Mitochondrial DNA sequence heterogeneity in circulating normal human CD34 cells and granulocytes

Mitochondria are the only organelles of animal cells other than the nucleus that contain DNA as well as their own machinery for RNA and protein synthesis.^1,2  Mitochondrial DNA (mtDNA) is present in multiple copies (usually 10³ to 10⁴ copies per cell).³  Human mtDNA is a circular molecule of 16 569 base pairs containing 37 genes, which include 2 ribosomal RNAs and 22 tRNAs, required for translation of the mtDNA, and 13 polypeptides that encode subunits of the respiratory chain.^2,3  Mitochondria function in intracellular signaling and apoptosis, in intermediary metabolism, and in the metabolism of amino acids, lipids, cholesterol, steroids, heme, and nucleotides.¹  Mitochondrial genetics differs from Mendelian genetics of the nuclear genome in 3 major characteristics: maternal inheritance, heteroplasmy, and mitotic segregation.²  In comparison to the nuclear genome, mtDNA has a modified genetic code,⁴  a paucity of introns, and lack of histone protection.⁵  The repair capacity of mtDNA is limited, and the proximity of mtDNA to sites of reactive oxygen species generation suggests that mitochondrial DNA may be more susceptible to mutation than in nuclear DNA. Although the limited repair capacity hypothesis has been validated experimentally in some experimental systems, recent data have shown that base excision repair mechanisms do occur in mammalian mtDNA.^6,7 

Because of its abundance, inherent variability, and ability to survive extreme environmental conditions, mtDNA has been widely used for forensic identification and in anthropologic studies. Furthermore, hundreds of human diseases have been associated with maternally inherited, specific mtDNA deletions and mutations.⁸  Somatically acquired mtDNA mutations also have been linked to aging and degenerative diseases, cancer, and autoimmunity.⁹  A large deletion of mtDNA is a hallmark of Pearson syndrome, a constitutional disorder that includes sideroblastic anemia.¹⁰  Mutations of mtDNA were reported in apparently acquired sideroblastic anemia and in myelodysplastic syndromes in general.¹¹  Although we were unable to confirm these results by amplification and direct sequencing of the entire mtDNA genome in patients and healthy control subjects, we coincidentally observed numerous sequence changes in bulk samples of bone marrow (BM) cells from our healthy control subjects as well as in patients.¹²  When we undertook to investigate the possibility that mtDNA mutations might accumulate in human CD34 cells by examination of a portion of the mtDNA control region in individual clonogenic cells, we unexpectedly found marked heterogeneity among marrow CD34 cells, not present in umbilical cord blood.¹³  Age-dependent accumulation of mtDNA mutations and the replacement of one DNA base pair by another, leading to heteroplasmy and then homoplasmy of mtDNA sequences in a single cell, appeared to be relatively common in marrow progenitor and presumably stem cells.¹³  Although aging has been linked to mitochondrial senescence, mutant mtDNA genomes have been assumed to be lost by dilution from rapidly dividing tissues such as bone marrow.⁸  Single cell analyses have indicated similar high levels of mtDNA heterogeneity and homoplasmy in other tissues.^14,15 

We hypothesized that mtDNA mutations might arise and become homoplasmic in hematopoietic progenitor and stem cells during life, and mutations might then become fixed after clonal expansion of individual CD34 cells. In the current study, we show that the mutational spectra of marrow hematopoietic progenitor cells is preserved in circulating CD34 cells and present direct evidence that the clonal expansion of age-related mtDNA mutations occurs in hematopoietic progeny.

BM and peripheral blood (PB) from 6 healthy adult donors were collected after informed consent was obtained following protocols approved by the Institutional Review Board of the National Heart, Lung, and Blood Institute (Tables 1 and 2). Sequence analyses of the mtDNA control region from BM CD34 cells of these healthy donors have been published¹³  and are included in the current manuscript for comparative purposes only.

