Genome-wide association study identifies a single major locus contributing to survival into old age; the APOE locus revisited in Helsinki, Finland. Quinta Helmer, Maris Kuningas, Lene Christiansen, Dennis Kremer, Ruud van der Breggen, H. Eka D. Suchiman, Nico Lakenberg, Erik B. van den Akker, Willemijn M. Passtoors, Henning Tiemeier, Diana van Heemst, Anton J. de Craen, Fernando Rivadeneira, Eco J de Geus, Markus Perola, Frans J. van der Ouderaa, David A. Gunn, Dorret I. Andre G. Uitterlinden, Kaare Christensen, Cornelia M. van Duijn, Bastiaan T. Heijmans, Jeanine J. Houwing-Duistermaat, Rudi G. J. Westendorp and P. Eline Slagboom.

14 Unilever Discover, Colworth Science Park, Sharnbrook, Bedfordshire MK44 1LQ, UK.

Summary:
The aim of this study was to identify mechanisms that contribute to healthy aging by studying the loci that contribute to human longevity. For this purpose, a genome-wide association study (GWAS) was conducted comparing 403 unrelated nonagenarians from long-living families included in the Leiden Longevity Study (LLS) and 1670 younger population controls. The strongest candidate SNPs from this GWAS were analyzed in a meta-analysis of nonagenarian cases from the Rotterdam Study, Leiden 85-plus study and Danish 1905 cohort. Only one of the 62 prioritized SNPs from the GWAS analysis (P < 1 × 10^-4) showed genome-wide significance with survival into old age in the meta-analysis of 4149 nonagenarian cases and 7582 younger controls [OR = 0.71 (95% CI 0.65-0.77), P = 3.39 × 10^-17]. This SNP, rs2075650, is located in TOMM40 at chromosome 19q13.32 close to the apolipoprotein E (APOE) gene. Although there was only moderate linkage disequilibrium between rs2075650 and the ApoE e4 defining SNP rs429358, we could not find an APOE-independent effect of rs2075650 on longevity, either in cross-sectional or in longitudinal analyses. As expected, rs429358 associated with metabolic phenotypes in the offspring of the nonagenarian cases from the LLS and their partners. In addition, we observed a novel association between this locus and serum levels of IGF-1 in women (P = 0.005). In conclusion, the major locus determining familial longevity up to high age as detected by GWAS was marked by rs2075650, which tags the deleterious effects of the ApoE e4 allele. No other major locus was found.

Introduction:
The increase in mean life expectancy worldwide in the past two centuries is mainly due to environmental factors such as improved hygiene, nutrition, and health care. However, the large variation in healthy lifespan among the elderly has prompted research into the determinants of aging and lifespan regulation. The genetic contribution to human lifespan variation was estimated at 25-30% in twin studies. The most prominent genetic influence is observed in families in which the capacity to attain a long lifespan clusters. Exceptional longevity can be reached with a low degree of age-related disability, raising the question whether protective mechanisms against disease exist in long-lived subjects.

Methodology:
The Leiden Longevity Study (LLS) families offer a route to identify genetic variants that influence human longevity. A genome-wide association study (GWAS) was performed comparing nonagenarian siblings from the LLS and younger population controls. Emerging candidate SNPs were subsequently investigated in nonagenarian cases from the Rotterdam Study, the Leiden 85-plus study, and the Danish 1905 cohort.

Results:
A GWAS was performed in nonagenarian participants from the LLS and middle-aged controls from the Rotterdam Study (RS). Genotype data for 516,721 SNPs that passed quality control thresholds were analyzed in a comparison of 403 unrelated nonagenarians (94 years on average) and 1670 controls (58 years on average). None of the SNPs reached genome-wide significance (P < 5 × 10^-8). Hypothesis-free genome-wide approaches have also been undertaken. Genome-wide linkage scans reported evidence for linkage with longevity on chromosome 4q25, 3p24-22, 9q31-34.12q24 (Boyden & Kunkel, 2010). However, the evidence for these loci is still very weak as the results obtained in centenarians and their families could not be replicated in nonagenarian sibling pairs (Beekman et al., 2006) or have yet to be tested in other studies. A meta GWAS of survival to 90 years or older in 1836 cases and 1955 controls did not find any significant genome-wide associations (Newman et al., 2010). Thus far, hypothesis-free approaches have not identified any loci involved in longevity.

Replication studies: We prioritized the SNPs that had the most significant association with survival into old age according to the analysis of stage 1 (P < 1 x 10^-4, Table S1). For 58 of the 62 selected SNPs, successful genotyping was obtained in the replication cohorts. In stage 2, these 58 SNPs were tested for association comparing 960 RS replication cases (mean age of 93 years), 1208 Leiden 85-plus replication cases (mean age of 92 years), and 1578 Danish replication cases (mean age of 93 years) with appropriate middle-aged population controls (Table 1). Meta-analysis for the 58 SNPs, comprising a total of 4149 nonagenarian cases and 7582 younger controls (from the LLS GWAS, RS replication, Leiden 85-plus replication, and Danish replication studies), was performed.