The analysis of haplogroups showed that haplogroups H2 and T2 are associated with longevity in females. HaplogHowever, complex I exhibited a higher mutation frequency in the 90+ 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 southern Europeans (suggesting that mutations in the subunits of complex I may be detrimental). On the other hand, for complexes III and V, Danes and southern Europeans had a similar trend with more nonsynonymous mutations in controls than in ultranonagenarians, 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 a 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 mutations is greatly increased by the concomitant occurrence of mutations 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 populations but not in others. In populations where J subhaplogroups with 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 not present (Carelli et al., 2006). Indeed previous analyses had shown that 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). Based on our results, we can hypothesize that mutations in complex I may decrease the rate of OXPHOS. Possibly by slowing the initiation rate of the electron transport chain, thus leading to lower production of both ATP and ROS, as suggested in an in vitro study by Bellizzi et al. (2012). In contrast, the impairment of supercomplex III (or complexes I and V) can affect the activity of OXPHOS by decreasing (or, at worst, blocking) the initiated electron transport chain. Leading to higher ROS production and/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 90+ participants when compared with controls. But they are limited to explaining 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 shown to affect mitochondrial transcription and copy number in cybrid models and is typical of haplogroup J (Suissa et al., 2009). Within GEHA, families with two or more siblings 90 years old or older were identified with different strategies depending on the available information on the population. Once identified, individuals were contacted, and an appointment was made to visit the individual in his or her usual place of residence. During the visit, a structured interview was conducted based on a standardized questionnaire, and blood samples consisting of three 7.5 mL EDTA tubes of blood were collected from each participant. For this study, we used only the older brother of each sibship. It is also worth mentioning that the availability of complete sequences allowed us to evaluate the frequency of mutations previously reported to be associated with degenerative diseases in 90+ and controls (Table S3). Unexpectedly, such mutations are present in the 90+ group sometimes at higher frequencies than in controls. For instance, the 4336T>C mutation in the tRNAgln has been found to be more frequent in patients with AD than in controls by a number of studies between 1993 and 2010 (Shoffner et al., 1993; Hutchin & Cortopassi, 1995; Brown et al., 1996; Santoro et al., 2010). We found this mutation to be twice as frequent in 90+ than in controls. Similarly, many other supposed deleterious mutations have been found to be quite frequent in 90+. These results support the idea that certain mtDNA-inherited mutations could contribute to longevity or disease according to population- and individual-specificIn this study, the DNA molecule of both northern and southern European populations was examined. The mtDNA subhaplogroup was determined for 2947 samples (1449 ultranonagenarians and 1498 controls) to verify haplogroup association with longevity and to compare haplogroup distribution among different European countries. Complete sequencing was performed using two different methods that were compared for reliability of results by parallel testing samples. A total of 125 sequences (109 Calabrians and 16 Greeks) were obtained using the MitoALL Resequencing kit (Applera, Foster City, USA) with four alternative primer pairs introduced to improve the cation rate. Each amplification step was followed by purification with EXOSAPit (U.S. Biochemical, Cleveland, Ohio), and the sequencing reaction was conducted using the BigDye Kit, version (Applera), and M13 universal primers (forward and reverse). The obtained consensus sequence was aligned and compared with the Cambridge Reference Sequence (rCRS, NCBI: NC_012920.1 106) (Andrews et al., 1999) using SEQSCAPE, version 2.5 software (Applera), which allows