several mitochondrial DNA haplogroups that are correlated with extreme longevity in Japan. The study analyzed data from supercentenarians, centenarians, healthy non-obese males, obese young males, and patients with diabetes, Alzheimer's disease, and Parkinson's disease. The analysis used two different methods to identify correlations between mitochondrial polymorphisms and longevity. The results showed a progressive enrichment of certain beneficial patterns in the D4a haplogroup for centenarians and semi-supercentenarians. However, the correlations were found to be entirely due to population structure rather than functional mtDNA SNPs. The study suggests that hitch-hiking on autosomal events is a possible explanation for the observed correlations. The analysis was limited to mtDNA sequences, as genome-wide data is not yet available. The study was approved by the Committees on the Ethics of Human Research and written informed consent was obtained from each participant or a member of their immediate family.The mtSNP 14979C (Cytb: Ile78Thr) in D4a has been found to be correlated with extreme longevity. To investigate whether this mtSNP might be hitchhiking on nuclear SNPs, we searched for association signatures of enrichment in the Japanese D clade samples in HapMap [5]. However, the SNP density in HapMap is too low to allow any definite conclusions.

Materials and Methods:

Figure 1 displays the age distribution of the samples used in the present study. The data comes from two sources. The first source is Tanaka et al [6], consisting of 672 complete mitochondrial DNA sequences divided into sets of 96 samples in each of seven phenotypes: JD or diabetic patients with severe angiopathy (mean age ...). The sequences are attached in FASTA format in Data S1. The other sequences used in this paper can be downloaded from http://mtsnp.tmig.or.jp/mtsnp/search_mtDNA_sequence_e.html. The centenarian (i.e., non-target or NT) groups correspond to healthy non-obese males, obese young males, diabetics with and without angiopathy, Alzheimer's and Parkinson's disease patients.

The statistics L are motivated by an analogy with the t-test. We pairwise aligned the 672 sequences from [6] and the 112 new semi-super-centenarian sequences relative to the revised Cambridge Reference Sequence rCRS (http://www.mitomap.org/mitoseq.html) using the Miller-Meyers [8] algorithm as implemented in the Stretcher module in EMBOSS (http://emboss.sourceforge.net/). We applied a robust technique developed in [9] to the 672 sequences, which identified all robust mutations in the data using Principal Component Spectral Analysis [10-21] to identify the mutations that represent most of the variation in the data. Following this, we separated the samples into haplogroup clusters by applying an ensemble consensus clustering technique, which averages over probabilistic, agglomerative, and hierarchical clustering methods and many data perturbations to build an agreement matrix from which a robust set of clusters is determined along with patterns of discriminatory mutations [9]. The optimum number of clusters (kmax) was estimated using the silhouette score [11] and gap statistics [12]. We validated the robustness and statistical significance of the clusters and mutations by averaging over ten 2:1 training/test bootstrapped datasets on which we repeated the complete clustering analysis. Only 90% or higher sample assignments into clusters and predictive mutations were accepted.

This analysis clustered the 672 Japanese samples into 31 robust haplogroups after discarding samples with ambiguous membership in the clustering analysis because of small sample size. The 31 haplogroups, as defined in Tanaka et al. [6] and confirmed by our unsupervised consensus ensemble clustering technique [9], were: A, B4a, B4b, B4c, B5, D4a, D4b1a, D4b1b, D4b2a, D4b2b, D4d1a, D4d1b, D4e1, D4e2, D4g, D4h, D5a, D5b, F1, F2, M10, M7a, M7b, M8, M9, C, G1, G2, Z, N9a and N9b. We also found 261 SNPs in the patterns of mutation that are characteristic markers for these haplogroups. The 112 semi-supercentenarian sequences were classified into haplogroups based on the patterns of these 261 characteristic SNPs.

A similar score that measures haplogroup enrichment for longevity is defined as:

This measure is defined for each haplogroup H. fT is the H fraction of samples in the target (longevity) group (either centenarians or semi-supercentenarians) that belong to the haplogroup H, fC is the fraction of samples in the control group S (healthy non-obese males) that belong to the haplogroup H, and sNT is the standard deviation of these fractions across the 6 non-H target groups.