Detection and quantification of a radiation-associated mitochondrial DNA deletion by a nested real-time PCR in human peripheral lymphocytes

Abstract

In this study we implemented a new assay using a nested real-time polymerase chain reaction (PCR) to detect radiation-induced common deletion (CD) in mitochondrial DNA (mtDNA) of human peripheral lymphocytes. A standard curve for real-time PCR was established by applying a plasmid DNA containing human normal mtDNA or mutated mtDNA. Human peripheral lymphocyte DNA was amplified and quantified by real-time PCR using primer sets for total damaged or mutated mtDNA, plus probes labeled with the fluorescent dyes. The first-round PCR generated multiple products were used as the template for a second-round PCR. We herein describe a nested real-time PCR assay capable of quantifying mtDNA bearing the CD in human peripheral lymphocytes following exposure (in vitro) to ¹³⁷Cs γ-rays in a dose range of 0.5 up to 5 Gy. The reproducibility of this assay was evident for both unirradiated and irradiated samples by examining human blood lymphocytes from 14 donors. This technique was sensitive enough to detect deletions in mtDNA at low dose levels, as low as 0.5 Gy, and higher levels of CD mtDNA were evident at higher doses (≥1 Gy), however, there was no consistent dose–response relationship.

Introduction

Mitochondrial DNA (mtDNA), which is the only extra-nuclear source of functional DNA in human cells, is a 16,569 bp double-stranded, circular DNA molecule that encodes 37 genes: 13 proteins of the mitochondrial respiratory chain, plus the 22 transfer ribonucleic acids (mt-tRNAs) and two ribosomal ribonucleic acids (mt-rRNAs) required for the translation of these proteins [1], [2]. mtDNA does not contain introns; the coding sequences are either continuous or have very few bases between them. Since 95% of mtDNA is coding (versus only 3% of nuclear DNA), any damage on mtDNA is likely to affect a coding region [3]. In addition, mtDNA lacks protective histones and does not have an effective DNA repair system [4], [5]. Within a given cell, the mtDNA may be a mix of both wild-type and mutant genotypes; this is known as heteroplasmy. Mutations to mtDNA are functionally recessive, with the wild-type molecules largely compensating for the deleterious effects of the mutant genomes (complementation). In general, cellular dysfunction only occurs when the ratio of mutated to wild-type mtDNA exceeds a threshold level [3], [6].

Accumulation of mtDNA damage is believed to result from oxidative stress (e.g., from superoxide and hydrogen peroxide), as mtDNA appears to be more prone to oxidative damage than nuclear DNA [7]. Excited oxygen species (e.g., the superoxide radical, hydrogen peroxide and hydroxyl radical) are formed during aerobic metabolism and exposure to ionizing radiation, which is a potent genotoxic factor that may contribute to the excessive mutagenesis of nucleic acids. Thus, mtDNA could be particularly sensitive to ionizing radiation [5], [8]. Consistent with this notion, both point mutations and large-scale deletions in mtDNA are known to arise after exposure to ionizing radiation [5], [9], [10]. The most abundant large-scale deletion reported in mtDNA, the so-called common deletion (CD) is a 4977-base pair (bp) deletion (ΔmtDNA⁴⁹⁷⁷) specifically occurring from the GC-rich 13-bp direct repeats found at nucleotide (nt) 8470 of the mtDNA sequence and stretching to nt 13,446. CD can be used as a marker of oxidative or radiation-induced damage to mtDNA; it is considered to be a particularly sensitive marker because the lesion is amplified during mtDNA replication. Indeed, studies have suggested that CD may be a good indicator of total DNA damage [11], [12].

Fluorescence-based quantitative real-time PCR (qPCR) is the enabling technology that can be applied in molecular diagnostics, life sciences, and medicine [13], [14], [15], [16], [17]. Its conceptual and practical simplicity, together with its ability to combine speed, sensitivity, and specificity in a homogeneous assay, have made it the touchstone for nucleic acid quantification. The technique often involves using fluorescent dye-labeled oligonucleotides to assess fluorescence resonance energy transfer (FRET), wherein emission/quenching reflects the interaction between the electron-excitation states of two fluorescent dye molecules [17]. The technique makes use of a polymerase with exonuclease activity and a target-specific probe that anneals downstream from the forward primer. The probe contains a fluorescent reporter at one end and a quencher at the other. In the fluorogenic 5′-nuclease PCR approach, which is a real-time assay that uses fluorescence to monitor the amplification of PCR products at each cycle, the fluorescence increases proportionally with amplification, thereby making the assay quantitative [18], [19], [20].

As a reliable marker of cumulative radiation exposure in human peripheral blood lymphocytes, we have explored the novel idea of using mtDNA, rather than nuclear DNA, as a biomarker for radiation-induced DNA damage, as proposed in this study. This assay has been applied in several studies using different cell types of human skin fibroblasts [3], [9], [21]. To date, mtDNA has been primarily used as part of a biomarker panel aimed at the rapid assessment of radiation exposure and radiation-induced injury.

Here, we report the development of a PCR-based method, capable of amplifying small populations of deleted mtDNA, and confirm the identified deletions by comparing the fluorescence of the second-round PCR products. As a model system, human peripheral lymphocytes from 14 individuals exposed to a ¹³⁷Cs γ-ray source, and after 48 h isolated DNA is examined for the presence of the CD.