The analysis of haplogroups showed that haplogroups H2 and T2 are associated with longevity in females. Haplogroup J2 was also associated with longevity in males, showing an opposite trend with respect to Prnin previous studies (Niemi et al., 2003; Domınguez-Garrido et al., 2009). In fact, J2 turned out to be less frequent in 90+ than in controls. Although none of these associations held after multiple tests adjustment, it is interesting to note that the haplogroups H2, T (including T2) and J (including J2) are characterized by nonsynonymous mutations in subunits of OXPHOS complex I, which, as mentioned above, has previously, although not consistently, been suggested to be associated with longevity.

ing data, it proved once again to be unable to fully shed light on the correlation between mtDNA variability and longevity. The analysis of sequencing data showed that the pooled nonsynonymous variability of each of OXPHOS complexes I, III and V is associated with longevity. However,complexIexhibitedhighermutationfrequencyinthe90+group than in controls in Finns (suggesting that mutations in these subunits may be beneficial), but it showed more mutations in controls than in 90+ in Danish and in southern Europeans (suggesting that mutations in the subunits of complex I may be detrimental). On the other hand, also for complexes III and V, Danes and southern Europeans had a similar trend with more nonsynonymous mutations in controls than in ultranonage- narians, while Finns showed no differences between ultranonagenarians and controls for both complexes. The analysis of the simultaneous presence of mutations on both complexes I and III and in both complexes I and V suggests that the presence of mutation on complex I may be beneficial for longevity, while the co-occurrence of mutations on both complexes I and III or on both I and V might lower the individual’s chances for longevity. It might be worth mentioning in this context that studies on the molecular basis of LHON showed that the penetrance of LHON mutationsisgreatlyincreasedbytheconcomitantoccurrenceofmutations on complexes I and III defining specific J subhaplogroups, such as J1c and J2b (Carelli et al., 2006;Achilli et al., 2012). Accordingly, this result might explain why the J haplogroup is associated with longevity in some populationsbutnotinothers.InpopulationswhereJsubhaplogroupswith additional mutations in complex III (or complex V) are more frequent, we may expect to find no association between J and longevity, similarly to what has been reported regarding the absence of correlation between haplogroup J and LHON in populations where these subhaplogroups are notpresent(Carelliet al.,2006).Indeedpreviousanalyseshadshownthat haplogroup J is associated with longevity in Finns but not in southern Italians (De Benedictis et al., 1999; Niemi et al., 2003; Dato et al., 2004). Onthebasisofourresults,wecanhypothesizethatmutationsincomplexI maydecreasetherateofOXPHOS,possiblybyslowingtheinitiationrateof the electron transport chain, thus leading to a lower production of both ATP and ROS, as suggested in an in vitro study by Bellizzi et al. (2012). In contrast,theimpairmentofsupercomplexI–III(orofcomplexesIandV)can affect the activity of OXPHOS by decreasing (or, at worse, blocking) the initiatedelectrontransportchain,leadingtohigherROSproductionand/or hyperpolarization of the mitochondrial membrane (Wallace, 2005). This hypothesis takes into account available data but will certainly need to be studied in vivo using appropriate models with specific mutations. In fact, our results are based on the different genetic burden of NS mutations in

Discussion

Previous analyses on the correlation between mitochondrial DNA variability and longevity in humans have produced interesting but contradictory results (Passarino et al., 2010 and references therein). The large GEHA sampling of European ultranonagenarians with sex- and geographically matched controls, and the complete sequencing of a great number of mtDNA molecules allowed us to shed light on this contradictory scenario. The unprecedented number of samples collected and analyzed makes these results very relevant in the longevity and

Table 3 (a) Samples with 2 or more mutations in both complex I and III; (b) samples with 2 or more mutations in both complex I and V

Population (n. total sample) n. sample % (a) Denmark controls (429) 131 30.53 inland controls (166) 32 21.91 South Europe controls (71) 28 39.43 Total controls (666) 191 28.67* Denmark 90+ (423) 117 27.66 Finland 90+ (148) 23 15.54 South Europe 90+ (75) 22 18.66 Total 90+(646) 162 25.07* (b) Denmark controls (429) 55 12.82 Finland controls (166) 17 10.24 South Europe controls (71) 13 18.31 † Total Controls (666) 85 12.76 Denmark 90+ (423) 39 9.22 Finland 90+ (148) 11 7.43 South Europe 90+ (75) 7 9.33 Total 90+(646) 57 8.82†

*The co-occurrence of two or more mutations in complex I and III is more frequent in controls than in 90+ (P = 0.03). † The co-occurrence of two or more mutations in complex I and V is more frequent in controls than in 90+ (P = 0.02).

90+ participants when compared with controls, but they are limited to explain epistatic interactions and the real effect of the mutations. Future work will need to better characterize the molecular effects of single mutations. Moreover, it will be important also to better analyze the interaction between these mutations and mutations in the regulatory region affecting mtDNA expression, as the 295C/T transition which was showed to affect mitochondrial transcription and copy number in cybrid models and is typical of haplogroup J (Suissa et al., 2009).