Scientists have long questioned whether HGPS is truly a model of nor- mal human aging. Although displaying many features typically associated with normal aging (alopecia, skin wrinkling, atherosclerosis), HGPS appar- ently lacks other common features such as tumors or dementia. However, recent evidence obtained since the identification of mutations in LMNA as the cause of HGPS suggests that there may in fact be a molecular rela- tionship. For example, there is evidence that a very small amount of the mutant lamin A protein produced in HGPS, termed ‘progerin’, is pro- duced in normal cells (Scaffidi & Misteli, 2006; Cao et al., 2007). Levels of the progerin transcript increase with age in normal cells (Rodriguez et al., 2009), and work using a monoclonal antibody specific for progerin has demonstrated that progerin accumulates in human skin in an age-depen- dent manner (McClintock et al., 2007). Further work in normal human fibroblasts has demonstrated that progerin production is activated by progressive telomere damage during cellular senescence (Cao et al., 2011). Finally, it has been established that numerous other abnormalities present in HGPS are also common in cells from aged individuals. These

Karen Conneely,

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; e-mail: kconnee@emory.edu *Present address: Department of Dermatology, University of Pennsylvania, Philadelphia, PA,

, USA. Accepted for publication

February

include nuclear blebbing, particular epigenetic changes, and increased levels of DNA damage. Most remarkably, suppressing the production of the mutant progerin protein through the use of an antisense morpholino reversed all of these abnormalities in cell culture (Scaffidi & Misteli, 2006).

genetically matched younger controls, N = 443), we genotyped 16 SNPs covering two coherent linkage disequilibrium (LD) blocks, corresponding to the LMNA gene and promoter region (Fig. 1). A total of 2414 nonre- dundant haplotypes (i.e., haplotypes not in perfect LD with another hap- lotype) involving 2–16 SNPs were present with estimated frequency ‡ 0.05 in our Stage 1 analysis. We tested all SNPs and all 2414 haplotypes for association with longevity. SNP 6 showed the strongest single-SNP association (P = 0.0137) in Stage 1, but was not significant upon correc- tion for multiple testing via permutation tests. However, an excess of sig- nificant haplotypes over what is expected under no association was evident. We examined the proportion of haplotypes that were significant at a range of significance thresholds (a = 0.00005, 0.0001, 0.0005, 0.001, 0.005, 0.01, and 0.05) and in all cases observed significantly more than expected under no association. P-values for association tests of each of these haplotypes are plotted against their quantiles in Fig. S1 (Support- ing Information). The most significant haplotype, involving SNPs 6, 7, 13, and 15 (GTCT), remained significant after adjustment for multiple testing via permutation tests (P = 2.5 · 10)5, multiple-testing-adjusted P = 0.0045). Haplotypes involving SNPs 4 and 5 were also significant, and the most significant 6-SNP haplotype involved SNPs 4, 5, 6, 7, 13, and 15 (GAGTCT) (P = 4.0 · 10)5). One hundred and forty nonredundant haplo- types involving different combinations of 2–6 of these six SNPs were pres- ent with estimated frequency > 0.05.

reversed all of these abnormalities in cell culture (Scaffidi & Misteli, 2006). The corollary to this observation is the possibility that variants of LMNA that produce less progerin might be protective and thus promote a longer lifespan. To investigate further the potential links of HGPS, LMNA, and progerin with the normal aging process, we sought to determine whether common variants in the LMNA gene might be associated with extreme human longevity. Toward this end, we tested variants of the LMNA gene for association with extreme longevity in five independent samples of long-lived individuals (LLI) and control subjects.

Results

Characteristics of the five study samples are shown in Table 1. In the Stage 1 sample (US Caucasians with age ‡ 95 years, N = 873 and

Table 1 Samples tested

Sample N Age range Mean age Stage 1 (US) 873 LLI ‡ 95 101.5 443 controls < 50 38.6 NECS (US) 545 LLI 96–113 103.2 193 controls 55–92 76.8 French 557 LLI ‡ 99 103.1 546 controls 18–70 51.2 SICS (Southern Italy) 455 LLI 90–109 97.6 450 controls 18–46 32.9 Ashkenazi Jewish 354 LLI 95–109 97.9 348 controls 43–90 70.7

To follow up on these 140 haplotypes, we selected SNPs 4, 5, 6, 7, 13, and 15 for genotyping in our four Stage 2 samples (Table 1). One of the six SNPs was not successfully genotyped in the French sample, but for purposes of haplotype inference, we were able to impute genotypes for this SNP using MaCH 1.0 (Li et al., 2010) in conjunction with phased hap- lotype information from the HapMap CEU sample (estimated r2 between true underlying genotype and imputed genotype = 0.97). Additional LMNA SNPs beyond SNPs 4, 5, 6, 7, 13, and 15 were also genotyped based on convenience and were included in the SNP association analysis in Table 2 but not the haplotype analysis. SNP association results for all

LLI, long-lived individuals; NECS, New England Centenarian Study; SICS, Southern Italian Centenarian Study.

   Block 1 (2 kb) Block 2 (33 kb)

Fig. 1 Linkage disequilibrium (R2 in HapMap CEU sample) between SNPs in LMNA region. Labeled SNPs indicate SNPs tested in Table 2. Encircled SNP names indicate SNPs involved in GTCT haplotype in Table 3.