Finally, we have also shown that mtDNA mutations, previously to be associated with a number of degenerative disorders, are present ultranonagenarians and in some cases even at higher frequencies than controls. Again, this suggests that mtDNA mutations can be positive or negative according to other factors, most likely environmental factors as well as other mitochondrial and genomic background.

Experimental procedures

Sampling

Samples were collected within the framework of the GEHA research project (2004–2010) in 11 European Countries. Each institution provid- ing blood samples received the approval from its own ethical committee, and all the recruited subjects provided written informed consent for the use of their phenotypic and genetic data in studies on human aging (Skytthe et al., 2011).

The definition of the mtDNA subhaplogroups in the remaining 2947 samples was conducted by resequencing the D-loop region from nucleotide position (np) 16024 to np 576 followed by RFLP analysis in specific coding region traits as previously described (Torroni et al., 1996).

Author contribution

samples was conducted by resequencing the D-loop region from nucleotide position (np) 16024 to np 576 followed by RFLP analysis in specific coding region traits as previously described (Torroni et al., 1996). For the haplogroup and subhaplogroup assignment, we followed the Phylotree nomenclature (www.phylotree.org) (van Oven & Kayser, 2009). Considering the number of samples to classify, we developed a

N. Raule, F. Sevini, S. Lin, K. Majamaa, M. Perola, L. Bolund, H. Yang, G. Passarino and C. Franceschi designed and coordinated the study. T. E. Johnson collaborated in defining the design and assisted in the coordinationofthestudy.N.Raule,F.Sevini,S.Li,A.Barbieri,L.Lomartire, carried out laboratory analyses. F. Tallaro, D. Vianello, G. Rose, A. Montesanto, J. Moilanen carried out bioinformatic and statistic analyses. V.Bezrukov,H.Blanch(cid:1) e,A.Hervonen,K.Christensen,L.Deiana,E.Gonos, T.B.L.Kirkwood,P.Kristensen,A.Leon,P.G.Pelicci,M.Poulain,I.M.Rea,J. Remacle, J.M. Robine, S. Schreiber, E. Sikora, P.E. Slagboom, L. Spazza- fumo, M.A. Stazi, O. Toussaint, J.W. Vaupel, M. Perola, G. Passarino, C. Franceschi provided samples and opportunity to carry out the analysis. N. Raule, F. Sevini, G. Passarino and C. Franceschi wrote the Manuscript.

specific coding region traits as previously described (Torroni et al., 1996). For the haplogroup and subhaplogroup assignment, we followed the Phylotree nomenclature (www.phylotree.org) (van Oven & Kayser, 2009). Considering the number of samples to classify, we developed a custom tree search algorithm that, coupled with a highly-efficient SNP discovery pipeline, is able to define the haplogroup that better matches the sample mutational motifs, starting from the raw sequence in FastA format (Vianello et al., 2013).

Statistical analysis

R statistical software was used for all statistical analyses. mtDNA subhaplogroups and relevant frequencies were stratified by gender and were compared between 90+ subjects and their ethnically matched controls using the chi-squared test with Pearson correction or Fisher’s exact test. The comparison of frequencies in 90+ and younger controls of each mtDNA subhaplogroup was computed by applying Pearson’s chi- squared test with Yates’ continuity correction. Tests for statistical significance were two-sided with a = 0.05.

Conflict of interest

The authors declare no conflict of interest.

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