Heteroplasmic mitochondrial DNA mutations in normal and tumour cells

The presence of hundreds of copies of mitochondrial DNA (mtDNA) in each human cell poses a challenge for the complete characterization of mtDNA genomes by conventional sequencing technologies. Here we describe digital sequencing of mtDNA genomes with the use of massively parallel sequencing-bysynthesis approaches. Although the mtDNA of human cells is considered to be homogeneous, we found widespread heterogeneity (heteroplasmy) in the mtDNA of normal human cells. Moreover, the frequency of heteroplasmic variants varied considerably between different tissues in the same individual. In addition to the variants identified in normal tissues, cancer cells harboured further homoplasmic and heteroplasmic mutations that could also be detected in patient plasma. These studies provide insights into the nature and variability of mtDNA sequences and have implications for mitochondrial processes during embryogenesis, cancer biomarker development and forensic analysis. In particular, they demonstrate that individual humans are characterized by a complex mixture of related mitochondrial genotypes rather than a single genotype. Mitochondria are crucial in many basic cellular processes, and as a result of its unique maternal inheritance pattern and relatively high mutation rate, mtDNA is often used in studies of evolutionary biology and population genetics. These same attributes, combined with the high copy number of mtDNA in cells, makes mtDNA a favoured substrate for forensic analysis. In typical human cells, there are between 50 and hundreds of mitochondria per cell and five to ten copies of mtDNA per mitochondrion. The presence of multiple copies of mtDNA per cell leaves open the possibility that all the copies are not identical. Many studies have shown that mtDNA is homoplasmic in normal cells; that is, all of the mtDNA copies are identical not only in an individual cell but also among cells. However, there is apparently a low level of heteroplasmy in the mtDNA of various species, including humans. To evaluate this issue further, we have used massively parallel sequencing-by-synthesis approaches to thoroughly characterize the mtDNA of normal and neoplastic human cells. Two sets of PCR primers, each resulting in amplicons of about 650 base pairs (bp) in length, were designed to cover the mtDNA genome (Fig. 1a). Sequencing libraries for Illumina GAII made from the PCR products of normal colonic mucosa DNA (patient 1) yielded 8.5 million tags that matched the mitochondrial genome. Each mtDNA base was sequenced, on average, 16,700 times and fewer than 11 bases (0.07% of the 16,569 bp in the mtDNA genome) were represented fewer than 1,000 times (Supplementary Fig. 1a). This high coverage permitted us to identify heteroplasmic variants even when they were relatively rare—theoretically, when present in as few as 1 per 10,000 mitochondrial genomes. However, errors that had accumulated during the PCR and sequencing steps limited the sensitivity achieved. Control libraries made from PCR products of nuclear DNA demonstrated that the average proportion of mutations per base was 0.058%, with a standard deviation of 0.057%, and no base was mutated at greater than 0.82% frequency (Supplementary Information). We therefore made the very conservative assumption that all variants present in excess of twice this value (1.6%) represented true heteroplasmies rather than sequencing artefacts. Using these criteria, we detected 28 homoplasmic alleles and 8 heteroplasmic alleles in this sample of normal colonic mucosa (patient 1). Homoplasmic alleles were defined as any allele not present in the standard mtDNA reference sequence of humans but present in more than 98.4% (that is, 100%2 1.6%) of the mtDNA sequences analysed. All homoplasmic alleles identified in patient 1 had previously been identified in normal individuals. The less frequent (minor) alleles at the heteroplasmic sites represented 1.6–29.7% of the total alleles at that site (Table 1). All (100%) of these eight heteroplasmic alleles were listed as normal variants in mtDNA databases, whereas only 3,601