Mononuclear cells from BM and PB were separated by density gradient centrifugation and washed twice in phosphate-buffered saline (PBS). The number of cells suspended in PBS was adjusted to 2 × 10⁷ cells/mL. Anti-CD34 phycoerythrin (PE)-conjugated monoclonal antibody (10 μL) and anti-CD33 fluorescein isothiocyanate (FITC)-conjugated antibody (10 μL) (BD Bioscience, San Jose, CA) were added to each 12 × 75-mm tube containing 100 μL cell suspension. After incubation for 30 minutes at 4°C, cells were washed with cold PBS and resuspended in 0.5 mL PBS. Cell sorting was performed on a MoFlo Cytometer (Dako-Cytomation, Ft Collins, CO), using 100 mW of the 488 nm line of an argon laser (I-90; Coherent, Palo Alto, CA) for excitation. Forward scatter was the triggering parameter. Fluorescence of FITC was detected by using a 530/20 bandpass filter, and the fluorescence of PE was detected by using a 580/30 bandpass filter. Single cell deposition was accomplished by using the CyClone automated cloner (Dako-Cytomation) in the 0.5 single drop mode with gating based on forward scatter and fluorescence. Individual CD34 cells were placed into each well of a 96-well microplate (Nalge Nunc International, Rochester, NY) containing 100 μL culture media, and single granulocytes were deposited in each well of a MicroAmp optical 96-well reaction plate (Applied Biosystems, Foster City, CA) containing 30 μL1 × Tris (tris(hydroxymethyl)aminomethane) EDTA (ethylenediaminetetraacetic acid) (TE) buffer (Figure 1A).

Individual CD34 cells placed into separate wells of 96-well plates were cultured in serum-free medium containing 100 ng/mL stem cell factor, 100 ng/mL Flt-3, 100 ng/mL thrombopoietin, and 50 ng/mL granulocyte colony-stimulating factor (G-CSF) (all from Stem Cell Technologies, Vancouver, British Columbia, Canada). After culture for 5 days, each well of the microtiter plate was examined with an inverted microscope (Olympus IX50, Melville, NY) to determine growth and plating efficiency of the single CD34 cells. Growth was quantified and graded with the following scoring system according to cell number in each CD34 clone: grade 1, 5 or less cells/well; grade 2, 6 to 10 cells/well; grade 3, 11 to 20 cells/well; grade 4, 21 or more cells/well (Figure 1C). Plating efficiency was defined as the number of positive (cells were present) wells/total wells × 100. Each CD34 clone was harvested from the well by vigorous pipetting and dispensed into a 1.5-mL microcentrifuge tube and rinsed with 200 μL PBS. Cells were collected after centrifugation at 300g for 5 minutes, and then washed with PBS. Cell pellets were stored at -80°C.

The amount of 30 μL 1 × TE buffer was placed in each 1.5-mL tube containing one cell pellet. The cells were lysed by incubation at 95°C for 10 minutes with occasional shaking to liberate the total DNA.¹⁶  Following single granulocyte deposition into each well of a 96-well microplate containing 30 μL 1 × TE buffer, total DNA was extracted from single granulocytes by incubation at 95°C for 10 minutes in the 96-well reaction plate (Applied Biosystems) with use of the GeneAmp PCR system 9700 (Applied Biosystems). The lysate was briefly centrifuged and stored at -20°C.

Cell lysates of individual CD34 clones and single granulocytes were subjected to amplification of mtDNA with use of the LA PCR (polymerase chain reaction) kit (TaKaRa LA Taq, Madison, WI). Two-step nested PCR amplification was performed with outer and inner pairs of primers to generate sufficient template from CD34 clones and single granulocyte for sequencing of the mtDNA control region and CO1 and Cytb gene coding regions. Primer pairs for targeted mtDNA amplification are shown in Table 3. The primary PCR mixture contained 400 μM of each deoxynucleotide triphosphate (dNTP), 2 U LA Taq (TaKaRa LA Taq), 0.8 μM outer primers, 3 μL and 10 μL cell lysates from individual CD34 clones and single granulocyte, respectively. PCR amplification was carried out in a MicroAmp optical 96-well reaction plate (Applied Biosystems) with use of the GeneAmp PCR system 9700 (Applied Biosystems) as follows: 1 cycle of 95°C for 1 minute; then 35 cycles of 95°C for 30 seconds, 52°C for 50 seconds, and 72°C for 1 minute with 10-second increase per cycle; ending with 1 cycle of 72°C for 5 minutes. The secondary PCR was performed in 50 μL reaction mixture containing 400 μM of each dNTP, 2 U LA Taq, 0.8 μM inner nested primers, and 2 μL primary PCR product under the same amplification conditions as described earlier. Secondary PCR samples were electrophoresed on 1% agarose gels and stained with ethidium bromide to assess the purity and size of the DNA fragments and were subsequently purified by using the QIA quick PCR purification kit (Qiagen, Valencia, CA). Controls, reaction mixtures without DNA templates, were subjected to the same PCR amplification conditions and in all cases were confirmed to be negative. To prevent DNA cross-contamination, special precautions were taken during the procedures of cell harvesting, DNA extraction, PCR amplification, and DNA sequencing.

