In this study besides the high hits for the evolutionarily

In this study, besides the high hits for the evolutionarily hypervariable sites, we also noticed that certain mtDNA variants were shared by family members, pedigree-specific occurrence of mtDNA variants. This observation was unexpected but confirmed our previous speculation for a family-specific occurrence of mtDNA variants among siblings (Yao et al., 2007b). We hypothesized that this variation was shaped by family genetic background. Indeed, others have recently shown that somatic point mutations in the mtDNA control region are influenced by genetic background and associated with healthy aging (Rose et al., 2010). How this seemingly family-specific mtDNA mutation occurs and accumulates and whether it is restricted to CD34+ ace inhibitor but not other tissues are unknown. As the family differences for mutation accumulation and presence of family-specific variants are obvious in this study, one likely explanation for this observation is the maternal inheritance of low-level variants or maternal influence on occurrence of low-level variants. Direct experimental data is needed to confirm this speculation. Nonetheless, our observations contribute to an inference of complexity in the mechanism of mtDNA mutations in single cells. The pedigree-specific mtDNA variants could have potential forensic consequences and individual patterns of mutations could help to identify individuals instead of matri-lineages (Salas et al., 2007). The mtDNA control region plays a crucial role in regulating mtDNA replication and transcription (Falkenberg et al., 2007; Shadel and Clayton, 1997). Deleterious mutations in this region would be expected to have functional consequences (Bi et al., 2010). We do not know the exact role of the mutations that were frequently observed in the mtDNA control region of CD34+ cells. As some of these highly mutated sites, such as 146, 204 and 16129, are also hypervariable in the general populations, indicating that they are unlikely to be of deleterious significance. However, the accumulation of these mutations is unlikely to be a simple byproduct of oxidative damage, as we observed high frequencies of various mutations at both evolutionarily hypervariable sites and seemingly family-specific sites. For instance, the frequency of variant A215G is relatively high in pedigrees from a high radiation area (Forster et al., 2002), but this variant also was present in two out of six individuals in Family A. The age-associated variant T414G (Michikawa et al., 1999) was only found in two CD34+ cells from a 48-year old healthy donor in our previous study (Yao et al., 2007b) and in one 79-year old donor (C-2) in this study. Further work is required to clarify putative functional effects of these variants on mtDNA replication and mtDNA copy number within a cell (Falkenberg et al., 2007; Shadel and Clayton, 1997). Our current study has some limitations. First, due to the relatively low number of subjects per family and limited number of families analyzed, the family-related patterns as we observed here need to be further confirmed. Second, we did not perform any functional characterizations for those CD34+ cells with and without mtDNA mutations, to show the potential effect of mtDNA variants. It remains unknown whether there is a family-specific mutation accumulation in mtDNA coding region in CD34+ cells. Further study should be carried out to answer these questions. In summary, we provide, to the best of our knowledge, the mutation spectra of the human mtDNA control region in CD34+ hematopoietic cells from a large cohort of maternally related and unrelated individuals. The results in general support age-related accumulation of mtDNA mutations, but further discerned a family-specific rate of mtDNA mutation accumulation and mutational sites. The exact biological significance of accumulation of aging-related and family-specific mtDNA mutations in CD34+ cells awaits future study.
Acknowledgments We thank our blood donors for participating in this study and Thomas M. Herndon and Carol Boss for their help with sample collection. This research was supported (in part) by the Intramural Research Program of the NIH, NHLBI. Y.-G.Y was supported by the Ministry of Science and Technology of China (2011CB910900), Yunnan Province (2009CI119), and Natural Science Foundation of China (30925021).