Here, we present the results of a very large study, where over 2200 subjects older than 90 years, recruited within the framework of the Genetics of Healthy Ageing (GEHA) EU project (Skytthe et al., 2011) and coming from most of the European populations, were compared with a similar number of younger controls matched for sex and geographical origins. In this study, we also availed of the complete sequencing of a subgroup of GEHA samples [650 ultra nonagenarians (90+) and a comparable number of controls, coming from Denmark, Finland, southern Italy and Greece], to determine whether recurrent or sporadic mutations accumulated in specific genes not detected by haplogroup analysis, may influence longevity.

*is part of the internationally used designation of these groups of haplotypes. Haplogroups and subhaplogroups with frequencies higher than 1.5% are reported. OTHER includes N1a, N1b, N1c, N9a, A4, D5, C1d, M1, L1b1.

of OXPHOS encoded by mitochondrial DNA. No significant correlation was observed for either rRNA genes or tRNA genes.

controls to find an aggregate mtDNA variation associated with longevity. To avoid false-positive results as much as possible, the analysis of mtDNA sequences was carried out using a gene-based kernel approach (Wu et al., 2011), adjusted for genetic ancestry by means of principal component analysis according to Biffi et al. (2010) and corrected for multiple testing (number of ‘genes’ analyzed). In our case, we considered a ‘gene’ as all the subunits encoded by mtDNA and belonging to a single complex of OXPHOS (ND 1-6 and ND4L are in complex I, Cyt b in complex III, COI-III in complex IV, ATPase 6 and 8 in complex V). Table 2 reports the results of the gene-based analysis, while Fig. S1 shows the results of the underlying principal component analysis. Table 2 clearly shows that nonsynonymous mutations falling within Complex I, III and V are associated with longevity in our sample. By contrast, there was no correlation between synonymous mutations falling in different subunits

whether mutations falling in different complexes have a positive or negative effect, we compared 90+ and controls in different populations for nonsynonymous mutations in genes belonging to the mitochondrial complex that was significantly associated with longevity upon the gene- based sequence analysis above (Fig. 1). When we considered the subunits of complex I, all three populations showed a difference between 90+ and controls. However, the frequency of nonsynonymous mutations was higher in controls than in the 90+ belonging to Danish and southern European populations (respectively, P = 0.03 in Denmark and P << 0.001 in South Europe), while in the Finnish population, we =

Table 2 Results of sequence analysis according to sequence kernel association test (SKAT) to find aggregate gene-based associations for human longevity using the 1840 mtDNA SNPs

Nonsynonymous mutations Synonymous mutations Number of SNPs P-value Number of SNPs P-Value Number of SNPs P-value Complex I 712 200 0.02648710 512 0.39526309 Complex III 136 54 0.03876515 82 0.17760404 Complex IV 321* 85 0.51612941 235 0.36290044 Complex V 134 74 0.00057112 60 0.14215469 rRNA 154 0.43332840 tRNA 100 0.26393589 Control region 267 0.10580026

P-values were obtained, by bootstrap analysis (10 000 bootstrap), using the SKAT algorithm after adjusting for the first four principal components. *One position is noncoding (position 8269, in COII).

Frequency18 +1.61.4 41.20.80.60.4 40.2 4 **__x* **DK FI SE DK FI SE DK FI SECOMPLEX I(ND1-6)m@ CONTROLS90+ COMPLEX III COMPLEX V(Cytb) (ATP6-ATP8)

Fig. 1 Nonsynonymous mutation frequencies in 90+ and controls in mtDNA genes of OXPHOS complexes I, III and V, which showed a significant association with longevity after sequence analysis for pooled associations (Table 2). * indicates statistical significant differences between 90+ and controls. Frequency (y-axis) indicates the average number of mutation per subject every 100 bp of the relevant mtDNA region. DK, FI, and SE stand for Denmark, Finland, and South Europe populations,

ª 2013 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

For complex III and V, Danish and southern European populations showed a higher frequency of mutations in controls than in the 90+ group (P = 0.005 in Denmark and P = 0.048 in South Europe) (P = 0.0011 and P = 0.0023, respectively), while in the Finnish popula- tion, the mutation frequency did not differ between the 90+ and controls for both complexes.

mtDNA fields. The results are reinforced by the specific attention to the recruitment procedure aimed at minimizing population sampling bias stipulated by the GEHA project (see Experimental procedures).On the other hand, it must be acknowledged that cases and controls come from different birth cohorts, as it happens for studies on longevity, and this may represent a limitation.

Given the absolute linkage among mtDNA mutations, we investigated whether the different results obtained in our samples (with Finns showing a different trend with respect to other samples) could be due to a different distribution of the simultaneous mutations in subunits of different complexes. As most of the samples showed at least one mutation in both complexes I and III, for each sample, we counted the subjects where we observed two or more mutations in the subunits of both complex I and complex III (Table 3a). Indeed, we observed that, when controls are considered, both Danes and southern Europeans show a higher frequency of the simultaneous presence of two mutations in complex I and III than Finns (P = 0.04 and P = 0.006, respectively). The same result was obtained when complex I and V were considered (Table 3b). In both cases, the number of subjects with two or more mutations in complex I and III or in complex I and V was higher in controls than in ultranonagenarians (P = 0.03 and P = 0.02, respectively).