Recently, an increasingly important role for mTOR in directing T-cell activation and differentiation has become apparent (Derhovanessian et al., 2010). We therefore examined whether the difference in expres- sion levels of RPTOR and PRR5L between offspring and partners is influenced by the level of T-cell differentiation reflected in the distribution of different T-cell phenotypes. To this end, the frequency of na¨ıve (CD45RA+ CCR7+ CD27+ CD28+), central memory (CD45RA(cid:1)CCR7+ CD27+ CD28+), effector memory (CD45RA(cid:1)CCR7(cid:1)CD27(cid:1)CD28(cid:1)) and late-stage differentiated (CD45RA+CCR7(cid:1)CD27(cid:1)CD28(cid:1)) T cells was assessed in 71 offspring and 73 controls. The largest effect was seen when correcting for the relative amount of effector memory T cells present in this sample; it reduced the expression difference between offspring and controls for PRR5L by about 60% (the beta changed from (cid:1)0.44 to (cid:1)0.17, Supplementary Table S4). This indicated that differences in the proportion of effector memory T cells in these groups explained about half of the differential gene expression effect for PRR5L. No

in bold. Mean, mean relative expression level; SD, standard deviation of relative expression level; FC, fold change, above one indicated higher expression in long-lived individuals. P, raw P-value from linear regression model.

were expressed at a lower level, and EIF4EBP1, LAMTOR2, AKT1S1, PRR5L and RHOA at a higher level in nonagenarians.

Genetic analysis: nonagenarians vs. controls

Since we had GWAS data available in the LLS study, we analysed the set of GWAS-based SNPs within a 10-kb window around the 40 mTOR signalling 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 set- based test, we investigated 1,018 SNPs in the 40 mTOR signalling

middle-aged members of the longevity families as compared to similarly aged controls. The significant differential expression of RPTOR and suggestive differential PRR5L expression indicate that expression of these genes may not just mark old age but familial longevity per se.

Table 3 Linear regression results of gene expression of offspring compared with controls

Protein Genename N cases N controls Mean SD FC P 4EBP1 EIF4EBP1 320 303 2.03 0.295 1.03 0.311 FOXO1 FOXO1 311 296 2.18 0.238 1.00 0.878 p14 LAMTOR2 311 296 1.93 0.397 0.99 0.852 PRAS40 AKT1S1 311 296 1.67 0.278 0.96 0.282 Protor2 PRR5L 311 296 5.59 1.143 0.74 0.041 Raptor RPTOR 332 312 0.99 0.184 0.92 0.001 RhoA RHOA 311 296 2.15 0.265 1.00 0.927

We also compared nonagenarians and controls in a genetic approach. Variation in the mTOR gene set associated with longevity in the LLS due to the joint contribution of small effects in several genes. This association could not pinpoint the main effector genes or SNPs, so we conclude 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 the 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.

Significance level for P-value after Bonferroni correction for multiple testing is 0.00714. Genes significantly differentially expressed with at least 5% are depicted in bold. Mean, mean relative expression level; SD, standard deviation of relative expression level; FC, fold change, above one indicated higher expression in offspring of nonagenarians; P, raw P-value from linear regression model.

remarkable changes in effect size were found for RPTOR when for the above-mentioned 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.

Association of gene expression in whole blood with disease and glucose phenotypes

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 (Supple- mentary Table S6).