Stage 1 NECS French SICS Ashkenazi Jewish Meta- SNP Ref SNP ID (N = 1316) (N = 738) (N = 1103) (N = 905) (N = 702) analysis 1 rs2275075 0.54 0.73 0.78 2 rs3814314 0.017 0.81 0.039 3 rs11578696 0.21 0.93 0.39 4 rs4661146 0.12 0.17 0.42 0.65 0.72 0.97 5 rs10737170 0.11 0.016 0.84 0.54 0.45 0.075 6 rs915179 0.014 0.0086 0.96 0.66 0.59 0.020 7 rs2485662 0.054 0.11 0.81 0.82 0.35 0.026 8 rs547915 0.11 0.84 0.84 0.29 9 rs582690 0.078 0.42 0.35 10 rs6686943 0.51 11 rs508641 0.014 0.95 0.044 12 rs12117552 0.21 0.26 0.71 13 rs4641 0.50 0.14 0.61 0.30 0.45 0.98 14 rs12063072 0.36 0.060 0.065 15 rs1468772 0.034 0.35 0.41 0.14 0.0056 16 rs6669212 0.041 0.48 0.36 0.18

NECS, New England Centenarian Study; SICS, Southern Italian Centenarian Study.

five samples are shown in Table 2. When we combined results across samples, SNP 15 showed the strongest single-SNP association in the meta-analysis (OR = 1.17, P = 0.0056) with odds ratios ranging from 1.09 to 1.22 in the four samples tested, but was not significant after cor- rection for multiple testing via permutation tests (multiple-testing- adjusted P = 0.105).

The Stage 1 sample had previously been tested for the presence of pop- ulation stratification between LLI and control subjects (see Supporting information) (Geesaman et al., 2003). However, to ensure that the asso- ciation with the GTCT haplotype did not result from subtle population stratification, we refit the model including the principal components of genotypes from 73 ancestry informative markers (AIMs) from a published panel of European substructure AIMs (Tian et al., 2008). Inclusion of the first four principal components from the AIM genotypes as covariates in the analyses did not substantially diminish the strength of the association. For the 1242 individuals for whom AIMs were available, a similar associa- tion between the GTCT haplotype and longevity was observed before (OR = 1.47, P = 0.00039) and after (OR = 1.46, P = 0.00060) inclusion of the principal components. Although a Tracy–Widom test (Patterson et al., 2006) suggested that four principal components was sufficient to account for potential population stratification, the observed result was robust to the number of principal components included in the model (P ranged from 0.00057 to 0.00062 when 1–10 principal components were included). Similarly, the top two principal components from gen- ome-wide data were available for 671 of the NECS individuals; inclusion of these components as covariates in the analysis slightly strengthened the result. In both cases, the inclusion of principal components in the analyses affected the results only minimally, suggesting that the observed haplotype association in the Stage 1 and NECS samples is not driven by population stratification. Similarly, analysis of the first two principal com- ponents did not reveal evidence of population stratification in a genome- wide study of SICS cases and controls (Malovini et al., 2011). The French sample had been previously tested for population stratification on 57 randomly selected microsatellite markers. As described in Supporting

In replication analyses of the 140 haplotypes of SNPs 4, 5, 6, 7, 13, and 15 in the four Stage 2 samples, an excess of significant haplotypes was observed in the New England Centenarian Study (NECS) sample (Fig. S2; P < 0.01, Supporting information) for all significance thresholds exam- ined (a = 0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01, and 0.05), but not in the other three Stage 2 samples. When the results for all 140 followed-up haplotypes were combined across all five samples in a meta- analysis (Table 3 and Fig. 2), the original GTCT haplotype of SNPs 6, 7, 13, and 15 was again the most significant (P = 7.5 · 10)4) and remained significant after permutation-based adjustment for multiple testing in both stages of the analysis (multiple-testing-adjusted P = 0.037).

If the genetic load is greater for older individuals, restricting the defini- tion of LLI to include only centenarians should lead to a stronger genetic effect (Tan et al., 2008). For the four samples whose age and sex infor- mation was available [Stage 1, NECS, Southern Italian Centenarian Study (SICS), and the Ashkenazi sample], we reperformed the association test for the GTCT haplotype within each sample after restricting the definition of LLI to include individuals with age ‡ 100. However, the odds ratio for GTCT did not change substantially in any of the four samples. We also performed a sex-stratified version of the haplotype analysis. Similar results were observed in the sex-stratified analysis for each sample, and results were similar for men and women within each sample.

Sample N Case Control OR Z statistic P-value Adjusted P-value Stage 1 1316 0.235 0.163 1.56 4.21 0.000025 0.0045 NECS 738 0.239 0.163 1.60 3.05 0.0023 French 1103 0.208 0.207 1.01 0.074 0.94 SICS 905 0.175 0.157 1.14 1.05 0.29 Ashkenazi 702 0.114 0.139 0.80 )1.40 0.16 Full meta-analysis 1.18 3.37 0.00075 0.037 (two-stage)

NECS, New England Centenarian Study; SICS, Southern Italian Centenarian Study.