Discussion

A strong finding of this paper, that is consistent across many different analysis techniques, is that in Japanese individuals are more likely to live longer if they belong to the D4a haplogroup. This result is also supported by previous studies [6], which found that the frequency of D4a in Japanese samples (n = 1312) was 7.39%. The overall frequency of 14979C (a marker of D4a) in our control samples was 8.44% (477 of 5651); 9.6% in Gifu (263 of 2748) [22,23], 6.9% in Tokyo (103 of 1493) [unpublished data], and 7.8% in Gunma (111 of 1418) [unpublished data]. In our semi-supercentenarian samples, it was significantly enriched to 15.18%.

We attribute the reduction of the longevity effect seen in D4b2b [7] compared to D4a when moving from centenarians to semi- supercentenarians to the presence of 14979C (Cytb: Ile78Thr) in D4a. The hypothesis is that 5178A (ND2: Leu237Met) is associated with an overall protective effect for both groups, but the additional mutation at 14979C in D4a is correlated with a significant additional longevity effect in D4a.

A limitation of our study is the small sample size and lack of direct correlation with SNPs on nuclear chromosomes. We are currently trying to mitigate this problem by identifying nuclear SNPs associated with our samples on the complexes formed by proteins encoded by mtDNA. Adaptive selection of mitochondrial complex I subunits during primate evolution has been reported to support the concept of co-evolution of mtDNA-encoded and nDNA-encoded subunits of the OXPHOS complexes [25].

Our results suggest a possible scenario for the evolution of a longevity phenotype, which may develop by combinations of protective mutations. The signal for longevity in our data is strongest in D4a because it has the greatest number of protective mutations. Our spectral analysis suggests that the longevity

diseases such as type 2 diabetes, nephropathy, myocardial infarction etc. In conclusion, the nuclear-mitochondrial interaction model for longevity may be an attractive working hypothesis to be examined by resequencing projects targeting Aisan- or Japanese- specific nuclear genome variations. The resequencing projects focusing on mitochondrial genome in Japanese semi-supercente- narian reported here is a landmark for the start of investigation on longevity-associated nuclear genome polymorphisms.

Table 1. List of most frequent mtSNPs from enhanced patterns (see Figure 5) in semi-supercentenarians vs. healthy normals.

Haplogrooup SNP specificity rCRS Mutant Gene Sense 152C None T C HVS-II 489C M Clade, J T C HVS-III 709A B5, G, M10, B4c, G A 12S rRNA H13a 961C None T C 12S rRNA 3010A D4 and many G A 16S rRNA other groups 3206T D4a C T 16S rRNA 4883T D C T ND2 Syn 5178A D C A ND2 Nsyn 8414T D4 C T ATP8 Nsyn 8473C D4a T C ATP8 Syn 8701G M Clade A G ATP6 Nsyn 9540C M Clade T C CO3 Syn 10398G None A G ND3 Nsyn 10400T M Clade C T ND3 Syn 10410C D4a T C tRNA-Arg 10873C M Clade T C ND4 Syn 12705T None C T ND5 Syn 14668T D4 C T ND6 Syn 14783C M Clade T C Cytb Syn 14979C D4a T C Cytb Nsyn 15043A M Clade G A Cytb Syn 15301A M Clade G A Cytb Syn 16093C None T C HVS-I 16129A None G A HVS-I 16223T None C T HVS-I 16319A None G A HVS-I 16362C None T C HVS-I 16519C None T C HVS-I

section ‘Estimation of Mutation rates and coalescence times: some caveats’ in [29] for details). These are mainly concentrated in the hypervariable regions of the mtDNA control region. In particular, the locations 16129, 152 and 489 etc, which appear in Table 1 and Figure 5 are mutational hotspots in the hypervariable regions HVS-I, HVS-II and HVS-III respectively. They are commonly found in several unrelated haplogroups. 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.