that mESCs offer a suitable starting point for studying rare genetic variants linked to human longevity, allowing for the identification of promising variants to pursue in in vivo studies using model organism.

The response to lifestyle intervention studies is often heterogeneous, especially in older adults. Subtle responses that may represent a health gain for individuals are not always detected by classical health variables, stressing the need for novel biomarkers that detect intermediate changes in metabolic, inflammatory, and immunity-related health. Here, our aim was to develop and validate a molecular multivariate biomarker maximally sensitive to the individual effect of a lifestyle intervention; the Personalized Lifestyle Intervention Status (PLIS). We used 1 H-NMR fasting blood metabolite measurements from before and after the 13-week combined physical and nutritional Growing Old TOgether (GOTO) lifestyle intervention study in combination with a fivefold cross-validation and a bootstrapping method to train a separate PLIS score for men and women. The PLIS scores consisted of 14 and four metabolites for females and males, respectively. Performance of the PLIS score in tracking health gain was illustrated by association of the sex-specific PLIS scores with several classical metabolic health markers, such as BMI, trunk fat%, fasting HDL cholesterol, and fasting insulin, the primary outcome of the GOTO study. We also showed that the baseline PLIS score indicated which participants respond positively to the intervention. Finally, we explored PLIS in an independent physical activity lifestyle intervention study, showing similar, albeit remarkably weaker, associations of PLIS with classical metabolic health markers. To conclude, we found that the sex-specific PLIS score was able to track the individual short-term metabolic health gain of the GOTO lifestyle intervention study. The methodology used to train the PLIS score potentially provides a useful instrument to track personal responses and predict the participant's health benefit in lifestyle interventions similar to the GOTO study.