Deep-Coverage MPS Analysis of Heteroplasmic Variants within the mtGenome Allows for Frequent Differentiation of Maternal Relatives

Abstract

Distinguishing between maternal relatives through mitochondrial (mt) DNA sequence analysis has been a longstanding desire of the forensic community. Using a deep-coverage, massively parallel sequencing (DCMPS) approach, we studied the pattern of mtDNA heteroplasmy across the mtgenomes of 39 mother-child pairs of European decent; haplogroups H, J, K, R, T, U, and X. Both shared and differentiating heteroplasmy were observed on a frequent basis in these closely related maternal relatives, with the minor variant often presented as 2–10% of the sequencing reads. A total of 17 pairs exhibited differentiating heteroplasmy (44%), with the majority of sites (76%, 16 of 21) occurring in the coding region, further illustrating the value of conducting sequence analysis on the entire mtgenome. A number of the sites of differentiating heteroplasmy resulted in non-synonymous changes in protein sequence (5 of 21), and to changes in transfer or ribosomal RNA sequences (5 of 21), highlighting the potentially deleterious nature of these heteroplasmic states. Shared heteroplasmy was observed in 12 of the 39 mother-child pairs (31%), with no duplicate sites of either differentiating or shared heteroplasmy observed; a single nucleotide position (16093) was duplicated between the data sets. Finally, rates of heteroplasmy in blood and buccal cells were compared, as it is known that rates can vary across tissue types, with similar observations in the current study. Our data support the view that differentiating heteroplasmy across the mtgenome can be used to frequently distinguish maternal relatives, and could be of interest to both the medical genetics and forensic communities.

Validation of NGS for mitochondrial DNA casework at the FBI Laboratory

“…Additional GeneMarker HTS analysis options employed included filter settings (both region filters and variant filters) and amplicon settings. Settings were based on vendor and published paper recommendations [5,7,28,29] as well as preliminary evaluations of the PowerSeq assay. The region filters, which define the range of mtDNA sequence to be analyzed, were set to include nucleotide positions 16013-592 of the mtDNA CR.…”

“…At present, casework laboratories may use the number of mixed positions observed in a profile to distinguish between intraindividual (heteroplasmic) variation and a mtDNA mixturethe latter of which will not be interpreted. Although some studies have examined rates of heteroplasmy using detection levels below 10% [28,[51][52][53][54], the quality of such studies has varied [33]. Further basic research is still needed to better characterize the incidence and patterns of heteroplasmy and the number of heteroplasmic positions that may be expected per individual (which may vary by sequence range, by tissue type, by age of the individual, etc.)…”

“…at the levels of detection possible with NGS. Similarly, while the increased sensitivity of NGS may have the potential to enhance the value of heteroplasmic variation in standard sequence comparisons or for specific casework purposes [28], further work will be needed to develop confident match interpretation protocols that incorporate heteroplasmy.…”

A Forensic Genomics Approach for the Identification of Sister Marija Crucifiksa Kozulić

“…Although each haplotype was unique when heteroplasmy is considered, only two of the haplotypes shared the same haplogroup (H1bu) indicating a shared maternal ancestor. Moreover, the two H1bu haplotypes exhibit shared heteroplasmy of a private polymorphism, which is not unexpected among close maternal relatives [19,21]. This shared heteroplasmy was observed at two different proportions in the two individual sets of skeletal remains.…”

“…Based on the uniqueness of the haplotypes observed, skeletal elements could be reassociated (Figure 3), indicating a minimum of six individuals. The two individuals belonging to haplogroup H1bu, represented by samples 38/39 and 42/43, had similar but distinct haplotypes when considering heteroplasmy [19]. Both individuals shared a point heteroplasmy (PHP) at np 13327 (13327R), although variant proportions differed.…”