Learn how your comment data is processed. Skip to content Mitochondria, often thought of as powerhouses of the cell, are fascinating eukaryotic organelles with a double-layered membrane and their own genome. Investigative Genetics.
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Example of calcified tissue - A molar. Mitochondrial DNA is maternally inherited. The high sensitivity of mtDNA analysis allows forensic scientists to obtain information from old items of evidence associated with cold cases and small pieces of evidence containing little biological material. Additionally the maternal inheritance of mtDNA allows scientists to compare the mtDNA profile of a set of remains to that of reference samples from individuals such as the mother, brother s , sister s , or any other maternally related individuals of a missing person.
However, mtDNA is an excellent technique to use for obtaining information in cases where nuclear DNA analysis is not feasible. A fragmentation; B Adapters annealed; C Fragments bind to glass surface loaded with complementary primers; D Bridge formation; E Bridge amplification; F Dissociation; G Cluster formed from repeated bridge formation and amplification; H Sequencing by synthesis and fluorescence detection. Different dNTPs are introduced to the chip in sequence and when one binds to a complementary nucleotide a hydrogen ion is released, resulting in a change of pH.
MPS technology offers cost-effective sequencing of the whole mitochondrial genome mitogenome with the provision of increased discrimination and phylogenetic resolution along with an assessment of data quality.
Several groups have validated the different methodologies [ 9—11 ]. Missing person identification frequently makes use of mtDNA analysis, often because of the degraded nature of the remains, with significantly increased success rates when using MPS compared with Sanger sequencing [ 12 ].
In addition to improving discrimination power, the increased sensitivity of MPS can also reveal low-level variants not seen using Sanger sequencing but care must be taken to distinguish these from background noise and low-level contamination from intrinsic and extrinsic sources.
The extent of the data produced with MPS means that robust bioinformatic approaches are required when mixtures are observed to validate observations of heteroplasmy [ 13 ]. Published mitochondrial DNA mutation rates are very variable, possibly reflecting the size of the individual studies, but also relate to how the rate is measured, through phylogenetic inference or through direct counting empirical experiments which have been made more possible in the advent of high throughput sequencing.
In an Icelandic study [ 14 ] a rate of 0. A meta-analysis combining eleven pedigree-related studies estimated the rate per generation at 0. Although the lack of mtDNA repair mechanisms has often been cited as the reason for the relatively high mutation rate in comparison with nuclear DNA, the answer is much more complex and unclear.
Research has revealed multiple repair pathways such as single strand break repair and mismatch repair, all apparently encoded by nuclear genes [ 18 ] and an interesting study revealed that the abundant mitochondrial dGTP in most tissues potentially leads to a loss of proof-reading activity by the exonuclease domain of the POLG DNA polymerase subunit gamma protein that removes DNA base-pair mismatches in replication [ 19 ].
Normally all the mtDNA in a cell has the same sequence homoplasmy. Mitochondrial dysfunction due to mutations can impact on bioenergetic genes; those that are passed to the next generation can lead to severe inherited disease.
Maintenance of the homoplasmic state is therefore very important but the mechanism poorly understood. Evidence points to some form of genetic bottleneck so that the mature oocyte represents a single molecule, which is generally free of mutations [ 20 ]. However, because mitochondria contain many mtDNA molecules, and each mitochondrion can have several genomes, it is not uncommon for mutations to be present affecting just a proportion of the molecules in the cell.
Heteroplasmy is an example of a point mutation where a single base is substituted in portion of the genomes, demonstrating two different bases in the same position in the sequence Figure 5. Clinically, there appears to be a threshold effect of the proportional change from wild to mutant type in a particular tissue when these mutations impact on the health of the individual. Somatically, the level of heteroplasmy can be different in the different cells from the same tissue or organ, or between organs from the same person, or between individuals of the same family because different tissues have different bioenergetic thresholds [ 21 ].
Of forensic importance, variability in heteroplasmy can be seen between hairs, even from the same individual, probably relating to the developmental origins of individual hair follicles where there is localised cell division, in comparison with the lack of variability in blood cells in which the measured heteroplasmy will reflect the average seen across many stem cells. Rarely there may be a discordance between the haplotypes from a single hair root and from blood in the same individual with no demonstrable heteroplasmy; analysis of additional hairs should be undertaken in these very unusual circumstances [ 22 ].
Mitochondrial DNA is considered to be inherited through the maternal line such that an individual's mtDNA will be shared with that person's mother and all prior maternal ancestors. Luo in raised the possibility of biparental mtDNA inheritance [ 25 ] when they reported three families with unusually high heteroplasmy, stating that the patterns could be explained by a combination of mtDNA from both parents.
