Table 1. Results of the discovery phase, replication phase and joint analysis of cases aged ≥85

Locus Lead SNP Chromosome Position Candidate/closest gene EA Analysis n EAF OR 95% CI P I2 (%) P het Cases Controls Cases Controls 1q43 rs1625040 1 235 213 002 MTR, RYR2 A Discovery 7729 16 121 0.170 0.150 1.16 1.09–1.23 3.36 × 1026 Replication 13 027 60 914 0.178 0.182 1.02 0.98–1.07 0.216 Joint 20 756 77 035 1.07 1.03–1.10 3.50 × 1024 31.0 0.093 2q24.3 rs6432832 2 166 079 072 CSRNP3 A Discovery 7729 16 121 0.344 0.321 1.12 1.07–1.17 2.79 × 1026 Replication 13 019 60 824 0.346 0.339 1.03 1.00–1.07 0.029 Joint 20 748 76 945 1.06 1.03–1.09 8.73 × 1026 0.0 0.467 4q27 rs13114426 4 120 942 533 PDE5A, MAD2L1 T Discovery 7729 16 121 0.387 0.405 0.90 0.87–0.95 2.20 × 1025 Replication 13 024 60 932 0.364 0.351 1.00 0.97–1.04 0.711 Joint 20 753 77 053 0.97 0.94–0.99 0.033 46.5 0.012 5q33.3 rs2149954 5 157 753 180 EBF1 T Discovery 7729 16 121 0.388 0.360 1.12 1.07–1.17 5.98 × 1026 Replication 12 973 60 262 0.365 0.352 1.04 1.01–1.07 0.013 Joint 20 702 76 383 1.07 1.04–1.09 4.34 × 1026 28.2 0.118 8q13.3 rs10957550a 8 72 457 142 EYA1 A Discovery 7727 16 093 0.268 0.285 0.88 0.84–0.93 3.61 × 1026 Replication 10 056 56 262 0.236 0.244 0.95 0.92–0.99 0.012 Joint 17 783 72 355 0.92 0.90–0.95 1.41 × 1026 29.4 0.130 10q23.33 rs4466755 10 96 622 243 CYP2C19, CYP2C9 T Discovery 7729 16 121 0.454 0.443 1.12 1.07–1.16 2.72 × 1026 Replication 13 051 61 105 0.488 0.508 0.98 0.95–1.01 0.129 Joint 20 780 77 226 1.03 1.00–1.05 0.161 65.6 2.15 × 1025 17q23.3 rs17760362 17 58 772 399 TANC2 A Discovery 7729 16 121 0.252 0.233 1.13 1.07–1.19 5.38 × 1026 Replication 13 007 60 679 0.252 0.249 1.04 1.00–1.07 0.033 Joint 20 736 76 800 1.07 1.04–1.10 1.56 × 1025 0.0 0.473 19q13.32 rs4420638a 19 50 114 786 APOE G Discovery 7728 16 111 0.157 0.195 0.71 0.67–0.77 6.14 × 10219 Replication 10 165 57 126 0.180 0.202 0.87 0.83–0.91 2.12 × 10212 Joint 17 893 73 237 0.82 0.79–0.85 2.33 × 10226 80.2 4.35 × 10210 20q13.2 rs8126377 20 51 590 254 TSHZ2, ZNF217 G Discovery 7532 15 902 0.059 0.069 0.79 0.71–0.87 1.35 × 1025 Replication 12 974 60 647 0.058 0.054 1.01 0.94–1.08 0.901 Joint 20 506 76 549 0.93 0.88–0.99 0.020 51.1 0.006



EA, effect allele; EAF, effect allele frequency after pooling the data of all analyzed individuals; OR, odds ratio for the effect allele; 95% CI, 95% confidence interval; heterogeneity. aGenotyping of these SNPs with the Sequenom MassARRAY system for the replication phase was unsuccessful. The SNPs in bold overlap with Table 2.

A255APOE  1 2 3 4 s 6 7 8 9 10 1 12 13 14 15 16 171819202122Chromosome257APOE2074 :154 .104      7 es 9Chromosome     10 11 12 13 14 15 16 17 1819202122    

Gene set enrichment analysis (GSEA) of the meta-analysis ≥ results of the discovery-phase analysis of survival aged 90 years using Meta-Analysis Gene-set Enrichment of variaNT Associations(MAGENTA)(33),aswellasexaminationofinter- connectivity of implicated genes using Gene Relationships AcrossImplicatedLoci(GRAIL)(34)(SupplementaryMaterial, Fig.S3and TableS11),providedno firmcluesforpotential path- ways 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 identi- Py fied 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 exert its longevity-promoting effect. We did, however, find some promising functional impli- cation of this locus, i.e. the presence of multiple DNase I hyper- sensitivity 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, muscleandtestis.PhastCons44-wayalignmentsupportsconser- ′ vation of the transcription start site (TSS), 3 UTR and the third, fifth and last exon of the lincRNA transcript (Fig. 5). The tran- script does not align to the mouse genome, but orthologous tran- scripts are found in other primate genome sequences, suggesting that this is a primate-specific lincRNA.

Figure2.Resultsofthediscoveryphaseanalysis.Manhattanplotpresentingthe 2log P-values from the discovery phase analysis of cases aged ≥85 years (A) 10 and ≥90 years (B). The loci that showed a genome-wide significant association after the joint analysis of the discovery and replication phase (chromosome 19q13.32 and 5q33.3) are shown in red.

Material, Table S9). Despite the highly interesting association of the minor allele of rs2149954 with low blood pressure and a decreased risk for CAD, stroke and mortality, its association with decreased all-cause mortality was not influenced by ≥ blood pressure in two studies of participants aged 75 years (PROSPER and Leiden 85-plus study Cohort II; Supplementary Material, Table S10). This may indicate that at higher ages, this locus influences longevity via pathways additional to those involved in blood pressure regulation.

DISCUSSION

We have performed the largest genome-wide association meta-analysis for human longevity, in which a novel locus on chromosome 5q33.3 associating with survival beyond 90 years was identified.