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-specific genetic backgrounds (nuclear genome and combina- tion of mtDNA mutations), as well as stochastic events (Rose et al., 2001; Moilanen et al., 2003; Wallace, 2010).

The following inclusion criteria were applied to the recruitment of younger controls: (i) the participant should be between 50 and 75 years of age; (ii) he or she should be genetically unrelated to the old siblings to whom he/she was assigned as control; (iii) he or she should be of the same ethnicity and geographical background as the old siblings; (iv) whenever possible, he or she should be of the same gender as the oldest sibling in the sib pair or group. In cases where it was possible to contact the spouse of a child of an old sibling, he/she was asked to participate. Alternatively, control volunteers were recruited from the same geo- graphical area as the ultranonagenarians (further details on the sampling can be found on Skytthe et al., 2011).

DNA was recovered from fresh blood by automated and standardized protocol to guarantee quality and concentration uniformity among samples. The GEHA Partner in charge of the DNA extraction (KTL – Helsinki, Finland) provided 4 lg of genomic DNA for mtDNA variability analysis.

present only in specific populations, might be beneficial (or detrimental) for longevity and may explain part of the genetic component of longevity in that population, similarly to what has been suggested for private nuclear DNA polymorphisms (Beekman et al., 2013). On the whole, our study has allowed the reappraisal of the correlation between mtDNA variability and longevity. We show that complete mtDNA sequencing and analysis of its whole variability can highlight correlations between mtDNA variability and a specific phenotype, such as longevity which escapes standard haplogroup analysis. In fact, complete sequence analysis allows the appreciation of the effects of rare or repeated mutations unaccounted for in haplogroup analysis (Rollins et al., 2009). In addition, we have better highlighted the beneficial effects on longevity of mutations in the subunits of OXPHOS complex I and the detrimental effects of mutations in subunits of complex III or V (also in presence of mutations in complex I). It is important to underline that the very small effect of these mutations on longevity requires a very large sample size to be significantly highlighted.

A total of 4239 samples were available for mitochondrial haplogroup classification and for exploring the possible association between mtDNA- inherited sequence variation and longevity. Samples were distributed among ultranonagenarians (90+) and younger controls as reported in Table S4 and had the features reported in Table S5 (Supporting Information).

mtDNA Sequence Variation Screening

From the 4239 samples available, 1292 (646 ultranonagenarians and 646 controls) were selected for complete sequencing to analyze the differences between northern and southern European populations in the whole DNA molecule. For the remaining 2947 samples (1449 ultrano- nagenarians and 1498 controls), the mtDNA subhaplogroup was determined to verify possible haplogroup association with longevity and to compare haplogroup distribution among different European countries.

Complete sequencing was performed using two different previously compared for the reliability of results by parallel testing samples. A total of 125 sequences (109 Calabrians and 16 Greeks) obtained using the MitoALL Resequencing kit (Applera, Foster City, USA) introducing four alternative primer pairs to improve the cation rate (Table S6). Each amplification step was followed purification with EXOSAPit (U.S. Biochemical, Cleveland, Ohio). sequencing reaction was conducted using the BigDye Kit, version (Applera), and M13 universal primers (forward and reverse), followed purification of the sequences by ethanol precipitation. grams were analyzed with SEQSCAPE, version 2.5 software (Applera), allows the assembly of all 46 fragments belonging to the sample and align and compare the obtained consensus sequence with the Cambridge Reference Sequence (rCRS, NCBI: NC_012920.1 106) (Andrews et al., 1999). Two independent operators verified sequences for phantom mutations by reads of both strands. remaining samples were sequenced in Beijing Genomics Institute (BGI) previously described (Wang et al., 2008).