-R, PO Box

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RC Leiden, the

J. W. Vaupel

Max Planck Institute for Demographic Research, Konrad-Zuse-Str.

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Rostock, Germany

2004; Hurme et al. 2005; Rea et al. 2003). Another cytokine gene often suggested is the transforming β growth factor beta 1 gene (TGF 1), which is involved, among others, in regulation of cellular proliferation β and differentiation. In TGF 1, the +915 polymorphism (rs1800471) and a haplotype including rs1800471 have been reported to be of relevance for longevity (Carrieri et al. 2004).

1999; Blanche et al. 2001; Yang et al. 2009), rs1800562 in HFE (Carru et al. 2003; Coppin et al. 2003), and rs180113 in MTHFR (Bladbjerg et al. 1999; Khabour et al. 2009; Hessner et al. 2001; Brattstrom et − al. 1998) and 176C/G in IL6 (Pes et al. 2004; Wang et al. 2001). Furthermore, a few meta-analyses or pooled ε analyses have been published; for APOE 4 and the ACE I/D, insertion/deletion association was supported (McKay et al. 2011; Zajc et al. 2012), while for − the IL6 176C/G polymorphism, a North/South Euro- pean gradient of association was suggested (Di Bona et al. 2009). In any case, the involvement in longevity of the majority of these specific polymorphisms still remains uncertain.

We also explored the heat shock protein (hsp) encoding genes HSPA1A, 1L, and 14, all playing a role in folding of newly translated proteins and in stabilization of proteins against aggregation, processes reported to decrease during aging (Singh et al. 2007). The A/C-110 polymorphism in HSP1A and T2437C in HSP1L have been described to be independently as- sociated with longevity (Altomare et al. 2003; Ross et al. 2003; Singh et al. 2010), as well as in separate haplotypes (Li et al. 2009a; Singh et al. 2010).

The majority of candidate gene studies published to date have investigated only very few polymorphisms in each gene. To more thoroughly examine as much of the known variation in each of these genes as possible, we here apply a tagging SNP approach using a total of 102 tagging SNPs covering the 16 gene regions. With the exception of SIRT 1 and 3 (Flachsbart et al. 2006; Kuningas et al. 2007; Lescai et al. 2009), such an approach has, to our knowledge, not been applied before in a candidate gene study. Moreover, preceding studies – have generally used a case control study design, thus raising concerns of bias introduced by differences in characteristics of cases and controls, for example cohort – effects. Here we apply a case control approach using – two cohorts of 1,089 oldest-old (age 92 93 years) and – 736 middle-aged (age 46 67 years) Danish individuals, as well as a longitudinal approach with 11 years of follow-up survival data of the 1,089 oldest-old. Finally, we include replication data from Dutch and German study populations, as well as exploration of the initial – findings from the Danish case control study in the Danish longitudinal data and vice versa. Thus, the study presented here offers a good opportunity to confirm the putative association of variation in these major candidate

The sirtuins 1, 3, and 6 probably function as intra- cellular regulatory proteins affecting for example IGF1/insulin signaling and DNA repair, although their roles are not entirely clear (Olmos et al. 2011). In SIRT3, a G477T polymorphism (Rose et al. 2003) and a VNTR polymorphism (Bellizzi et al. 2005) have been put forward as longevity polymorphisms.

Finally, we examined three genes not belonging to any of these biological processes but still considered reliable candidates in previous studies: the highly dis- cussed candidate gene angiotensin I-converting en- zyme (ACE), for which the I/D insertion/deletion (tagged by rs4343 (Abdollahi et al. 2008)) has been reported by several authors to be associated with lon- gevity (Arias-Vasquez et al. 2005; Cellini et al. 2005; Frederiksen et al. 2003; Luft 1999; Novelli et al. 2008; Schachter et al. 1994; Zajc et al. 2012); the iron absorption regulatory protein hfe, for which the Cys282Tyr variation (rs1800562) was found to de- crease with age (Bathum et al. 2001; Lio et al. 2002); and, finally, MTHFR, which is of importance for the essential remethylation of homocysteine to methionine, where the C677T (rs1801133) polymor- phism has been suggested as associated with longevity (Stessman et al. 2005; Todesco et al. 1999).

Subjects

A considerable number of the associations men- tioned above could not be replicated in subsequent studies. These studies include rs2542052 in APOC3 (Novelli et al. 2008), rs5882 in CETP (Cellini et al. 2005; Novelli et al. 2008), I/D insertion/deletion in ACE (Agerholm-Larsen et al. 1997; Bladbjerg et al.

Detailed characteristics for the discovery cohorts were previously described in (Soerensen et al. 2010). In

short, the study population of oldest-old were 1,200 participants from The Danish 1905 Birth Cohort Study (Nybo et al. 2001), while the 800 younger controls were singleton participants from the Study of Middle Aged Danish Twins (Skytthe et al. 2002). The survival status information was retrieved from the Danish Central Population Register (Pedersen et al. 2006), and the vital status was followed until death or January 1, 2010. After data cleaning, the final sample size was 1,089 oldest-old and 736 middle-aged individuals. Permission to collect blood samples and usage of register-based information was granted by The Danish National Committee on Biomedical Research Ethics.

(see Online Resource 1). The chromosomal regions investigated are the gene regions plus 5,000 kb up- stream and 1,000 kb downstream. Tagging SNPs were identified via HapMap consortium database (http:// hapmap.ncbi.nlm.nih.gov/index.html.en, CEU cohort) and analyzed using the HaploView software (http:// www.broadinstitute.org/haploview/haploview; Barrett et al. 2005). Finally, for optimal genotyping, SNPs known to perform poorly on the Illumina GoldenGate genotyping platform were excluded.

Genotyping