The discovery-phase meta-analysis of cases aged 85 years (n=7729) showed genome-wide significant association with survival into old age at one locus, the previously identified TOMM40/APOE/APOC1 locus (17,21) (rs4420638 (G); odds ratio (OR)=0.71, P=6.14×10^-19; Table 1). No gender-dependent effects were observed in the sex-stratified analysis of the cases aged over 85 years (Supplementary Material, Table S4). The discovery-phase meta-analysis of the cases aged over 90 years (n=5406) showed a similar result, i.e. the TOMM40/APOEIn order to identify novel loci involved in lifespan regulation, a meta-analysis was conducted on GWAS data from 7729 long-lived cases (85 years) and 16,121 younger controls (<65 years) from 14 studies originating from 7 European countries (see Supplementary Material, Table S1). For each study, cases and controls were from the same country. Given the higher heritability of longevity at older ages (11,20), we performed a subset analysis in which we compared cases aged 90 years (n=5406) with 15,112 controls (<65 years) from the corresponding control cohorts. Replication was performed in 13,060 cases aged 85 years (of which 7330 were 90 years) and 61,156 controls from 6 additional studies, of which 3 originated from European countries not represented in the discovery phase meta-analysis (Supplementary Material, Table S1). Analysis of each study was performed using a logistic regression-based method, and results were adjusted for study-specific genomic inflation factors (λ) (Supplementary Material, Table S2). Meta-analysis was performed on 2,480,356 (<85 years) and 2,470,825 (<90 years) imputed SNPs using a fixed-effect approach, and results showed that nine loci had more than one SNP with P<1x10-5 and twelve loci had more than one SNP with P<1x10-4.

To determine the association of rs4420638 (TOMM40/APOE/APOC1 locus) and rs2149954 (chromosome 5q33.3 locus) with longitudinal survival, we performed a prospective meta-analysis of the two SNPs in 34,103 individuals aged 30-105 years from eleven different cohorts, of which 8582 had died after a mean follow-up time ranging from 2.2 to 17.4 years. Carriers of the minor allele of rs4420638 (G) showed significantly higher all-cause mortality (hazard ratio (HR) 1.07, P 0.019), whereas carriers of the minor allele of rs2149954 (T) demonstrated significantly lower all-cause mortality (HR 0.95, P 0.003; see Supplementary Material, Table S6). Furthermore, rs2149954 had an effect on non-CVD mortality (HR 0.86, P 0.002) (Supplementary Material, Table S7). The Coronary ARtery DIsease Genome-Wide Replication And Meta-Analysis (CARDIoGRAM) GWAS also showed a significant association of rs2149954 with a decreased risk for coronary artery disease (CAD) (OR 0.96, P 0.011) (Supplementary Material, Table S8). Additionally, two SNPs on chromosome 5q33.3 in high LD with rs2149954, rs9313772 (r2=0.928) and rs11953630 (r2=0.854), have previously been reported to associate with blood pressure and hypertension (24,25). As expected, examining rs2149954 in the International Consortium for Blood Pressure GWAS (24) showed a significant association of the minor allele with lower diastolic (P 3.46x10-25) and systolic (P 6.55x10-26) blood pressure (Supplementary Material).

To gain insight into the mechanism by which the chromosome 5q33.3 locus might promote human longevity, we analyzed the cause-specific mortality of rs2149954. Carriers of the minor allele of rs2149954 have a lower mortality risk for cardiovascular disease (CVD) (HR 0.86, P 0.004), which mainly appeared to be caused by protection from stroke (HR 0.60, Table 1). Results of the discovery phase, replication phase, and joint analysis of cases aged 85-90 years are summarized in Table 1. Gene set enrichment analysis (GSEA) of the meta-analysis results of the discovery-phase analysis of survival aged 90 years using the APOE gene is shown in Figure 1.Meta-Analysis Gene-set Enrichment of Variant Associations (MAGENTA) (33), as well as examination of interconnectivity of implicated genes using Gene Relationships Across Implicated Loci (GRAIL) (34) (Supplementary Material, Fig. S3 and Table S11), provided no firm clues for potential pathways involved in human longevity. 

Fine mapping and functional characterization: 

The newly identified longevity locus on chromosome 5q33.3 is located in an intergenic region on chromosome 5q33.3, 302 kb downstream of the EBF1 gene. To determine the functional impact of this locus, we first identified the SNPs in LD with rs2149954 (r2 > 0.8) using the 1000 Genomes CEU Phase 1 data implemented in HaploReg v2 (http://www.broadinstitute.org/mammals/haploreg/haploreg.php) (35). In total, we identified 25 SNPs, spanning a region of 22.3 kb (Supplementary Material, Table S12).

Subsequently, we examined the potential effects of these SNPs on gene expression using several eQTL databases. None of the SNPs showed an association with gene expression in the various examined tissues, so it is still unclear in which tissue(s) the locus exerts its longevity-promoting effect. We did, however, find some promising functional implications of this locus, i.e., the presence of multiple DNase I hypersensitivity sites, transcription factor binding sites, and enhancer histone marks, by exploring ENCODE data using HaploReg v2 (35) and RegulomeDB (http://www.regulomedb.org/) (36) (Supplementary Material, Table S12).

Very recently, a large intergenic non-coding RNA (lincRNA), RP11-524N5.1, has been annotated right on top of our locus. The poly(A) features of this lincRNA are supported by PolyA-seq reads from liver, muscle, and testis. PhastCons 44-way alignment supports conservation of the transcription start site (TSS), 3' UTR and the third, fifth and last exon of the lincRNA transcript (Fig. 5). The transcript does not align to the mouse genome, but orthologous transcripts are found in other primate genome sequences, suggesting that this is a primate-specific lincRNA.