Mitochondrial DNA spectra of single human CD34+ cells, T cells, B cells, and granulocytes

Previously, we described the age-dependent accumulation of mitochondrial DNA (mtDNA) mutations, leading to a high degree of mtDNA heterogeneity among normal marrow and blood CD34+ clones and in granulocytes. We established a method for sequence analysis of single cells. We show marked, distinct mtDNA heterogeneity from corresponding aggregate sequences in isolated cells of 5 healthy adult donors—37.9% ± 3.6% heterogeneity in circulating CD34+ cells, 36.4% ± 14.1% in T cells, 36.0% ± 10.7% in B cells, and 47.7% ± 7.4% in granulocytes. Most heterogeneity was caused by poly-C tract variability; however, base substitutions were also prevalent, as follows: 14.7% ± 5.7% in CD34+ cells, 15.2% ± 9.0% in T cells, 15.4% ± 6.7% in B cells, and 32.3% ± 2.4% in granulocytes. Many poly-C tract length differences and specific point mutations seen in these same donors but assayed 2 years earlier were still present in the new CD34+ samples. Additionally, specific poly-C tract differences and point mutations were frequently shared among cells of the lymphoid and myeloid lineages. Secular stability and lineage sharing of mtDNA sequence variability suggest that mutations arise in the lymphohematopoietic stem cell compartment and that these changes may be used as a natural genetic marker to estimate the number of active stem cells

Mitochondrial DNA sequence variation in single cells from leukemia patients

A high frequency of mtDNA somatic mutation has been observed in many tumors as well as in aging tissues. In this study, we analyzed the mtDNA control region sequence variation in 3534 single normal cells and individual blasts from 18 patients with leukemia and 10 healthy donors, to address the mutation process in leukemic cells. We found significant differences in mtDNA sequence, as represented by the number of haplotypes and the mean number of cells with each nonaggregate haplotype in a population of cells, in patients compared to controls. Patients with similar clinical leukemia types, particularly acute myeloid leukemia (AML), did not show a uniform pattern of sequence variation in single blasts. Some patients at relapse presented a complex shift of major haplotypes in single cells. Four patients showed high frequencies of cells containing mutations 189, 260, 16150, and 16488, respectively, as a result of clonal expansion and could be considered as potential markers for their respective disease progression. To our knowledge, this is the first large-scale study of mtDNA variation in single malignant cells. Our results suggest that the somatic mutation process in leukemia is complex, leading to diverse levels of genetic alterations due to either intrinsic aspects of leukemia pathophysiology or chemotherapy effects