MADT the Study of Middle Aged Danish Twins, 1905 the Danish 1905 birth cohort, LLI long-lived individuals, SD standard deviation

strohochsinaDdlo-tsedlodnadega-elddimfonosirapmoclortnoc –esacehtniytivegnolhtiwdetaicossayltnacifingissPNS2elbaT nosirapmoclortnoc –esacepytonegdnaelellA ycneuqerfniegnahCdetcerrocnuGCCP)IC%59(ROACCPACCPFAMnoitisoP/.rhC/roniMPNSeneG setogyzomohnommocfodetcerrocdetcerrocnu5091/TDAMenegninoitisopelellarojaM degaelddimmorf degadloot↑190.0007.0810.02000.0321.0/661.0cinortnI24810105/91G/A944967srEOPA )648.0 –975.0( ↓100.0849.1 4−01×0.1 01−01×24.4461.0/190.0cinortnI30547555/61A/C4583299srPTEC )904.2 –675.1(nosirapmoclortnoc –esacepytolpaH detcerrocHCCPdetcerrocnuHCCPRO5091qerFTDAMqerFepytolpaH3PNS2PNS1PNSeneGRHC 8020.0 5−01×86.956.0590.0141.0AGA944967sr644044sr905504srEOPA91 8300.0 5−01×53.207.1821.0380.0GCG7770081sr4583299sr2885srPTEC61 2300.0 5−01×11.296.1921.0480.0CGG4583299sr2885sr4770081sr dlobninwohssignitsetelpitlumfonoitcerrocretfatnacifingisgniebseulavGCCProACCP ,oitarsddoRO,eulavPnosirapmoclortnoc –esaccilellaACCP,trohoc5091eht5091,sniwThsinaDdegAelddiMfoydutSehtTDAM,ycneuqerfelellaronimFAM,emosomorhcrhC eulavPnosirapmoclortnoc-esaccipytolpahHCCP,ycneuqerfqerF,eulavPnosirapmoclortnoc –esaccipytonegGCCP,lavretniecnedifnoc%59IC%59





Table 3 Danish case–control study; gene-based test of PCCAs

Gene No. of SNPs Single-SNP-based test (PCCA<0.05) Gene-set-based genotyped SNP PCCA OR P-set CETP 20 rs1800777 0.020 1.517 1×10−5 rs289714 0.019 1.233 rs4784744 0.027 0.856 rs5883 0.022 1.385 rs9923854 4.42×10−10 1.948 rs9930761 0.004 1.460 APOE 3 rs405509 0.025 0.859 0.0016 rs769449 0.0002 0.700 HSPA14 6 rs12770830 0.011 1.346 0.0019 rs17268499 0.004 1.292 rs7905174 0.005 1.236 rs9787671 0.001 1.343 SIRT6 3 rs107251 0.001 1.348 0.0043

PCCA allelic case–control comparison P value, OR odds ratio, P-set set/gene-based test

up survival data on the oldest-old Danes were per- formed. After adjusting for multiple testing, only rs2069827 (IL6) obtained a corrected P value below 0.10 for the Number_of_days_lived variable: P- 0 0 uncorrected 0.0007, P-corrected 0.064, and only when applying a dominant model. In the gene- based analysis, IL6 showed a P-set value of 0.0068, yet this estimate reflected only rs2069827, since the other IL6 SNP (rs12700386) was insignif- icant in the single-marker analysis (data not shown). None of the remaining 15 genes showed P-set values below 0.05 (which was based on more than one signif- icant SNP) confirming the lack of association between variations in any other genes than IL6.

Replication studies in a German case– control population and in the Danish longitudinal data

When analyzing the APOE and CETP SNPs in a German population of 1,613 oldest-old and 1,104 middle-aged individuals, rs769449 (APOE) was sup- ported at the allelic level, whereas rs9923854 (CETP) showed borderline significance at the allelic level (95% CI (OR)0 0.960– 1.358), and was supported at the genotype level (see Table 4). In order to investigate whether the two SNPs also affected survival during old age, regression analysis on survival data on the oldest-old Danes (see below) was performed. We found no effect of rs769449 (APOE), while rs9923854 (CETP) showed nominal significance in females separately, with an estimate pointing in the same direction as observed in the case– control com- parison (the data are summarized in Table 4).

From the regression analysis, the quantitative effect of carrying at least one rs2069827 A allele was esti- mated to be 0.77 and 0.74 additional years of survival for AA/AC males and females, respectively, compared to a mean survival time of 2.91 and 3.74 years for rs2069827-CC males and females, respectively, hence indicating a considerable effect.

Longitudinal study in the cohort of oldest-old Danes

Replication studies in a Dutch prospective cohort – and in the Danish case control data

In order to evaluate the association of the 102 SNPs with survival at advanced ages and to esti- mate their quantitative effects, regression analyses of the survival variables using 11 years of follow-

Investigation of this single borderline significant IL6 SNP in the Dutch replication cohort using Cox regres- 0 sion supported an effect on late life survival; HR 0.74,

ydutslanidutignolhsinaDehtnidnanoitalupoplortnoc ydutslortnoc –esacnamreGydutslortnoc –esachsinaD ecnadrocnoCGCCP)IC%59(ROdetcerrocnuACCPsesac/slortnocFAM)IC%59(ROACCPsesac/slortnocFAMPNSeneG seidutsneewtebdetcerrocnudetcerrocnu seY190.0)625 –0 –153.0(034.0 71−01×02.5550.0/911.0)648.0 –975.0(007.02000.0321.0/661.0944967srEOPA seY b100.0)853.1 –069.0(241.1331.0911.0/601.0)904.2 –675.1(849.1 01−01×24.4461.0/190.04583299srPTEC ydutslanidutignolhsinaDydutslortnoc –esachsinaD seidutsneewtebecnadrocnoCeulavP)IC%59(ROdetcerrocnuACCPPNSeneG –50.0>)648.0 –975.0(007.02000.0944967srEOPA seY a50.0>)904.2 –675.1(849.1 01−01×24.44583299srPTEC eulavPnosirapmoclortnoc –esaccipytonegGCCP,lavretniecnedifnoc%59IC%59,oitarsddoRO,eulavPnosirapmoclortnoc –esaccilellaACCP )elbairavdevil_syad_fo_rebmuN(140.0 0P,)041.4 –980.0 0IC%59(411.2 0tneiciffeoc- βdna,)elbairavetal_ylrae(840.0 0P,)998.1 –200.1 0IC%59(083.1 0RO:selamefroF a 11.2 –74.1 0IC%59,67.1 0)setogyzomoherar(RO,85.1 –11.1 0IC%59,23.1 0)setogyzoreteh(RO b

qPCR investigation of IL6 expression

–esacnamreGaniydutslortnoc –esachsinaDehtmorfsgnidniflaitinifonoitacilpeR4elbaT

base on SNP function prediction locates rs2069827 to ′ a putative transcription factor binding site in the 5 untranslated region of IL6. Hence, the level of IL6 mRNAwas analyzed by qPCR using 198 representative samples from the middle-aged cohort, for which RNA was available. No significant difference in mRNA levels was observed between genotype groups for rs2069827 0 Δ (P value 0.532). The average C values in each t rs2069827 group are depicted in Fig. 2, clearly illustrat- ing no difference between heterozygotes and common homozygotes.

Discussion