Furthermore, analyses of longitudinal cohorts of Danish nonagenar- ians demonstrated that exceptional longevity does not result in exces- sive levels of disability (Christensen et al., 2008), suggesting a relationship between the general increase of life expectancy with a good maintenance of individual activities of daily living. This evidence supports the hypothesis that factors influencing the increased survival can also influence disability levels in older cohorts (Engberg et al., 2008) and so that longevity and lower disability levels may have com- mon causes at organismal level.

Permissiontocollect blood samples andusageofregister-basedinfor- mation was granted by The Danish National Committee on Biomedical Research Ethics, both for initial and follow-up study.

Inthisstudy,we investigatedtheassociationbetween311 singlenu- cleotide polymorphisms (SNPs) at 38 genes belonging to the oxidative stress pathway with functional status at very old age through a gene- based approach. To this purpose, we analyzed 1089 Danish nonagenar- ians, drawn from the longitudinal 1905 Cohort Study, a well character- ized (Christensen et al., 2009; Nybo et al., 2001, 2003) and very suitable sample for genotype–phenotype association studies (Dato et al., 2010; Soerensen et al., 2010, 2012a,b, 2013) for the associations with physical and cognitive parameters, respectively represented by HG strength, ADL, WS, MMSE and CCS.

2.2. Genetic data

Genetic data analyzed in this work were collected in the frame of a project on the study of genetic determinants of human longevity, de- scribed in Soerensen et al. (2012b), where 148 genes (1273 SNPs) be- longing to three candidate pathways (GH/IGF-1/Insulin signaling, DNA damage signaling and repair and pro–anti-oxidant pathways) were in- vestigated in two cohorts of middle aged and oldest old Danes. Briefly, candidate genes were chosen by integrating a comprehensive analysis of literature and database search. SNP selection was carried out through a dedicated protocol, which takes into account both candidate SNPs for aging and age-related diseases in each analyzed pathway and coding or potentiallyfunctionalSNPs (non-synonymous SNPs,SNPs located in po- tential splice sites or transcription factor binding sites and SNPs poten- tially inducing frame shifts or nonsense-mediated mRNA decay). Finally, HAPMAP tagging SNPs were added for reasonably covering the genetic variability in the gene region. Supplementary Table 1S reports the complete list of the 311 SNPs relative to 38 genes belonging to the pro–anti-oxidant pathway analyzed in this study.

Moreover, for verifying a possible effect of each gene on mortality, we tested the association with survival after the ninth decade of age.

2. Materials and methods

2.1. Samples

1089 Danish samples were analyzed (313 males and 776 females), belonging to the “Danish 1905 Cohort Study”. General characteristic of this population and post-survey mortality data are described in Table 1.

2.3.3. Cognitive functioning

Screening of cognitive impairment was carried out by MMSE and Composite Cognitive Score tests. MMSE is a 30-point cognitive scale which evaluates several different areas of thinking including memory, judgment, calculation, abstraction, language, and visual–spatial ability (Folstein et al., 1975). MMSE scores range from 0 (lowest cognitive function) to 30 (highest cognitive function). Since the test is affected by age and educational status, the MMSE scores were normalized for these variables.

2.3. Functional parameters and survival outcome

2.3.1. Disability

ThemanagementofactivitiesofdailylivingorADL(toileting,getting up from bed, raising from a chair, walking around) was assessed by using a modification of an international and widely used scale, the Katz' Index of activities of daily living (ADL) (Katz et al., 1970). The as- sessment was based on what the subject was able to do at the time of the visit. Each activity was scored as 0 when the subject was unable to perform the activity analyzed and 1 when able to perform such activity. In the following analysis, ADL scores were dichotomized as 1 if the sub- ject was independent in all items, and 0 otherwise.

Composite cognitive score is computed by aggregating five single scores: a fluency test, forward digit span, backward digit span, immedi- ate recall and delayed recall of a 12-item list. Single items were individ- ually standardized to a mean of 0 and standard deviation of 1, as suggested by McGue and Christensen (2001), for facilitating the inter- pretation of results.

2.3.4. Survival outcome

2.3.2. Physical performance

The time to event was defined as the time elapsed from the visit until death. The average time to event is 1290 days. The data were not affected by censoring, i.e., all the subjects included in the analysis after the quality controls were dead at the time of the analyses.

WS performances. HG was measured by a handheld dynamometer (SMEDLEY's dynamometer TTM, Tokyo, Japan) while the subject was sit- tingwiththearmclosetohis/herbody.Thetestwasrepeatedthreetimes withthestrongerhandandthemaximumofthesevalueswasusedinthe analyses. When a test was not carried out, it was specified if it was due to physicaldisabilitiesorbecausethesubjectrefusedtoparticipate.Walking timewasmeasuredasthebestperformance(shortesttimeinseconds)of twowalksalongadefineddistance.Sincetwodifferentwalkingdistances (3 or 4 m) were measured, in order to make these two performances comparable we adopted the participant's usual gait-speed (in m/s) as a measure of performance, defined in this paper as WS.

2.4. Statistical analyses

2.4.1. Quality-control

After genotype calling, in order to eliminate possible bias on the analysis due to a low calling in subject and/or SNP data, the dataset was subjected to a battery of quality-control (QC) tests. On the subject

Table 2