There are two ways in which any group of polymorphisms might associate with a phenotype. The most direct 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 the 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, the ones most correlated with patterns enriched in the longevity group. Note that these are either non-specific 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 the 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 suggest that this mitochondrial subhaplogroup may be compatible with the 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 chr18:49397848 CC[66.66%] CT[20.00%] TT[13.33%] 2.99E-05 rs4799386 chr18:31341066 C18orf37 Promoter - AA[56.25%] AC[43.75%] CC[0.0%] 3.63E-05 rs7791070 chr7:17174267 CC[6.25%] CT[37.5%] TT[56.25%] 4.08E-05 rs12232754 chr18:31309143 C18orf37 Intron - CC[56.25%] CT[43.75%] TT[0.0%] 4.74E-05 rs2066853 chr7:17152350 AHR exon R/K AA[12.5%] AG[31.25%] GG[56.25%] 4.95E-05 rs10269143 chr7:17172223 AA[12.5%] AT[31.25%] TT[56.25%] 5.02E-05 rs10274243 chr7:17176936 AA[12.5%] AG[31.25%] GG[56.25%] 5.02E-05 rs7780687 chr7:17178958 AA[56.25%] AG[31.25%] GG[12.5%] 5.02E-05 rs17137566 chr7:17133761 AHR Intron - CC[6.25%] CT[37.5%] TT[56.25%] 5.33E-05 rs3910440 chr12:29568570 ARG99 Intron - AA[18.75%] AG[6.25%] GG[75.0%] 5.79E-05 rs299454 chr12:29579764 ARG99 Intron - CC[18.75%] CT[6.25%] TT[75.0%] 5.79E-05 rs299467 chr12:29592557 ARG99 Intron - AA[75.0%] AG[6.25%] GG[18.75%] 5.79E-05 rs299470 chr12:29593452 ARG99 Intron - AA[75.0%] AG[6.25%] GG[18.75%] 5.79E-05 rs299479 chr12:29600357 ARG99 Intron - CC[75.0%] CT[6.25%] TT[18.75%] 5.79E-05 rs148898 chr12:29606383 ARG99 Intron - CC[75.0%] CG[6.25%] GG[18.75%] 5.79E-05 rs299487 chr12:29608184 ARG99 Intron - CC[75.0%] CT[6.25%] TT[18.75%] 5.79E-05 rs10169728 chr2:65917282 FLJ16124 Intron - AA[75.0%] AC[6.25%] CC[18.75%] 5.79E-05 rs733312 chr12:41827766 AA[81.25%] AG[18.75%] GG[0.0%] 5.81E-05 rs8096007 chr18:2199867 AA[75.0%] AG[18.75%] GG[6.25%] 8.55E-05 rs11188799 chr10:98250196 TLL2 Intron - CC[12.5%] CT[18.75%] TT[68.75%] 9.16E-05 rs9528775 chr13:63976625 CC[31.25%] CT[12.5%] TT[56.25%] 9.89E-05 rs7996275 chr13:64055128 CC[31.25%] CT[12.5%] TT[56.25%] 9.89E-05

We compare the D clade samples with the rest of the Japanese samples. Enrichment of the SNP was determined using a chi-squared test at 0.0001 significance level. The choice of mtSNPs in Hapmap was too sparse to resolve the D samples into subhaplogroups. We also cannot correlate SNP 14979C with autosomal SNPs because no D4a defining markers were available in the HapMap dataset. doi:10.1371/journal.pone.0002421.t002

Author Contributions

Table S2 This table shows the number of samples in each robust haplogroup in each of the 8 phenotypes. Ambiguous sequences, which could not be classified based on both our clustering system and standard markers in the literature are labelled with *. Thus, M* labels those samples which can be classified into the M clade but not further and so on. Found at: doi:10.1371/journal.pone.0002421.s003 (0.09 MB

Conceived and designed the experiments: MT GB. Performed the experiments: NF HU YN YF. Analyzed the data: MT RR GB GA EB AR. Contributed reagents/materials/analysis tools: MT MI YA NH. Wrote the paper: MT GB EB.