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 signalling genes on familial longevity is very small.

compared with controls N N Protein Genename cases controls Mean SD FC P 4EBP1 EIF4EBP1 76 303 2.37 0.048 1.19 2.1 3 10(cid:1)8 AKT AKT1 76 303 2.27 0.103 1.18 0.030 ATG101 C12orf44 71 296 1.02 0.020 1.00 0.644 ATG13 ATG13 71 296 1.10 0.019 0.99 0.095 Deptor DEPTOR 70 296 0.05 0.001 1.00 0.001 eIF4B EIF4B 70 295 0.47 0.018 1.00 0.572 eIF4E EIF4E 71 296 1.10 0.045 0.94 0.012 FIP200 RB1CC1 71 295 2.41 0.153 1.29 0.005 FOXO1 FOXO1 71 296 1.50 0.034 0.90 1.0 3 10(cid:1)11 FOXO3 FOXO3 71 296 1.73 0.057 1.05 0.166 HIF1a HIF1A 71 296 4.05 0.322 1.90 0.005 mLST8 MLST8 71 296 0.42 0.010 1.00 0.446 MP1 MAPKSP1 71 296 1.64 0.039 0.97 0.112 mSIN1 MAPKAP1 70 294 1.42 0.018 0.98 0.002 mTOR MTOR 68 294 0.71 0.013 0.97 6.0E-06 p14 LAMTOR2 71 296 1.50 0.049 1.08 9.1 3 10(cid:1)5 p18 CDKN2C 70 295 0.71 0.020 1.00 0.630 PGC1a PPARGC1A 66 287 0.02 0.003 1.00 0.762 PKCa PRKCA 71 296 0.57 0.016 0.95 5.4 9 10(cid:1)9 PPARg PPARG 70 294 0.03 0.002 1.01 8.4 9 10(cid:1)5 PRAS40 AKT1S1 70 296 0.87 0.049 1.14 1.9 3 10(cid:1)4 Protor1 PRR5 71 296 4.47 0.197 0.82 0.035 Protor2 PRR5L 71 296 7.03 0.211 1.81 4.9 3 10(cid:1)5 RagA RRAGA 71 295 1.09 0.038 0.95 0.009 RagB RRAGB 71 296 0.67 0.019 0.99 0.028 RagC RRAGC 71 296 1.69 0.041 1.02 0.231 RagD RRAGD 70 296 0.66 0.015 1.02 0.035 Raptor RPTOR 79 312 0.70 0.037 0.92 0.001 Rheb RHEB 71 296 0.73 0.028 0.97 0.060 RhoA RHOA 71 296 2.15 0.037 1.08 5.6 3 10(cid:1)5 Rictor RICTOR 71 296 6.24 0.182 0.99 0.874 S6 RPS6 71 295 1.92 0.073 0.93 0.055 S6K1 RPS6KB1 76 303 3.05 0.110 0.94 0.648 SGK SGK1 71 296 0.79 0.019 1.01 0.247 SKAR POLDIP3 67 260 1.00 0.020 1.05 1.3 9 10(cid:1)4 SREBP SREBF1 71 296 0.72 0.031 1.00 0.758 TSC1 TSC1 71 296 1.02 0.020 0.98 0.019 TSC2 TSC2 71 296 0.78 0.021 1.04 3.8 9 10(cid:1)4 ULK1 ULK1 76 303 2.47 0.081 1.19 0.002 YY1 YY1 71 296 1.73 0.037 1.01 0.740 Significance level for P-value after Bonferroni correction for multiple testing is

Gene expression in whole blood: offspring of nonagenarians vs. 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 nonage- narians 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, the 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 the nonagenarians, but decreased in their offspring. The gene expression differences between offspring and control groups may be due to variation in gene expression level indeed or could (partially) be explained by differences in between the groups in blood cell subsets or in disease prevalence.

Relation of gene expression in whole blood with cell subtypes

Whole blood consists of several cell subtypes and subsets of differen- tiated 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 leucocytes, 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 the gene expression with familial longevity.

Significance level for P-value after Bonferroni correction for multiple testing is 0.00125. 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 long-lived