Functional characterisation of rare variants in genes encoding the MAPK/ERK signalling pathway identified in long-lived Leiden Longevity Study participants

Human longevity, which is coupled to a compression of age-related diseases, has been shown to be heritable. However, the number of identified common genetic variants linked to this trait remains small. This may indicate that longevity is, at least to some extent, determined by rare genetic variants that are potentially even family-specific. We therefore investigated whole-genome sequencing data of long-lived families from the Leiden Longevity Study for family-specific variants. We focussed on variants residing in genes involved in the MAPK/ERK signalling pathway, a lifespan-associated pathway emerging from studies in model organisms. We then used CRISPR/Cas9 to generate transgenic mouse embryonic stem cells (mESCs) and fruit flies harbouring these variants and conducted in vitro and in vivo functional characterisation. Two variants, located in NF1 and RAF1 , show opposite effects on MAPK/ERK signalling pathway activity in mESCs. At the proteomic level, we observed prominent changes that are shared between the variants (e.g. upregulation of the PI3K-AKT signalling pathway) and RAF1-specific (e.g. downregulation of oxidative phosphorylation). The variant in RAF1 also improved resistance to endoplasmic reticulum and oxidative stress in vitro . Conversely, the variant in NF1 shortened lifespan when introduced in fruit flies. In conclusion, our findings suggest that mESCs offer a good starting point for in vitro characterisation of rare genetic variants linked to human longevity. However, given the complex regulation of the MAPK/ERK signalling pathway across tissues and organisms and the large evolutionary distance between flies and humans, future studies will likely benefit from functional characterisation in human cells and model organisms evolutionary closer to humans. ### Competing Interest Statement The authors have declared no competing interest.