Genotyping of the German replication samples were performed using either Sequenom MassARRAY iPLEX®Gold technology (Seque- nom®, Inc., CA, USA) with iPlex primers designed via the MassARRAY Design Software, or via an automated platform using predesigned TaqMan® SNP genotyping assays (Applied Biosystems) (Hampe et al., 2001); PCR was performed using the GeneAmp PCR System 9700 (Applied Biosystems) under standard conditions and the ABI Prism 7900HT Sequence Detection System and the Sequence Detec- tion System Software 2.0 (Applied Biosystems) were used for allelic discrimination.

In order to cover as much of the common genetic variation within each gene region as possible, a number of tagging SNPs in each gene re- gion were added, obtained through the HapMap consortium database (http://hapmap.ncbi.nlm.nih.gov/index.html.en) for the CEU cohort, using the HapMap Data Rel 23a/phase II Mar08, on NCBI B36 assembly, dbSNPbuild126criteria.GenotypedatawereanalyzedusingtheHaplo- View software (http://www.broadinstitute.org/haploview/haploview and Barrett et al., 2005), by applying the ‘pair wise tagging only’, r2>0.8, LOD=3, minor allele frequency (MAF)>5%, and a minimum distance between SNPs=60 bp criteria. Finally, for optimizing the

The Dutch replication samples were genotyped via Sequenom MassARRAY iPLEX®Gold technology (Sequenom®, Inc., CA, USA), using primers designed via the SpectroDESIGNER (Sequenom®, Inc., CA, USA) and the high plex reaction protocol (www.sequenom.com/ iplex). Since rs207444 and rs3211994 were not compatible with the iPLEX format, genotyping was conducted using assay-on-demand TaqMan® SNP genotyping assays (Applied Biosystems); PCR was performed on the GeneAmp PCR system 9700 (Applied Biosystems, CA, USA) under universal conditions and the Applied Biosystems 7900HT Fast Real-time PCR System and SDS2.3 Software (Applied Biosystems, CA, USA) were used for allelic discrimination.

Table 1

Characteristics of (A) the Danish discovery cohorts and (B) the German and Dutch rep- lication samples.

2.5. Statistical analysis

(A) The Danish discovery cohorts MADT 1905 Number of individuals 736 1089 Average age in years at intake (range) 50.6 (46.0–55.0) 93.2 (92.2–93.8) Male:female ratio (%) at intake 50.4:49.6 28.7:71.3 Mean follow up time (SD) – 3.53 (2.5) Follow up time (years) – 11.4 (B) The replication samples German case–control Dutch longitudinal study study Controls LLI Leiden 85-plus Study Number of individuals 1104 1613 563 Average age at intake (SD) 67.2 (4.1) 98.4 (2.7) 85.0 (0) Male:female ratio (%) at intake 25.6:74.3 26.8:73.2 33.4:66.6 Mean follow up time (SD) – – 5.49 (3.0) Follow up time (years) – – 10.4

In the case–control study of the Danish discovery cohorts single- SNP case–control comparisons of allele frequencies (CCA) were performed using the Plink statistical program (http://pngu.mgh. harvard.edu/purcell/plink, Purcell et al., 2007) by two-tailed Chi- square test. Replication of the findings from the Danish case–control study was conducted by CCA in the German case–control samples in

Approximately 1 in 20 members of the 1905 birth cohort in Denmark had survived until 1998 (start of the Danish 1905 Birth Cohort Study) and approximately 1 in 20 of these members survived until 100 years of age, i.e. reaching age 92–93 (the inclusion age of the Danish 1905 Birth Cohort Study participants) is only “halfway to be- coming a centenarian” in terms of selection. In the longitudinal sur- vival analysis of the oldest-old from the Danish 1905 Cohort Study, two survival variables were used. First, we wanted to investigate the genetic contribution to surviving for only a short period of time as opposed to surviving for a longer period of time during the ninth

Notes: MADT: the Study of Middle Aged Danish Twins, 1905: the Danish 1905 Birth cohort, LLI: long lived individuals, SD: standard deviation.

1/INS-SNPs rs1207362 (KL), rs2267723 (GHRHR) and rs572169 (GHSR), as well as the pro/antioxidant-SNP rs1002149 (GSR) and the DNA-repair-SNP rs1805329 (RAD23B) decreased in frequency from middle-aged to old-aged (ORb1), whereas the GH/IGF-1/INS- SNPs rs3842755 (INS) and rs9456497 (IGF2R) and the DNA-repair- SNPs rs11571461 (RAD52), rs13251813 (WRN) and rs2953983 (POLB) increased in frequency (OR>1). As sex-specific differences previously have been suggested for association of SNPs with longevity (Candore et al., 2006; Franceschi et al., 2000; Li et al., 2009; Soerensen et al., 2010) a sex-stratified analysis was also performed; the analysis supported the associations of the 10 SNPs mentioned above for both genders (data not shown), and in addition identified 1 DNA-repair- SNP in males; the minor allele of rs3211994 (NTLH1) increased in fre- quency from middle-aged to old-aged. The data are summarized in Table 2.