Two-step nested PCR amplification of mtDNA CO1 and Cytb genes was performed in the same manner as described earlier; corresponding primers for each gene amplification are listed in Table 3.

The amplified mtDNA PCR products were directly sequenced by using the BigDye Terminator v3.1 ready reaction kit (Applied Biosystems) and the ABI Prism 3100 Genetic Analyzer (Applied Biosystems). Sequencing primers used for each mtDNA product are shown in Table 3. Experimentally obtained mtDNA sequences were compared with the Revised Cambridge Reference Sequence (RCRS) (http://www.mitomap.org/mitomap/mitoseq.html)¹⁷  by using the Blast2 program (http://www.ncbi.nlm.nih.gov/blast/bl2seq/bl2.html) and the database search tool, MitoAnalyzer (http://www.cstl.nist.gov/biotech/strbase/mitoanalyzer.html, 2001)¹⁸  to determine the polymorphisms and mutations that differed from the Revised Cambridge Reference Sequence and whether the differences caused amino acid changes in the resultant polypeptides. All differences detected through automated procedures were manually confirmed. To exclude potential artifacts, PCR amplifications from original cell lysates were replicated 1 or 2 additional times and resequenced. When the same nucleotide changes were reproduced in all independent PCR amplifications, they were considered to be confirmed. Several sequence differences, which were not reproducible on replicated PCR of mtDNA extracted from CD34 cells and granulocytes, were discarded, although they may have represented mutation events at very low copy number.

In other experiments to develop a heteroplasmic Standard Reference Material, 285-bp amplicons were generated by gene amplification of wild-type mtDNA and mtDNA altered in a single base and then mixed in varying proportions.¹⁹  By sequencing the mixtures, one could detect the minor species of mtDNA when it was present above about the 20% level. Therefore, mixed nucleotide signals on sequencing electropherograms, when observed in the current study, were assumed to represent at least 20% heteroplasmy but could have also resulted from gene amplification artifacts. To confirm heteroplasmy and mixed nucleotide signals in the mtDNA control region, PCR products were directly inserted into the pCR 2.1-TOPO vector and transformed into competent Escherichia coli (TOP10 cells) using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA). Recombinant plasmids isolated from 8 to 12 white colonies were sequenced.

A chi-square test was used to determine statistical differences in the frequency of heteroplasmy in adult bone marrow and circulating blood mtDNA. The one-way ANOVA (analysis of variance) test was performed to examine whether the total rate and unique differences observed in cord blood CD34 clones, adult BM CD34 clones, circulating CD34 clones, and single granulocytes produced significant statistical differences in mtDNA heterogeneity; a P value less than .05 was considered significant.

After sorting, single CD34 cells from PB were cultured in individual wells of 96-well plates in serum-free medium containing selected hematopoietic growth factors. CD34 cell-derived colonies were classified according to the cell number per well (Figure 1C). Although there was some variation of plating efficiency of CD34 cells obtained from 6 healthy donors, overall average plating efficiency in PB was 45% ± 9.3% (mean ± SD), comparable and not statistically different from our previous results with BM from these same donors (36% ± 3.6%; Table 1).

Individual granulocytes from the 6 donors were isolated by sorting, and the mtDNA control region was amplified by using a nested PCR procedure (Table 1). From 4704 granulocytes, 355 (8%) yielded a PCR product of the correct size; however, there was marked variation of PCR efficiency from the various donors. Any observed polymorphisms and mutations were confirmed by reamplification and sequencing (described in “Materials and methods”).

