No abstract

We have reported marked mitochondrial DNA (mtDNA) sequence heterogeneity among individual CD34 clones from adult bone marrow (BM) and the age-dependent accumulation of mtDNA mutations in this mitotically active tissue. Here, we show direct evidence of clonal expansion of cells containing mtDNA mutations and that the mtDNA sequence may be easily determined by using peripheral blood (PB) as a CD34 cell source. Analysis of 594 circulating CD34 clones showed that 150 (25%) had mtDNA sequences different from the same donor's corresponding aggregate sequence. Examination of single granulocytes indicated that 103 (29%) from the same 6 individuals showed mtDNA heterogeneity, with sequences distinct from the corresponding aggregate tissue sequence and from the sequences of other single granulocytes. Circulating and BM CD34 cells showed virtually identical patterns of mtDNA heterogeneity, and the same changes were seen in progeny granulocytes as in their progenitors, indicating that blood sampling could be used in studies to determine whether mtDNA reflects an individual's cumulative or recent exposure to mutagens; as a marker of individual hematopoietic progenitors, stem cells, and their expansion; and for the detection of minimal residual disease in hematologic malignancies of

Abnormalities of mitochondrial DNA (mtDNA) are responsible for a variety of inherited syndromes and have been broadly implicated in aging, cancer, and autoimmunity diseases. Mutations in mtDNA have been reported in myelodysplasia and leukemia, although their pathogenic mechanism remains uncertain. We have described age-dependent accumulation of mtDNA mutations, leading to a high degree of mtDNA sequence heterogeneity among normal marrow and blood CD34 cells as well as in granulocytes (Shin M et al, Blood101:3118 [2003], 103:553 [2004], 103:4466 [2004]). In order to examine mtDNA heterogeneity in detail, we developed a method for analysis of the mtDNA control region from single cells that were sorted by flow cytometry. Highly purified populations of CD34 cells, T cells, B cells, and granulocytes were obtained from five healthy adult donors. The sequence of the individual cells’ mtDNA was compared to the aggregate mtDNA for the respective cell type and differences were expressed as a measure of mtDNA heterogeneity among cells. Overall, heterogeneity was high: for circulating CD34 cells, 38±3.4%; for T cells, 37±14%; B cells, 36±10.8%; and for granulocytes, 48±7.2% (the value for granulocytes statistically differed from CD34 cells; p = 0.03). Most intercellular heterogeneity was due to polyC tract length variability; however, mtDNA base substitution mutations were also prevalent: 15±5.5% in CD34 cells; 15±9.0% in T cells, 15±6.7% in B cells; and 33±2.4% for granulocytes (granulocytes were significantly higher than other cells; p < 0.01). The higher rate of base substitution in granulocytes may reflect their greater exposure to reactive oxygen species. Surprisingly, for both polyC tract length differences and point mutations, the specific mtDNA abnormalities and the proportion of circulating cells characterized by these changes were similar among different cell lineages and relatively constant over time (~2 years) in the same donors. One inference from these results is that mtDNA heterogeneity during development is fixed in the primitive lymphohematopoietic stem cell compartment. In contrast to normal adults, circulating CD34 cells from patients obtained even years after successful allogeneic stem cell transplantation showed a remarkable level of mtDNA homogeneity, similar to the uniformity we have previously observed in cord blood CD34 cells and consistent with limited numbers of stem cells active in these individuals. Leukemic blast cells (from patients with AML-M2, AML evolving from CMML, and T-PLL) also showed a high degree of homogeneity. We propose that mtDNA sequence of single cells may be utilized as a natural genetic marker of hematopoietic progenitors and stem cells; to detect minimal residual disease in leukemia; and as a measure of the accumulation of mutagenic events in mammalian cells in vivo and in vitro.