Relation of gene expression in whole blood with cell subtypes
Whole blood consists of several cell subtypes and subsets of differentiated immune cells that may have different mRNA expression profiles. To investigate whether the proportions of different blood cell types could explain differential expression levels of RPTOR and PRRL5, the comparisons between offspring and controls were corrected for the relative counts of leukocytes, thrombocytes, neutrophils, lymphocytes, monocytes, basophils, and eosinophils present in the whole blood samples (see Experimental procedures). We observed that variation between cell counts in offspring and controls did not influence the associations of gene expression with familial longevity.

Since we had GWAS data available in the LLS study, we analyzed the set of GWAS-based SNPs within a 10-kb window around the 40 mTOR signaling genes for differences in variation between 417 unrelated nonagenarian participants and 476 younger controls from the LLS (Supplementary Table S1) (Deelen et al., 2011a). Using the PLINK setcorresponding proteins of both complexes, as well as the upstream regulators of mTORC1 and mTORC2. Finally, we added the proteins that interact with mTORC1 and mTORC2, which are involved in the regulation of autophagy, mRNA translation, metabolism, cell cycle and apoptosis (Wullschleger et al., 2006).

We performed a genetic approach to compare nonagenarians and controls. The mTOR gene set variation was associated with longevity in the LLS due to the contribution of small effects in several genes. This association couldn't pinpoint the main effector genes or SNPs; therefore, we concluded that this finding must be replicated in much larger studies to test whether genetic variation in the pathway and specific genes truly associate with familial longevity.

Decreased mTORC1 protein activity is associated with lower glucose levels and a lower prevalence of diabetes in humans (Liu et al., 2004; Krebs et al., 2007), while impaired mTORC2 is associated with glucose intolerance and insulin resistance (Lamming et al., 2012). The offspring of nonagenarian siblings in the LLS have a lower prevalence of diabetes and lower glucose levels than similarly aged controls. Since adjustment for glucose levels and excluding diabetic patients provides similar associations, differences in the prevalence of type 2 diabetes or plasma glucose levels between offspring and controls did not explain the association of RPTOR and PRR5L mRNA level with familial longevity.

Remarkable changes in effect size were found for RPTOR when considering T-cell differentiation markers (Table S4). Our results could be explained if RPTOR gene expression in blood marks familial longevity and metabolic health in middle-age and would thus be an early marker of familial longevity. The altered gene expression levels in the longevity family members may be the consequence of aging, or merely a trait shared by the long-lived families, or may truly contribute to human longevity. In view of the findings in animal models (Vellai et al., 2003; Kapahi et al., 2004; Kaeberlein et al., 2005; Harrison et al., 2009), it is not unlikely that a familial and genetic background contributing to low mTORC1 signalling activity (and preservation of low mTOR throughout life) contributes to metabolic health and an extended life expectancy across species.

Since in the literature expression levels of mTOR pathway proteins have been implicated in the pathogenesis of type 2 diabetes (Newgard et al., 2009) and progression of cancer (Guertin & Sabatini, 2005; Stanfel et al., 2009) and since the prevalence of diabetes differed between LLS offspring and controls (Westendorp et al., 2009), we tested whether RPTOR and PRR5L expression were simply marking differences in disease prevalence. We therefore performed the same analysis between offspring and controls without known diabetics and cancer patients, which yielded similar associations to those described above (Supplementary Table S6).

In our study, we found RPTOR, AKT1S1, and EIF4EBP1 to be associated with old age and/or familial longevity, and the directions of association indicate a transcriptional down-regulation of mTORC1. Even in middle-age, a down-regulation of RPTOR mRNA was associated with familial longevity, providing another example of the highly conserved role mTOR signalling has in lifespan regulation across different species.

Because glucose levels differed between offspring and controls (Supplementary Table S5), we investigated whether the mRNA differences of RPTOR and PRR5L depended on glucose levels in non-diabetic participants. The association of familial longevity with RPTOR was not affected by adjustment for glucose levels, and the association with PRR5L expression gained significance (Supplementary Table S7). Thus, the expression level of the RPTOR gene in blood marks familial longevity independent of the prevalence of type 2 diabetes and cancer and glucose levels. Due to the influence of T-cell differentiation, glucose levels, and diabetes prevalence on its gene expression, the association of PRR5L with familial longevity is more complex.