there are any age-related changes in gene expression that are not associated with familial longevity. 

Gene expression analysis was conducted on the mTOR pathway to investigate its association with human longevity. The mTOR pathway consists of two complexes, mTORC1 and mTORC2, with different functions. The main functions of mTORC1 include translation initiation, protein synthesis, and autophagy, while those of mTORC2 include cytoskeletal organization. The two complexes are connected in that mTORC2 regulates Akt phosphorylation, part of an upstream pathway that controls activation of mTORC1. Inhibition of the mTOR signaling pathway in model organisms results in lifespan extension and improved metabolic function. Additionally, reduced mTOR signaling is associated with a reduction in age-associated cancer incidence and growth rate. 

The study investigated whether transcriptional variation within the mTOR pathway is associated with human longevity using whole-blood samples from the Leiden Longevity Study, a cohort of Dutch families with extended survival across generations and beneficial metabolic profiles in middle-age. The mRNA levels of nonagenarians and middle-aged controls were compared, and the mTOR signaling gene set was found to associate with old age. Seven of 40 mTOR pathway genes had a significant differential expression of at least 5%, and the RPTOR (Raptor) gene was found to be differentially expressed also when the offspring of nonagenarians was compared with their spouses, indicating association with familial longevity in middle-age. This association was not explained by variation between the groups in the prevalence of type 2 diabetes and cancer or glucose levels. 

In conclusion, the study shows that the mTOR pathway not only plays a role in the regulation of disease and aging in animal models but also in human health and longevity.Differential expression of these genes does not simply indicate old age but is a defining characteristic of the long-lived families in middle age. Finally, we examined whether the association between the expression of mTOR signaling genes and longevity is independent of metabolic health parameters, namely plasma glucose levels and the prevalence of type 2 diabetes and cancer, as these factors are known characteristics of long-lived families and are suggested to be influenced by mTOR signaling.

Gene expression in whole blood: nonagenarians vs. controls
We measured gene expression levels for each of the 40 mTOR signaling genes using RT-qPCR on whole blood samples from 87 LLS nonagenarians, 337 of their middle-aged offspring, and 321 middle-aged LLS controls (Table 1). When comparing long-lived individuals to younger controls, differentially expressed genes may associate either with calendar age, familial longevity or both. We tested the total gene set for such association using the globaltest method (Goeman & Oosting, 2011). We observed that the gene expression levels of the mTOR signaling gene set did associate significantly with age and/or familial longevity in these families (P = 4.6 x 10^-7).

Second, to investigate which genes are primarily responsible for this association, single-gene analysis using linear regression was performed on all 40 mTOR signaling genes (Table 2). After Bonferroni correction for multiple testing, seven genes showed significant differential expression with at least a 5% expression difference: FOXO1 and RPTOR.

Table 1: Analyzed samples: Gene expression
Results
Samples Number Mean age Age range Men/women Long-lived individuals 87 94.3 89-102 40/47 Offspring 337 61.3 34-78 194/143 Controls 321 61.2 32-81 146/175

Selection of mTOR pathway genes
For the mTOR pathway, we selected 40 genes encoding proteins that belong to the well-described core of the mammalian mTOR pathway.

Table 2: Linear regression results of gene expression of long-lived individuals compared with controls
Genes simultaneously in one test (P-value < 0.05 for significance) (Supplementary Table S2). We observed a significant association of genetic variation in the mTOR pathway as a whole with familial longevity (P = 0.009). However, the single-gene analysis (of 40 SNP sets), for which the significance level for P-values after Bonferroni correction for multiple testing is 0.00125, did not result in significant associations. (Supplementary Table S3), suggesting that effects of single common variation in mTOR signaling genes on familial longevity are very small.

Table 3: Linear regression results of gene expression of offspring compared with controls
To investigate whether the differences in expression of the seven genes differentially expressed between nonagenarians and controls are a characteristic of the long-lived families and not just a marker of old age, we compared their expression in the middle-aged offspring of nonagenarians to the controls of similar age (Supplementary Table S5). While the association for six genes was not significant after correction for multiple testing, as compared to the controls, the offspring expressed significantly less RPTOR mRNA (Table 3). Thus, in two generations, RPTOR gene expression was decreased as compared to controls. As association of PRR5L was borderline significant and displayed the largest effect size (FC = 0.74), we also included PRR5L for further follow-up in this article. Gene expression levels of PRR5L were increased in nonagenarians but decreased in their offspring. The gene expression differences between offspring and control groups may be due to variation in gene expression level or could (partially) be explained by differences between the groups in blood cell subsets or disease prevalence.