In conclusion, we have shown that the deleterious effect of the ApoE e4 allele, tagged by rs2075650, is the single major hit in our GWAS study for longevity, indicating that no other major longevity locus was present among these nonagenarians. We confirmed the previously reported associations of the ApoE e4 allele with lipid metabolism parameters and report an additional effect on IGF-1 signaling in women. To identify genetic variants with smaller and protective effects on human lifespan, a meta- GWAS for longevity with a larger sample size is merited.

Netherlands twin registry

From the Netherlands Twin Registry (NTR), 2090 unrelated par- ticipants of European descent for whom DNA was available were selected as control samples (Boomsma et al., 2008) (Lei- den 85-plus replication controls). The substructure in the NTR has been reported before (Sullivan et al., 2009), and in this study, we included samples aged between 15 and 70 years at the time of blood sampling, without known family relations (i.e., those without any substructure).

Experimental procedures

Study populations

Danish 1905 cohort

The participants in this study are from the Danish 1905 birth cohort recruited in 1998 (Nybo et al., 2003) when they were aged 92–93 years. From this cohort, 3,600 subjects were still alive, of whom 2262 participated in the study. Participants were subjected to a home-based interview on health and lifestyle parameters, physical and cognitive function tests, and the col- lection of biological material. The current genetic study com- prises a total of 1578 of these individuals (Danish replication cases). Survival was followed up until January 2010. Ninety-nine percent (1561 subjects) of subjects died in the 12 years of fol- low-up. Control samples were 1997 twins (one twin for each pair) between 46 and 68 years of age collected from all over

Leiden longevity study

For the LLS, long-lived siblings of European descent were recruited together with their offspring and the partners of the offspring. Families were included if at least two long-lived sib- lings were alive and fulfilled the age criterion of 89 years or older for men and 91 years or older for women, representing <0.5% of the Dutch population in 2001 (Schoenmaker et al., 2006). In total, 944 long-lived proband siblings were included with a mean age of 94 years (range 89–104), 1671 offspring (61 years, 39–81), and 744 partners (60 years, 36–79). DNA from the LLS was extracted from samples at baseline using con- ventional methods (Beekman et al., 2006). For the GWAS, 403 unrelated LLS siblings (one sibling from each sibling pair) were included (LLS GWAS cases).

The cases in all three replication cohorts originate from popu- lation-based cohort studies from a genetic background similar to the LLS (Heath et al., 2008). All the participants in these stud- ies have signed an informed consent.

Rotterdam study

The Rotterdam Study (RS) is a prospective population-based study of people aged 55 years and older, which was designed to study neurological, cardiovascular, locomotor, and ophthalmo- logical diseases (Teichert et al., 2009). The study consists of 7983 participants from the baseline cohort (RS-I) and 3011 par- ticipants from an independent extended cohort formed in 1999 (RS-II) from which DNA was isolated between 1990 and 1993 (RS-I) or between 2000 and 2001 (RS-II). For the GWAS, 1731 participants from the combined cohort who were below 60 years of age and for whom GWAS data were available were included as controls (RS GWAS controls). For the replication study, 960 cases above 90 years at time of recruitment (RS repli- cation cases) and 1825 controls between 60 and 65 years at baseline (RS replication controls) from the combined cohorts, for whom GWAS data were also available, were included.

Genotyping

Genome-wide association study (GWAS)

Leiden Longevity Study GWAS cases were genotyped using Illu- mina Infinium HD Human660W-Quad BeadChips (Illumina, San Diego, CA, USA). The RS-I and RS-II cohorts were genotyped using Illumina Infinium II HumanHap 550K Beadchips and Illu- mina Infinium II HumanHap550-Duo BeadChips (Illumina), respectively (Teichert et al., 2009).

For the GWAS, we selected 551 606 SNPs for analysis because these were genotyped in both the LLS GWAS cases and (some of) the RS GWAS controls. Of these 551 606 SNPs, 34 885 SNPs were excluded on the basis of the following criteria: SNP call rate <0.95 or MAF <0.01 in RS GWAS controls or LLS GWAS cases (n = 8908 and n = 24 586, respectively), and P < 10)4 in RS HWE GWAS controls (n = 1355). In addition, SNPs with a between-

Leiden 85-plus study

In the Leiden 85-plus study, two prospective population-based cohorts were recruited from inhabitants of Leiden (Weverling- Rijnsburger et al., 1997; der Wiel et al., 2002). Between 1987

APOE e2⁄e3⁄e4 polymorphism

chip effect in the RS GWAS controls were removed using genotype trend test comparing the RS GWAS controls from with RS-II (n = 36), leaving 516,721 SNPs for statistical The Illumina clusterplots of the SNPs with P < 1 · 10)4 (n = were visually inspected to confirm high-quality genotyping, 9 SNPs were excluded on the basis of bad clustering in the GWAS cases or RS GWAS controls.

The APOE e2⁄e3⁄e4 defining SNPs, rs429358 (Cys112Arg; e4) and rs7412 (Arg136Cys; e2), were genotyped in the LLS GWAS cases, Leiden 85-plus replication study, and Danish replication study controls using pre-designed TaqMan SNP genotyping assays (C___3084793_20 and C____904973_10, respectively). For the RS GWAS controls and Danish replication study cases, previously measured data were used (Slooter et al., 1998; Jac- obsen et al., 2010).