To identify mtDNA heterogeneity in individual CD34 clones and single granulocytes, we first determined the aggregate cell mitochondrial genotype of each donor, as compared to the Revised Cambridge Reference Sequence. As we previously observed, there was marked variation in the number of nucleotide changes among individual donors, with a range from 6 (donor 1) to 24 (donor 4) (11.8 ± 5.8; Table 4). A total of 71 mtDNA sequence variants were found in aggregate cells from the 6 volunteers; 69 variants were already listed in the polymorphism database (http://www.mitomap.org/cgi-bin/mitomap/tbl4gen.pl), and the 2 new nucleotide variants (478A>G and 517A>G) were found in donors 2 and 6, respectively. Donors 2 and 3 had length variations of the homopolymeric C tracts at nucleotide positions 16183 to 16193 (16189T>C, 12C) and 303 to 315 (Table 4). Most of the substitutions were transitions, but a few were transversions, in agreement with findings in the literature.²⁰ 

A total of 17 mtDNA nucleotide changes (5.7 ± 3.2) were noted in the CO1 and Cytb genes among 3 donors (Table 5), 15 of these nucleotide variants were already listed in a published polymorphism database (http://www.mitomap.org/cgi-bin/mitomap/tbl4gen.pl), and 2 new sequence variations that were not previously recorded, including unpublished mtDNA polymorphisms, were found. These 2 mutations were silent changes and not predicted to produce any amino acid change (Table 5).

To assess the polymorphic spectra of the individual cell mtDNA sequences and their clonal expansion among CD34 clones and in single granulocytes, we first examined the 1121-bp mtDNA control region, known to contain multiple mutational hot spots (Figure 1B).^20,21  An average of 100 CD34 clones per donor was subjected to sequencing, resulting in a total number of 594 PB CD34 clones from the 6 donors (Table 2). A total of 355 single granulocytes from the same 6 donors were used directly for sequencing analysis to determine evidence for clonal expansion of progenitor cells bearing polymorphisms (Table 2). These sequences were compared with the 611 BM CD34 clones examined in our previous work.¹³ 

Analysis of 594 circulating CD34clones revealed that a total of 150 clones (25.3% ± 8.2%) had mtDNA heterogeneity distinct from the donor's corresponding aggregate mtDNA sequences (Tables 6-7). The frequent patterns of mtDNA heterogeneity in circulating CD34 clones consisted of 1 or 2 nucleotide changes (substitutions, insertions, or deletions) in addition to the polymorphisms detected in the respective aggregate mtDNA. Among them, most differences were due to single nucleotide transitions at various positions and length alterations in the homopolymeric C tract localized between nucleotides 303 to 315 (Figure 2A). The heterogeneous mtDNA of circulating CD34 clones from the 6 donors was classified into several polymorphic patterns according to the following nucleotide changes; 5, 3, 2, 5, 9, and 6 patterns in donors 1 to 6, respectively (Table 6). The mean proportion of unique polymorphisms in the mtDNA among PB CD34 clones was 5.1% ± 2.4% (Table 6).

One hundred three of 355 single granulocytes (29.0% ± 9.2%) from the same 6 donors showed mtDNA heterogeneity distinct from the mtDNA sequences of the donor's corresponding aggregate and other single granulocytes (Table 6). The mean proportion of mtDNA polymorphic pattern among single granulocytes was 15.2% (15.2% ± 5.2%).

We chose CO1 and Cytb as representative coding regions to determine whether the detected mtDNA heterogeneity was restricted to the hypervariable regions or present throughout the mtDNA genome, as well as to assess the possible functional significance of mtDNA mutations in hematopoietic cells. Analysis of 569 CD34 clones from BM (285 clones) and PB (284 clones) of 3 of the 6 donors showed a prevalence of 4.4% (25 clones) with sequences different from the donors' corresponding aggregate sequences (Table 8). All these mutations were base substitutions and mostly transitions. The mean proportion of unique mtDNA mutations was 3.9% (22 clones); half of these substitutions led to amino acid changes (Tables 7-8).