In this study we aimed to add further support to the association of UCP3 variability with hand grip strength, by investigating four Single Nucleotide Polymorphisms (SNPs) and their relevant haplotypes in a different population, composed by two large cohorts of middle aged and oldest old Danes, well characterized by a phenotypic point of view. Moreover, taking advantage of the longitudinal study design of the oldest cohort, we investigated the association of UCP3 variations with survival at old ages.

2.4.1. Single-locus analysis

In order to assess phenotype–genotype associations we used the F statistics max proposed by Lettre et al., 2007. Briefly, this approach allows obtaining a robust association test for quantitative traits within a linear regression framework using thebestFstatistics(F max)observedfordifferentgeneticmodels(additive,dominant and recessive) taking into account also possible covariates’ effects.

Inthepresentstudy,theF testhasbeenappliedtoinvestigatetherelationship max between UCP3 polymorphisms and the HG performances. The variables age at intake,genderandBodyMassIndex(BMI)wereusedasadjunctivecovariatesinthe regression analysis related to the MADT Cohort, while gender and BMI were includedinthecaseof1905Cohort.Apermutationapproach(10,000permutations) was used to evaluate the significance of the statistics obtained.

2. Materials and methods

2.4.2. Haplotypic analysis

Two Danish samples were analyzed, for a total of 1616 (621 men and 995 women) subjects. The oldest old sample included 908 subjects 93 years old (265 males and 643 females), drawnfromthe Danish1905 Cohort, a population-based nationwidesurveyofallDanishpeopleborninDenmarkin1905(Nyboetal.,2001, 2003; Christensen et al., 2008). Briefly, 2262 people were initially recruited in 1998,whentheywere92–93 yearsofage.Vitalstatuswasascertainedeveryyear, from the first visit to January 1st 2010 or until death whichever came first, resulting in a mean follow-up time for survivors of 11.4 years (range: 11.2–11.6). Information on death for all the cohort members was retrieved from the Danish Central Population Register, which since 1968 keeps a record of all those living in Denmark and which is continuously updated (Pedersen et al., 2006). The participation rate of the survey was 63% and comparisons of demographical characteristics of participantswith non participants demonstrated that recruited people were a fairly non-selected group of the 1905 Cohort (Nybo et al., 2001). Theyoungersample(meanage50.5)included708subjects(356malesand352

In order to model the effect of the UCP3 haplotypes on the HG performances, we used a haplotype-based association analysis within the Generalized Linear Model (GLM) framework, that allows to handle ambiguous haplotypes. The haplo.score function of haplo.stat package in R has been used to obtain the GLM-based score statistics for testing both global and individual haplotype effects on the HG performance(Schaidetal.,2002).Inthismodeltheeffectsofthedifferenthaplotypes were assessed by assuming an additive model. As in the previous case, the variables ageatintake,genderandBMIwereusedasadjunctivecovariatesintheMADTCohort, while gender and BMI were included in the case of 1905 Cohort. A permutation approach has been used to evaluate the significance of the scores obtained.

2.4.3. Survival analysis

In order to evaluate if the detected effects of the analyzed UCP3 polymorphisms ontheHGperformancemightfinallyresultindifferentialpatternsofsurvivalofthe differentrelevantgenotypes,weevaluatedsurvivalafter10 yearsfromthebaseline visit in the 1905 Cohort by using Accelerated Failure Time (AFT) models (Bradburn et al., 2003). Sex, BMI and the four UCP3 SNPs (a SNP each time) were considered as covariates. As reported in the paper by Dato et al. (2011), AFT models can be preferred over the more commonly used Cox regression model (Cox, 1972), particularly when, as in the case of the variable ‘‘sex’’ for the 1905 Cohort, proportional hazard assumptions are not respected (Schoenfeld, 1982, see Supplementary materials). Moreover, with respect to the less intuitive hazard functionprovidedbyCoxmodels,AFTmodelsallowobtainingaclearerexplanation of covariate effects, by directly linking the estimation of survival time with the values of covariates: a positive regression coefficient indicates that the covariate is associated with higher survival chances; conversely for negative coefficients. In addition, recent study demonstrated the usefulness of AFT models also in aging research (Swindell, 2009). Then, from the fitted AFT models, sex-adjusted survival curveswereobtained,analyzingeachSNPindependentlyfromeachother.Fittingof AFT models was carried out by assuming a Weibull distribution. Subjects alive or immigrated after 10 years from the baseline visit were considered as censored.

(Gaist et al., 2000). Recruitmentwas started in 1998, when 2640 intact twin from 22 consecutive birth years (1931–1952) were chosen via the Danish Population Register. The sample analyzed in this study includes only one from each twin pair.

Both surveys included multidimensional face-to-face interviews, aimed at the collection of socio-demographic information, assessment of physical, cognitive, depressive status, sensory impairments, medications, self-reported health status andDNA sampling. Permission tocollect blood samples and usageof register based information was granted by The Danish National Committee on Biomedical Research Ethics.

2.2. Measurement of hand grip strength

Handgrip(HG)strengthwasmeasuredbyahandhelddynamometer(SMEDLEY’s dynamometer TTM, Tokyo, Japan) while the subject was sitting with the arm close to his/her body. The test was repeated three times with the stronger hand; the maximumofthesevalueswasusedintheanalyses.Whenatestwasnotcarriedout, it was specified whether it was due to physical disabilities or because the subject refused to participate.