A stuayDiscoveryCEPH centenarian cohortDanish longevity study |GEHA DanishGEHA DutchGEHA FrenchGEHA ttalyGEHA UKLeiden 85-plus study |LLSNewcastle 85+ StudyRotterdam Study |TwinGeneCombinedReplicationDanish longevity study IldeCODECombinedCombined  Odds ratio0.450.530.510.560.560.840.580.690.600.770.860.640.640.510.790.770.7295% Cl(0.35; 0.58](0.38; 0.74][0.37; 0.69][0.32; 1.00][0.34; 0.90](0.51; 1.37](0.31; 1.08][0.50; 0.94](0.48; 0.74](0.63; 0.94][0.69; 1.06](0.44; 0.94][0.59; 0.70](0.38; 0.67](0.74; 0.84)(0.72; 0.82][0.68; 0.76]B stuayDiscoveryCEPH centenarian cohortDanish longevity study |GEHA DanishGEHA DutchGEHA FrenchGEHA talyGEHA UKLeiden 85-plus study |LLsNewcastle 85+ StudyRotterdam Study |TwinGeneCombinedReplicationBELFASTCalabria cohortDanish longevity study ildeCODEGerman longevity studyLeiden 85-plus study IlCombinedCombined05   4.07 [1.03; 1.12]140 [1.06; 1.14]

Figure4.Forestplotsforrs4420638andrs2149954.Forestplotsrepresentingtheoddsratioswith95%CIofrs4420638(A)andrs2149954(B)forthecohortsanalyzed in the discovery and replication phase (≥90 years). The size of the boxes represents the sample size of the cohort.

rightontopofourlocus(RP11-524N5.1)andshowsevidencefor expression in liver, muscle and testis. LincRNAs are involved in chromatin modification and transcriptional regulation (39) and seem to play a role in human disease (40). However, the newly annotated lincRNA is not yet available in the large eQTL data- bases, and the effect of SNPs in the chromosome 5q33.3 locus on expression of this transcript still needs to be determined. Hence, further functional studies are required to illuminate the mechanism by which this locus influences human longevity.

Rs2149954 is located in an intergenic region on chromosome 5q33.3 between CLINT1 and EBF1. The presence of several regulatory elements in this region implies that transcription factor binding and/or expression of (nearby) genes could be influenced. The currently available eQTL databases did not provide evidence for such effects, which might be due to the limited tissue diversity of the databases. The effects of the chromosome 5q33.3 locus on human longevity might be exerted throughthe lincRNA, which hasrecently been annotated

9 Chr5: 157,743,991-157,839,081 ss [ 1RP11-524NS.1 [ LtI4—i + +t 1LDelockaccordingto Halos [ Sasssziasese [ |Poly(A) features [| Ipocmesrwrmemes | Ald wt des wt Dd Ebi ih dd

Figure5.Chromosomalregionaroundrs2149954.TheregioncontainsalincRNA(RP11-524N5.1)forwhichthepoly(A)featuresaresupportedbyPolyA-seqreads ′ fromliver, muscleand testis. RP11-524N5.1 is transcribed fromthe negative strand, and the phastCons44-way alignmentsupportsconservationofthe TSS,3 UTR and the third, fifth and last exon of the transcript. Rs2149954 and the 25 SNPs in high LD (r2 ≥ 0.8, according to HaploReg v2 (35)) are located in the first intron of RP11-524N5.1.

why the novel locus on chromosome 5q33.3 was only genome- ≥ wide significant in the subset analysis of cases aged 90 years. For the same reason, a large number of individuals from the control groups (up to 50%, depending on the gender and year of birth of the individuals and demography of the cohort) will live ≥ to ages 85 years. In 2011, the mean life expectancy at age 65 in Europe was 21.3 years for women and 17.8 years for men (http://epp.eurostat.ec.europa.eu/portal/page/portal/product_deta ils/dataset?P_product_code=TSDDE210), which makes selec- tionofpropercontrolsachallengingissue.Themostidealcontrols would be individuals from the same birth cohort as the long-lived cases that survived to the mean age of death of that birth cohort. However,formostoftheseindividualsthereisno DNA available. Alternatively, we selected controls that have not yet reached the age of 65 years at inclusion to represent the frequency of variants inthegeneralpopulationandminimizeselectionowingtomortal- ity. Hence, the low contrast between cases and controls likely has reduced our probability of identifying longevity loci.

genome-wide significant hits for human longevity or mortality besides the well-known TOMM40/APOE/APOC1 locus (17–19). TheFOXO3A locus,forwhichthelongevityeffectismostpromin- ≥ ent in individuals aged 100 years (41), showed only moderate ≥ evidence for association with survival 90 years in the discovery phase of our GWAS (lowest P ¼ 1.35 × 1024 (rs1268161)). Sebastiani and colleagues suggested that human longevity might be explained by a signature consisting of 281 SNPs (42). However, none of the SNPs (except the already known SNP rs2075650 in TOMM40) was significant after adjustment for mul- tiple testing (P , 1.78 × 1024 (0.05/281)). In addition, we did not observe an enrichment of significant SNPs from their signature in our data (l¼ 1.004, Supplementary Material, Fig. S4). Because the association of SNPs other than the TOMM40/ APOE/APOC1 locus could not be replicated in this, much larger, GWAS, we have doubts that these signature SNPs are indeed candidate SNPs influencing human longevity. Although we detected merely one novel genome-wide significant locus, the current GWAS had sufficient power, based on our results, to detect lifespan-regulating loci with relatively small effects (OR ,0.9 and .1.1).