A High Incidence of Mitochondrial DNA Heteroplasmy in Hypervariable Region 1 in Normal Human Tissues: Implications for Forensic Casework

Mitochondrial DNA (mtDNA) sequence analysis of the hypervariable control region has been shown to be an effective tool for human identification. The high copy number and maternal mode of inheritance make mtDNA analysis particularly useful when advanced age or degradation of biological evidence prohibits the detection of nuclear DNA polymorphisms. Heteroplasmy, or the existence of subpopulations of mtDNA genomes within an individual, has been found to occur in the control region of normal humans. However, the frequency of heteroplasmy, as well as its impact on interpretation of forensic data, have not been established. In order to better understand the occurrence and patterns of heteroplasmy in human tissues, we have implemented a denaturant gradient gel electrophoresis (DGGE) system designed to examine heteroplasmy in the hypervariable region 1 (HV1) portion of the mtDNA control region. DGGE separates DNA molecules based on their sequence, as opposed to their size, and our system was shown to be capable of detecting heteroplasmic variants at levels as low as 1%. We used this DGGE assay to screen for heteroplasmy in a total of 104 postmortem tissues, including bone, blood, hair, brain, liver, and muscle, from 21 normal adult individuals. When heteroplasmy was detected, the heteroplasmic position was identified by excising the DGGE bands, and eluting, re-amplifying, and sequencing the DNA. Additionally, the DNA from all heteroplasmic tissues was sequenced to examine the success of heteroplasmy detection by direct sequencing of PCR products. Heteroplasmy in one or more tissues was observed in 11 individuals, or 52.4% (95% C.L. 31.0-73.8). The majority of heteroplasmic variants occurred at very low levels and were not detected by direct sequencing of original PCR products. Additionally, individuals with heteroplasmy at position 16093 demonstrated “flipped” heteroplasmic ratios, that is, the predominant species in one tissue was found to be the minor species in another. This study demonstrated that HV1 heteroplasmy is a common occurrence in tissues from normal individuals, and should be considered in forensic cases where two samples appear to differ at a single nucleotide position by direct sequencing.