Table 1 lists the most frequent mtSNPs from enhanced patterns in semi-supercentenarians vs. healthy normals. These mtSNPs are mainly concentrated in the hypervariable regions of the mtDNA control region, with locations 16129, 152, and 489 being mutational hotspots in HVS-I, HVS-II, and HVS-III, respectively.Similarly, the locus 3010 is also a mutational hotspot found in many disparate haplogroups. Because of this, it is difficult to convincingly demonstrate and/or validate any possible association of these hotspot polymorphisms with longevity. It is difficult to say whether their appearance in our analysis is due to chance or reflects some deeper epigenetic effect in association with other polymorphisms on autosomes. The enriched loci identified in this study are 3206T, 8473C, 14979C and 10410C, of which the first three are defining markers for D4a and the fourth (10410C) is enriched (80% to 20%) in D4a compared to its wild-type form 10410T. This polymorphism is a known identifying marker for subhaplogroup D4a1 [30]. Its identification in our analysis may reflect a further refinement of the association of longevity within D4a. However, our sample size is inadequate to pursue this further in the present study. There are two ways in which any group of polymorphisms might associate with a phenotype. The most direct way is if their presence alone confers the phenotype. An indirect way is if the polymorphisms are merely associated (correlated) with the phenotype because of population structure. Our spectral analysis suggests that the latter association is what is actually realized in the data. This does not mean that the association between D4a and longevity is spurious; only that the correlation between the defining SNPs for D4a and longevity is spurious. The longevity phenotype appears to correlate with D4a because it appeared due to factors other than the defining SNPs of D4a. Whereas there are many other sources of association (diet, lifestyle, etc.), one possibility is that the D4a haplogroup is enriched in autosomal SNPs that confer the longevity phenotype. Whereas our paper raises this as a strong possibility, our efforts to identify such autosomal correlations in the HapMap dataset were unsuccessful because of the limited sample size of the D haplogroup and the limited SNP coverage in the HapMap study. The hypothesis of association between D4a and longevity due to autosomal polymorphisms can be tested by a study involving more individuals in the D haplogroup in Japan and elsewhere. It is particularly important in this context to include D4a individuals outside Japan to rule out associations due to diet and lifestyle. Column 2 shows the haplogroup specificity for the SNP. The next two columns show the nucleotide in rCRS and the mutation. The last two columns are for the corresponding gene and the sense of the mutation, synonymous or nonsynonymous. SNPs shown in bold blue are the six SNPs in the top left corner of Figure 5, which are most correlated with patterns enriched in the longevity group. Note that these are either nonspecific or markers for haplogroup D4a. The locations 152 and 16129 are known mutational hotspots in the hypervariable regions HVS-II and HVS-I respectively, and hence their correlation with longevity cannot be considered significant. doi:10.1371/journal.pone.0002421.t001 Experiments with xenocybrids carrying human nuclear genome and various primate mitochondrial genome indicate that mismatches between mtDNA and nDNA gene products within the OXPHOS complexes result in decreased enzyme activities and/or increased production of reactive oxygen species from the mitochondria [26-28]. The finding that subhaplogroup D4a is one of the most frequent ones in Japanese suggests that this mitochondrial subhaplogroup may be compatible with major variants of the nDNA-encoded subunits of the OXPHOS complexes. Over a longer term, we plan to apply the techniques discussed in this paper to more extensive genome-wide association studies using SNP chips to find pathways correlated with complex Supporting Information Table 2. The top enhanced SNPs in the D clade in the Japanese samples in Hapmap [5]. #SNP rs# SNP position Gene(s) Role aa change genotypes p-value rs12960296 chr18:49408898 GG[68.75%] GT[18.75%] TT[12.5%] 1.11E-05 rs17439885 chr18:49417508 AA[68.75%] AG[18.75%] GG[12.5%] 1.11E-05 rs5766424 chr22:43823070 AA[6.25%] AG[81.25%] GG[12.5%] 1.57E-05 rs3802082 chr7:17143421 AHR Intron - AA[62.5%] AT[31.25%] TT[6.25%] 1.63E-05 rs13381188 chr18:493(2006) Price AL, Patterson NJ, Plenge RM, Weinblatt ME, Shadick NA, et al. (2006) Principal components analysis corrects for stratification in genome-wide association studies. Nat Genet 38: 904-909. 
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