An influence of MnSOD on aging and life span has been observed in several model organisms. Over expression of Sod2 (the MnSOD homologue) leads to increased life span in Drosophila melanogaster (Sun et al., 2002) and in Mus musculus (Hu et al., 2007), while Saccharomyces cerevisiae and D. melanogaster Sod2 null mutants show a decreased life span (Duttaroy et al., 2003; Fabrizio et al., 2003). Sod2 knock out mice display neonatal lethality (Lebovitz et al., 1996; Huang et al., 2002), while mice expressing 50% of the normal Sod2 level have increased suscept- ibility to oxidative stress, severe mitochondrial dysfunction, cardiomyopathy and degeneration of central nervous system neurons (Van Remmen et al., 2003; Hinerfeld et al., 2004). Gpx1 (the GPX1 mouse homologue) knock out mice on the other hand develop normally, yet show increased sensitivity to oxidative stress-inducing agents and have increased lethality when exposed to high doses (de Haan et al., 1998). In addition, mouse GPx1(cid:3)/(cid:3) cells in culture show senescence-like changes (such as reduced proliferation and DNA synthesis) as compared to GPx1+/+ cells (de

2. Materials and methods

2.1. Subjects

Studyparticipants weretheDanish1905cohort,whichincludesall Danesbornin 1905(Nyboetal.,2001a).Thecohortmemberswereassessedforthefirsttimein1998 andsubsequentlyassessedeverysecondyear.Participantswerefolloweduntildeath or the 29th of January2008,which ever came first. The cohort is now nearly extinct. The survey included multidimensional face-to-face interviews, assessment of functional and cognitive abilities and DNA sampling. DNA was available from a totalnumberof1650participants.Duetonationalcivilregistersthereis100%follow- upregardingmortality.Forevaluationofthefunctionalabilityoftheelderly,selfrated health and Activity of Daily Living (ADL) scores were used. Self reported health was determinedbyaskingtheparticipantstoratetheirpresenthealthas‘‘excellent/good/ acceptable’’ or ‘‘poor/very poor’’,while the 11 item ADLstrength score was based on theabilitytowalk,goupstairsandcarryweights(Christensenetal.,2000;Nyboetal., 2001b).ThescaleoftheADLstrengthscorewas1to4(1:cannotdo,2:candowithaid ormajor difficulty,3:can do with fatigue or minor difficultiesand4:cando without fatigue). For evaluation of the cognitive functioning of the elderly the Mini Mental StateExamination(MMSE)(Folsteinetal.,1975)scoreswereused,rangingfrom0to 30(30beingmaximalcognitivefunction).Forallthesethreephenotypes,valuesfrom intake(baseline)wereused.Permissiontocollectbloodsamplesandusageofregister based information on the oldest old was granted byTheDanish NationalCommittee on Biomedical Research Ethics.

The human gene encoding MnSOD is designated SOD2 (http:// www.genenames.org/data/hgnc_data.php?hgnc_id=11180) and contains a c.47T>C single nucleotide polymorphism (SNP) (also referred to as rs4880 (http://www.ncbi.nlm.nih.gov/SNP/snp_ref.c- gi?rs=4880)).TheSNPresultsinanvaline > alaninesubstitutionand islocatedwithinthemitochondrialtargetingsequence,correspond- ingly at position 16 of the precursor protein and position -9 of the processed mature (active) protein (Rosenblum et al., 1996). Hence, the SNP is in the literature also referred to as p.V16A and p.-9V/-9A. The SNP possibly poses an effect on the localization and activity of the variant proteins (Shimoda-Matsubayashi et al., 1996; Sutton et al., 2003). In this paper we will refer to the SNP as the rs4880 MnSOD SNP. The rs4880 MnSOD SNP has been found to be associated with several diseases: the T allele with cardiomyopathy (Hiroi et al., 1999), atherosclerosis (Kakko et al., 2003), and lung cancer (Wang et al., 2001), while the C allele has been associated withbreast,prostateandcolorectalcancers(Ambrosoneetal.,1999; Stoehlmacher et al., 2002; Woodson et al., 2003), hypertension (Hsuehetal.,2005),sporadicmotorneurondisease(VanLandeghem et al., 1999), Parkinson’s disease (Shimoda-Matsubayashi et al., 1996), and Alzheimer’s disease (Wiener et al., 2007).

2.2. Genotyping

DNAwasisolatedfromcheekswabsorbloodspotsusingtheQIAampDNAMiniKit (Qiagen).TheDanish1905 cohortwasgenotyped forthe rs4880 MnSOD SNP and for thers1050450GPX1SNPusingTaqmantechnology.rs4880MnSODSNPprimers:50- GGCTGTGCTTTCTCGTCTTCA-30 (forward) and 50-TCTGCCTGGAGCCCAGATAC-30 (reverse) and rs1050450 GPX1 SNP primers: 50-CATCGAAGCCCTGCTGTCT-30 (for- ward) and 50-CACTGCAACTGCCAAGCA-30 (reverse) were purchased from DNA Technology A/S (Denmark), while rs4880 MnSOD SNP probes: 50-Vic-CGAGGCC- GAAACCC-30 and 50-Fam-ACCGAGGCCAAAACC-30 and rs1050450 GPX1 SNP probes: 50-Vic-ACAGCTGGGCCCTT-30 and 50-Fam-ACAGCTGAGCCCTT-30 were purchased from Applied Biosystems (UK).

DNA was amplified in a total volume of 10 ml containing 5 ml Taqman Universal MasterMix(AppliedBiosystems),900 nMofeachprimer,200 nMofeachprobeand 10 ngtemplateDNA.PCRwasperformedintheABIPrism17700SequenceDetector (Applied Biosystems) using the conditions recommended by the manufacturer and the PCR data was analyzed using the Sequence Detector software version 1.7 (Applied Biosystems). 37 of the 1650 cohort members could not be genotyped with certainty for the rs1050450 GPX1 SNP due to poor quality of the DNA sample.