Results

the oxidative phosphorilation (OXPHOS) are encoded by the mitochon- drial DNA (mtDNA). Thus, many studies have addressed the effects of nuclear and mitochondrial DNA mutations (and of the interaction between the two genomes) on mitochondrial functioning and its effect on aging in model organisms and in humans (for a review, see Lagouge & Larsson, 2013; Bratic & Larsson, 2013; Greaves & Turnbull, 2009). In particular, many studies on humans have addressed the role played by the common variability of mitochondrial DNA in modulating the processes influenced by mitochondrial activity, and their effects on degenerative diseases and aging (Brown et al., 1997; Ivanova & Lepage, 1998; De Benedictis et al., 1999; Ruiz-Pesini et al., 2000; Ross et al., 2001; Rose et al., 2002; Niemi et al., 2003; van der Walt et al., 2003; Wallace, 2005; Montiel-Sosa et al., 2006; Santoro et al., 2006). These studies have been carried out mainly by taking advantage of the haplogroup classification of mtDNA molecules. Using this approach, we previously reported a beneficial effect of haplogroup J, present across Europe and the Near East, on longevity, being more frequent in centenarians than in ethnically matched younger controls in northern Italy (De Benedictis et al., 1999), Ireland (Ross et al., 2001), and Finland (Niemi et al., 2003). Intriguingly, haplogroup J was also reported to be strongly associated with LHON (Leber’s hereditary optic neuropathy), increasing the penetrance of specific mutations that are significantly less harmful when occurring in mtDNA molecules of different haplogroups (Brown et al., 1997; Torroni et al., 1997; Man et al., 2004). Similarly, the Asian haplogroup D (characterized by mutations affecting complex I, such as haplogroup J) has been found to be overrepresented in Japanese centenarians (Tanaka et al., 1998, 2000). Accordingly, it was proposed that the mutations defining the J haplogroup (falling in the protein subunits of OXPHOS complex I) decrease OXPHOS activity, putting the cell in a vulnerable situation where a further mutation (such as in LHON) would be more likely to be harmful. However, if the nuclear response stimulated by higher ROS production is adequate, the low OXPHOS activity may become advantageous and lead to lower ROS levels, resulting in more healthy aging (Rose et al., 2001). Consistent with this hypothesis, cells with mtDNA molecules belonging to haplogroup J were recently found to produce less ATP and less ROS than cells with different mtDNA molecules (Bellizzi et al., 2012). However, some studies have failed to find any association between mtDNA and longevity (Dato et al., 2004; nos Pinos et al., 2012; Collerton et al., 2013), suggesting that the possible effect of mtDNA on longevity might be population specific, but also that such an effect might be minimal, and require a very large number of samples to reach statistical significance. In addition, it is possible that the analysis of mitochondrial DNA haplogroups may not be a suitable approach to uncover the effects of mtDNA given that different sporadic mutations suggested to be important for human longevity (see Beekman et al., 2013) fall along mtDNA molecules but are not associated with any of the haplogroups. Here, we present the results of a very large study, where over 2200 subjects older than 90 years, recruited within the framework of the

Haplogroup analysis

The frequencies of mtDNA haplogroups in 90+ and controls are summarized in Table 1 and, separately for men and women, in Table S1. We observed that in females, the haplogroups H2 and T2 are more frequent in the 90+ group than in controls (P = 0.002 and 0.02, respectively). On the other hand, among male controls, the frequency of haplogroups H1 and J2 was higher than in the 90+ group (P = 0.023 and 0.004, respectively). No association was detectable when multiple test corrections were used.

Sequence analysis

The mtDNA sequences have been deposited in GenBank, with accession numbers JX152783 – JX154074.

To analyze the mtDNA sequences, we first compared all complete sequences with the reference sequence (rCRS). A summary of all identified mutations is shown in Table S2. The analysis of single mutations did not highlight any specific mutation that could consistently account for an advantage in longevity.

Thus, we analyzed the possibility that nonsynonymous mutations may alter specific genes and affect the chances of attaining extreme longevity. We analyzed mtDNA sequences of ultranonagenarians and

Table 1 Distribution of mtDNA haplogroups in the Genetics of Healthy Ageing (GEHA) samples

Controls 90+ (n = 2153) (n = 2086) Subhaplogroups N % N % P-value HV* 1042 48.4 1052 50.43 0.187 HV0* 68 3.16 83 3.98 0.159 H* 924 42.92 927 44.44 0.322 H1 325 15.1 292 14 0.317 H2 23 1.07 42 2.01 0.013 H3 85 3.95 71 3.4 0.370 H5 63 2.93 68 3.26 0.536 H6 47 2.18 52 2.49 0.542 I 44 2.04 46 2.21 0.750 J 210 9.75 183 8.5 0.289 J1 153 7.11 144 6.9 0.810 J2 57 2.65 39 1.87 0.099 K 161 7.48 137 3.98 0.254 K1 140 6.5 117 5.61 0.247 T 211 9.8 223 10.69 0.362 T1 53 2.46 43 2.06 0.410 T2 152 7.06 174 8.34 0.120 U 313 14.54 272 13.04 0.167 U2 39 1.81 38 1.82 1.000 U4 48 2.23 34 1.63 0.181 U5a 109 5.06 90 4.31 0.276 U5b 56 2.6 51 2.44 0.770 W 39 1.81 51 2.44 0.166 X 41 1.9 42 2.01 0.825 OTHER 92 5.53 80 5.08 0.484