The 16 SNPs in the second discovery phase effort were analyzed in the two study samples using an additive model. The results were added to the previous meta-analysis using a fixed-effects model as described earlier. Finally, using the top 24 results, we conducted a pathway analysis with the Database for Annotation, Visualization and Integrated Discovery (http://david.abcc.ncifcrf.gov/).

Participants in the Danish 1905 Cohort Survey are from the Danish 1905 birth cohort ascertained in 1998 when they were aged 92-93 years. Of the 3,600 participants alive from that cohort, 2,262 participants enrolled in the study. Participants underwent a home-based interview on health and lifestyle parameters, physical and cognitive tests, and collection of biologic material. The current genetic study comprises a total of 1,644 participants from this survey, mean age 93 years (range 92-93 years), 28% men. A comparison group included 2,007 Caucasians who were twins (one twin per pair) collected from all over Denmark, with a mean age of 57 years (range 46-68 years), 45% men.

The iPLEX genotyping assay developed for use with the MassARRAY platform was used to genotype the selected SNPs in the second discovery stage. The iPLEX genotyping assay is based on mass spectrometry and enables genotyping of 2536 custom SNPs on a sample in a single reaction. For quality control purposes, the system first automatically calls the genotypes and then generates cluster plots for all SNPs that are inspected individually by experienced technicians who check whether the plots show clear separation of the genotype clusters.That did not pass quality control, and two SNPs where no heterozygotes could be detected; thus, lack of Hardy-Weinberg equilibrium was the quality control. Negative controls were included in the genotyping procedure (8 per 384-well plate), and importantly, 4% of samples were genotyped twice to confirm reproducibility (reproducibility was 99.7%).

Results

Table 1 provides the characteristics of the persons achieving longevity and the comparison group in each of the four CHARGE discovery cohorts at the time of DNA collection. In line with the design of the study, persons achieving longevity were 10-20 years older than participants in the comparison group at baseline and were more likely to be women. Between 45% and 83% of those achieving longevity were still alive at the time that longevity status was ascertained. Among those who had died, the distributions of causes of death differed between those achieving longevity and the comparison group. Whereas 6%-12% of those achieving longevity died of cancer, more than 30% of the comparison group had death attributed to cancer. The prevalence of diabetes and a history of ever smoking were higher in the comparison group than in persons achieving longevity. The baseline prevalence of other cardiovascular risk factor levels showed substantial overlap between the two groups.

Statistical Analysis

Using logistic regression, each imputed and observed HapMap SNP was tested for association with the longevity outcome using an additive genetic model adjusting for sex. The mean dosage of one of the alleles (a value between 0 and 2) was the predictor for imputed SNPs. The CHS additionally adjusted for field study site in the regression model, and the FHS used generalized estimating equations to account for familial correlations.

We used the ratio of observed to expected variance in the imputed SNP genotype counts as a quality control metric for imputed SNPs (43). This ratio, multiplied by the sample size, is an estimate of the effective sample size. In the imputation software MaCH, this ratio is called r2 as it is an estimate of the allelic correlation between the imputed genotypes and the true genotypes for the SNP. A total of 2,287,520 SNPs that had average minor allele frequency greater than 0.01 and were genotyped or imputed in all studies with variance ratio greater than 0.1.

The genomic control inflation factor lambda (λ) for each cohort was less than 1.05 (45). After meta-analysis, overall inflation of the meta-analysis p-values was minor (λ = 1.034; Figure 1). None of the SNP longevity associations achieved the pre-specified level of genome-wide significance of p < 5x10^-8 (Figures 1 and 2). There were 273 SNP associations with meta-analysis p < 10^-4, and of these, 7 SNP associations had p < 10^-5 (Supplementary Table 2). Under the null hypothesis that there are no associations in the genome, we would expect 0.0001 x ~2.3 million = ~230 hits.

Table 2 shows the top 24 independent SNPs associated with longevity along with the number of supporting SNPs (additional SNPs with linkage disequilibrium r^2 > .80 and p < 10^-4). Thus, for example, there were 19 supporting SNPs on chromosome 15 in or near the longevity assurance homologue 3 (LASS3) gene, with the strongest association (OR = 0.79, p = 1.2x10^-5) noted for rs8029244. The study-specific ORs for the 24 SNP associations shown in Table 2 were in the same direction and were of similar magnitude across the four cohorts (Figure 3; Supplementary Table 3).

Of the 24 strongest independent regions shown in Table 2, the 22 SNPs tested in all four CHARGE cohorts were selected for further evaluation, and 16 were successfully genotyped in the second stage cohorts. Only 1 of the 16 SNPs had a smaller p-value after including the replication. 

Observed -log10(p-value)
lambda=1.032
Expected -log10(p-value)
    7
8
9
10
12
14
16
18
21
Coordinate

Figure 2. Plot of genome-wide association study for longevity meta-analysis (persons surviving to age 90 years, n = 1,836, and comparison group, n = 1,955) showing the log (p values) based on the fixed-effects meta-analysis by chromosome. Line indicates threshold for genome-wide significance of 5x10^-8.