The frequencies of the enriched patterns in various haplogroups are shown in Figure 4. The ordinate is defined as the proportion of beneficial patterns in the haplogroups = 1006 (Number of positive patterns found in haplogroup)/(Total number of patterns found in haplogroup). One of the haplogroups most enriched for longevity is again identified as D4a together with many haplogroups in the D4 clade followed by a smaller signal in other haplogroups outside the D clade. These results confirm our previous observation [17] that the longevity phenotype is enhanced for many of the D clade haplogroups.

The results of the agreement matrix analysis of mtSNPs in enriched patterns are shown in Figure 5. This heat map shows pairs of mtSNPs found in positive patterns across all haplogroups. Note the presence of a strong red block of SNPs corresponding to

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Figure 2. The significance scores for the 1386 maximal patterns of mutations in the data. The red region represents 124 patterns with q- value,0.05. The pattern enrichment score used here is L . P doi:10.1371/journal.pone.0002421.g002

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Figure 3. Significance score L at q-value,0.05 for the enrichment of SNPs in the three data sets – 96 centenarians, 96+112 S centenarians and semi supercentenarians, 112 semi-supercentenarians compared to healthy normals. 3206T, 14979C and 8473C are markers for D4a. SNP 10410C is enriched 80% in D4a vs its wild-type form 10410T. doi:10.1371/journal.pone.0002421.g003

tion. The low density of SNPs in HapMap and the limited coverage of mtDNA polymorphisms limits our ability to do a more detailed study. The defining SNP 14979C for D4a was not measured in the HapMap project, precluding us from analyzing its possible correlation with autosomal SNPs.

test. This result suggests that there is no significant correlation between the individual genetic information and the phenotype.

If instead, we consider the sub-space of the 40 top eigenvalues, we find, in agreement with the previous analysis, that there are many mtSNPs that correlate with the phenotype. After correcting for multiple hypotheses (which indicated a cutoff abs(r).0.15 for p- value,0.02) we found that the following 45 mtSNPs were significantly correlated with longevity: 103A, 194T, 199C, 489C, 1382C, 3010A, 3206T, 4048A, 4071T, 4164G, 4883T, 5178A, 5351G, 5460A, 6455T, 6680C, 7684C, 7853A, 8020A, 8251A, 8414T, 8473C, 8784G, 8964T, 9296T, 9824A, 10104T, 10345C, 10400T, 10410C, 12405T, 12811C, 13651G, 13708A, 14133G, 14605G, 14668T, 14783C, 14979C, 15043A, 15301A, 15524G, 16129A, 16297C, and 16362C. The mtSNPs undelined in this list were also identified in the analysis described in the previous section.

We now apply Principal Component Spectral Analysis [10,18– 21] to the SNP-SNP covariance matrix to split the mtSNPs between those due to population structure (highest eigenmodes) and those with functional significance (the lowest eigenmodes). Using the cutoff value x= 0.95 for the Tracy-Widom distribution [21] we found that the top 40 eigenvalues correspond to population structure. Projecting the observed mtSNP matrix into the subspace orthogonal to that of these 40 eigenvalues and computing correlations, we find that the most correlated mtSNP now is 489C with r =0.107 with p-value = 0.4 by the permutation P

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Figure 4. Proportion of beneficial patterns in haplogroups =1006 (Number of positive patterns found in haplogroup)/(Total number of patterns found in haplogroup). In this analysis, we used the 124 positive patterns from Figure 2 with q-value,0.05. doi:10.1371/journal.pone.0002421.g004

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Figure 5. Heat map of the agreement matrix for SNPs across significant patterns. Note the presence of a group of 6 SNPs (upper left hand red box) strongly associated with longevity. These are: 3206T, 10410C, 8473C, 14979C, 152C and 16129A. The ones in bold are the same as in Figure 3 and are markers for 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.g005

This analysis suggests that there is no functional mtSNPs significantly correlated with longevity. All significant correlations seem to be associated only with population structure (phylogenetic tree), suggesting that the significant longevity enrichment found in D4a (which is not in doubt) has causes other than mtDNA SNPs alone.

phenotype must require protective mutations on nuclear chromo- somes in addition to the defining mtDNA SNPs for D4a. Our rather limited association study on Japanese sequences in HapMap [5] identified a few coding SNPs associated with the D clade. The most enriched of these corresponds to the gene AHR [23] which is an Aryl hydrocarbon receptor and regulates several cytochrome P450 genes. It is not clear how these mutations affect longevity. Larger data sets with denser SNP coverage will be necessary to validate our results and generate hypothesis about possible mechanisms and pathways that contribute to longevity.

Discussion