The samples were collected within the framework of the GEHA research project (2004-2010) in 11 European countries. Each institution providing blood samples received 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).

The haplogroup and subhaplogroup assignment followed the Phylotree nomenclature (www.phylotree.org) (van Oven & Kayser, 2009). Since there were many samples to classify, a custom tree search algorithm was developed 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).

R statistical software was used for all statistical analyses. mtDNA subhaplogroups and relevant frequencies were stratified by gender and 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.

The authors declare no conflict of interest.

References:
Achilli A, Iommarini L, Olivieri A, Pala M, Hooshiar Kashani B, Reynier P, La Morgia C, Valentino ML, Liguori R, Pizza F, Barboni P, Sadun F, De Negri AM, Zeviani M, Dollfus H, Moulignier A, Ducos G, Orssaud C, Bonneau D, Procaccio V, Leo-Kottler B, Fauser S, Wissinger B, Amati-Bonneau P, Torroni A, Carelli V (2012) Rare primary mitochondrial DNA mutations and probable synergistic variants in Lebers hereditary optic neuropathy. PLoS ONE 7:e42242.

Beekman M, Blanche H, Perola M, Hervonen A, Bezrukov V, Sikora E, Flachsbart F, Christianse D rae J,Kirkwoo B ,Re M,Poulai ,Robin M,Valensin S, Stazi MA, Passarino G, Deiana L, Gonos ES, Paternoster L, Srensen TI, Tan Q, Helme vande kke B,Deele ,Martell ,Cordel J,Ayer L,Vaupe W,Tornwall O, Johnson TE, Schreiber S,Lathpopulation was used to perform rare-variant association testing for sequencing data. The aim was to identify rare mitochondrial DNA (mtDNA) variants that contribute to human longevity. To achieve this, the SKAT algorithm was employed as a supervised and flexible regression method to test for associations between rare variants in a gene or genetic region and continuous/dichotomous traits. The SKAT algorithm is implemented in the SKAT package of R and is capable of dealing with variants that have different directions and magnitudes of effects, allowing for covariate adjustment and avoiding arbitrary selection of a threshold in burden tests. Gene-based analysis was adjusted for the first four principal components derived from principal component analysis, as described previously. To compute the SKAT P-values, a resampling procedure based on 10,000 bootstraps was used. The function that measures the genetic similarity between two random subjects in the analyzed region via the P selected markers was the weighted linear kernel function. As suggested by Wu et al. (2011), the diagonal weight matrix was chosen using the beta distribution density function with parameters a1 = 1 and a2 = 25 evaluated at the sample minor-allele frequency or MAF (default option of SKAT package of R).

Acknowledgments: The study received support from the GEHA Project, which was funded through Priority 1 (Life Sciences, Genomics and Biotechnology for Health) of European Union's FP6, Project Number LSHM-CT-2004-503270. The research also received funding from the European Union's Seventh Framework Programme (FP7/2007-2011) under Grant Agreement No 259679.

Several studies have associated mitochondrial DNA inherited variants with successful aging and longevity in humans. These studies include Domínguez-Garrido et al. (2012), Greaves and Turnbull (2009), Hutchin and Cortopassi (1995), Ivanova and Lepage (2011), Lagouge and Larsson (2013), Man et al. (2004), Moilanen et al. (2003), Montiel-Sosa et al. (2006), Niemi et al. (2003), Passarino et al. (2001), Pinós et al. (2014), Ross et al. (2001), Ruiz-Pesini et al. (2000), Santoro et al. (2006, 2010), Shoffner et al. (1993), Skytthe et al. (2011), Suissa et al. (2009), Tanaka et al. (1998, 1999), Torroni et al. (1999, 2001), Vianello et al. (2013), and Wallace (1999a, 1999b). These studies have explored the role of mitochondrial DNA mutations, haplogroups, and variants in several diseases and conditions related to aging, such as Alzheimer's disease, Parkinson's disease, Leber hereditary optic neuropathy, and schizophrenia.