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

study (Table 1). Notably, the association with GTCT was observed in two of the three samples that had ascertained older sets of LLI (mean age > 100); Tan et al. (2008) has argued that the power of association studies investigating exceptional longevity markedly increases with the use of centenarian subjects compared to nonagenarians. However, in the four samples where individual ages were available, we observed similar results when the definition for cases was narrowed to include only centenarians. Another potential source of heterogeneity is environmental differences across samples owing to region of origin. The association between GTCT and longevity was observed in two of the three US samples, but not in the European samples. One region-specific environmental exposure that could interact with aging-related genes to affect longevity is diet. Dietary differences between Europe and the US have been proposed as explana- tions for regional differences in rates of coronary mortality (Renaud & de Lorgeril, 1992; de Lorgeril et al., 1994), and caloric restriction and several dietary antioxidants, including resveratrol, found in red wine, can alter expression of aging-related genes in the hearts and brains of mice (Park et al., 2009).

Stage 1:NECS:French:SICS:Ashkenazi:Meta-analysis: 06Odds ratioLa=tl08 10 «120 «141.618 20 22

Replicating genetic association results across diverse populations has generally been difficult in the longevity literature (Christensen et al., 2006). For example, a strong association between longevity and a FOXO3A SNP (Willcox et al., 2008) replicated in some samples (Flachsbart et al., 2009; Li et al., 2009), including men from the SICS (Anselmi et al., 2009), but was not present in others (Kuningas et al., 2007) (Paw- likowska et al., 2009; Soerensen et al., 2010), including women from the SICS (Anselmi et al., 2009), although two of these studies reported asso- ciations with other FOXO3A SNPs. Reasons proposed for the lack of repli- cation in some samples included those discussed above, as well as lack of gender stratification in some analyses (Anselmi et al., 2009), although, as described in Results, this does not appear to be an issue in the present study.

Fig. 2 Forest plot of odds ratios for meta-analysis of haplotype GTCT on SNPs 6, 7, 13, and 15 across five samples. For each subsample, the black box is centered at the estimated odds ratio, and the gray line represents a 95% confidence interval. Area of the black box varies in proportion to the precision of each estimate. For the meta-analysis, the black diamond is centered at the estimated odds ratio and spans the 95% confidence interval for the meta-analysis odds ratio.

Information, v2 £ 1.00 in all cases, indicating that the French LLI and con- trols were well matched.

Discussion

We observed evidence for association with longevity involving a 4-SNP haplotype, and an excess of significant associations among the same set of correlated haplotypes, in an initial sample of centenarians and younger controls. This result was replicated in one of four follow-up samples, and the association was sufficiently strong in those two samples to remain sig- nificant when combined in a meta-analysis of all five.

The meta-analysis results presented here complement a growing body of experimental evidence (Scaffidi & Misteli, 2006; McClintock et al., 2007; Cao et al., 2011) in suggesting a potential role for LMNA and prog- erin in aspects of normal aging. However, given the incomplete replica- tion we observed, further work is needed to validate and interpret the results reported here. One of the four SNPs in our reported haplotype, rs4641, is a coding SNP for which allele-specific expression has been dem- onstrated (Rodriguez & Eriksson, 2011), with 2.3-fold greater expression for transcripts with the C allele, which is part of the GTCT haplotype asso- ciated with greater longevity in our meta-analysis. Another SNP in our reported haplotype, rs915179, associated with longevity in a recent gen- ome-wide association study (Sebastiani et al., 2012), although this study included most of our NECS cases and controls along with other subjects. Additional association studies of LMNA variants in independent samples and additional experiments focusing on functional consequences of the associated variants would be particularly valuable in establishing and elu- cidating the role of LMNA in the normal aging process.

Had our initial haplotype association been unreplicated in all four fol- low-up samples, it would have been difficult to argue against the possibil- ity of a false positive. However, the probability of observing a second false positive of similar strength is low (< 0.005): in the same 10 000 per- mutations used to adjust the meta-analysis P-value for multiple testing, the best haplotype observed in the Stage 1 sample was followed up with an association in the same direction with |Z| > 3.05 in one or more of the four follow-up samples only 46 times.