We tested whether the number of disease alleles was different between long-lived individuals and younger controls, because the cumulative effect of disease alleles is expected to be more pro- nounced than that of individual SNPs, with a modest effect on disease risk (9, 10). On average the nonagenarian siblings carried ± 26.7 0.19 disease risk alleles, which was virtually identical to the number carried by younger controls (27.0 ± 0.12; P = 0.127; Fig. 1A ), and the same was observed for the number carried by ± sporadic long-lived subjects (26.8 0.11) and younger controls ± P B (26.8 0.10; = 0.847; Fig. 1 ). The distribution of the number of risk alleles was the same in highly aged and young subjects, C D even in the tails of the distribution (Fig. 1 and ). Similar results were obtained when the 19 alleles associated with meta-

Authorcontributions:M.B.,B.T.H.,andP.E.S.designedresearch;C.N.,H.E.D.S.,D.K.,R.v.d.B., and N.L. performed research; P.E.S., A.J.M.d.C., R.J.G.W., D.I.B., and E.J.C.d.G. contributed newreagents/analytictools;M.B.,W.G.A.,andJ.J.H.-D.analyzeddata;andM.B.,B.T.H.,and P.E.S.wrotethepaper.

Mean number of disease risk allelesMean number of disease risk alleles2520302520a     30Wm Controls Mmm Controlsa i Familial long-lived cases [5 Familial long-lived cases23S2=&53 2Qa°Q=o 15<2=— > 10o25» | mall [ Li.Total risk alleles Metabolic risk alleles Cancer risk alleles <18 19-20 21-22 23-24 25-26 27-28 29-30 31-32 33-34 35-36 37+Number of disease risk alleles30Ms Controls me Controls[55 Sporadic long-lived cases Sporadic long-lived cases23c2@Ss 20a°QaSo 8 —ce2tcO° 10a° —2|5: —     Total risk alleles Metabolic risk alleles Cancer risk alleles<18 19-20 21-22 23-24 25-26 27-28 29-30 31-32 33-34 35-36 37+Nuirmher of diceace rick allelec  

ee ee:Total risk alleles Metabolic risk alleles Cancer risk alleles

 Total risk alleles Metabolic risk alleles Cancer risk alleles

Fig. 1. GWAS-identified disease risk alleles in nonagenarians from long-lived families and sporadic long-lived individuals. (A) Average number of risk alleles among nonagenarian siblings and controls of the LLS. (B) Average number of risk alleles among sporadic long-lived individuals of the Leiden 85 Plus Study and NTR controls. (C) Distribution of the number of disease risk alleles among nonagenarian siblings and controls of the LLS. (D) Distribution of the number of disease risk alleles among sporadic long-lived participants of the Leiden 85 Plus Study and NTR controls.

cardiovascular disease, cancer, and type 2 diabetes by delaying onset or decreasing the severity may be prevalent in this group. To identify loci essential for delay of disease and a GWAS for survival into old age may be an approach by protective alleles could also be identifi ed.

cancer between long-lived individuals (aged 85 y and older) and the middle-aged general population, in whom the disease risk alleles are generally discovered.

Despite the fact that cardiovascular disease, cancer, and type 2 diabetes contribute to the majority of deaths in modern societies, the cumulative effects of this set of 30 risk alleles do not restrain the study group from surviving into old age. This observation, together with the observations that the offspring of the nonage- narian siblings and centenarians have a decreased prevalence of cardiovascular disease, hypertension, and type 2 diabetes as compared with controls (5, 14), suggests that the absence of these disease loci does not explain the lower morbidity in the offspring. Also, offspring of centenarians have lower mortality from cancer despite the same cancer prevalence as compared with controls (4). fi It may be possible that other factors, such as unidenti ed (rare) genetic variants that associate with fatal disease, are lacking in the study group. Alternatively, alleles protecting individuals from

The sum of risk alleles has commonly been used to estimate a cumulative effect of the risk alleles to the disease prevalence of – type 2 diabetes and cancer (9, 15 21). Previous studies reported substantially increased risk among the upper 10% of the pop- ulation carrying the highest number of risk alleles as compared with the 10% with the lowest number of risk alleles [e.g., type 2 diabetes, OR= 8.7 (9); prostate cancer, OR = 9.5 (19)]. Of 20 known variants associated with type 2 diabetes, it has been esti- – mated that they would account for 5 10% of an inherited pre- disposition for type 2 diabetes (22). The SNP sets that have been fi investigated for their cumulative effect on the risk for speci c diseases in these studies overlap with the SNP set investigated in

the current study. No effect of a subset of SNPs or among SNPs was detected in the current data, which are agreement with previous observations that additive variance accounts for almost all genetic variance (23). Putative are thus best described with a multiplicative model in line – previous disease-focused studies on GWAS SNP sets (9, 15 An accurate estimation of a predicted cumulative effect of SNPs on longevity was hardly possible because the genetic ciations used for this estimation derive mostly from studies fi on disease risk and not on the effect of disease-speci c mortality.

NTR as Population Controls. From the NTR, 1,203 unrelated participants of European descent with mean age of 41 y (range: 18–80 y) were selected as young population controls for whom DNA was available (8). The sub- structure in the NTR control group has already been reported (28), and in our study, we included the samples of Dutch descent without known family relations (i.e., those without any substructure).