This hypothesis significantly challenged a long-held biological tenet and has led to both supporting [ 26 ] and critical responses [ 27 , 28 ], the latter making relevant comments about the methodology and the importance of confirmation in experienced and fully independent laboratories. In each of the families at least one novel apparent NUMT was seen in the fathers, absent from the mothers. The observation of two full mitogenomes in an individual investigated for a possible contamination event revealed a megaNUMT in eight maternally related members of the family, in combination with the expected mtDNA haplogroup throughout the pedigree.
The megaNUMT was absent from hair shaft, where nuclear DNA will be missing, offering a mechanism to investigate future claims of paternal mitochondrial inheritance [ 30 ]. Mutations in mitochondrial sequences occur continuously and will affect the germline on occasions resulting in a heteroplasmy that is transmitted through the proposed genetic bottleneck.
Over time, through genetic drift, the wild:variant balance can alter and when the sequence change is agnostic, or beneficial to the individual, can become fixed homoplasmic in the population.
The uniparental inheritance and lack of recombination has led to mitochondrial variants being restricted to particular populations in different parts of the world. Different mitochondrial types, or haplogroups, are described according to a theoretical phylogenetic tree that offers evidence as to how these have evolved from a series of common ancestors.
Understanding the nature of these variants has enabled population geneticists to track the matrilineal inheritance of humans back to an origin in Africa and has helped describe the spread of humans across the world. Mitochondrial Eve belonged to haplogroup L. The main haplogroups and likely migration routes over time are illustrated in Figure 6.
The phylogenetic tree is continuously updated [ 32 ] to reflect new information at www. MtDNA sequencing provides an additional useful tool to characterise biological evidence. While its ability to identify an individual is limited because of the lack of recombination, it offers significant advantages when confirmation of maternal lineage is sought or where nuclear DNA is limited such as in the analysis of bones, teeth and hair, because of its high copy number.
Understandably it is often used in the analysis of ancient DNA and in the triage of disaster victim identification DVI. The rarity of a haplotype can be determined by simple counting of how many times the sequence is observed amongst samples of a database. As there may be various databases, both their representativeness and the quality of the sequences then becomes very important.
EMPOP is probably the most comprehensive mtDNA database worldwide and forensically the most useful, not only because of the large number of world populations that are represented, but also because of its emphasis on high-quality data.
EMPOP uses a string-based search algorithm SAM2 [ 35 ] that converts sequences into alignment-free strings to ensure that haplotypes are properly defined regardless of alignment differences. In addition to providing the haplogroup, the tool clarifies nomenclature in the presence of phylogenetically unstable positions and takes block indels into account.
Superficially straightforward apparent differences in nomenclature may lead to false exclusions and a defined reporting process is vital. While both point heteroplasmy discussed above and length heteroplasmy are associated with mtDNA typing, reporting the latter is not required in forensic typing and it has no impact on the haplogroup.
Samples with point heteroplasmy differences do not necessarily provide exclusionary evidence and sequences with a one-base difference should also be evaluated further with regard to their rate of mutation. The guidance highlights the importance of controls and avoidance of contamination, although they recognise that in mitochondrial analysis low-level contamination is difficult to avoid and suggest validated thresholds. Considering nomenclature, they recommend a blended application of rule-based and phylogenetic approaches and the use of the EMPOP database, although they advocate caution when considering historic data which might have been interpreted differently.
Rules are provided to advise about homopolymeric C-stretches, particularly important in MPS workflows, and they provide nomenclature recommendations on substitutions and indels, transitions and transversions [ 39 ].
Frequency reporting is done with simple counting in an appropriate recognised population database. An updated series of thirteen recommendations is provided by the ISFG to supplement that provided in Like SWGDAM they highlight methods to avoid errors and deal with potential low-level contamination, with recommendations for independent confirmation and participation in appropriate proficiency tests.
They continue to recommend reporting with reference to the rCRS reference sequence but with the addition of information on the sequencing range used, while recommending that the complete mtDNA control region should be typed.
Guidance and tools for nomenclature decisions are also provided [ 40 , 41 ]. Forensic laboratories are advised to establish their own interpretation and reporting guidelines for both length and point heteroplasmy because of the differences in technologies now being used with a choice as to whether length heteroplasmy is reported.
Because of the high incidence of mtDNA sequence interpretation errors [ 38 ] they recommend the use of software quality tools to check that the phylogeny is as expected. Use of alignment-based database searches, facilitated through EMPOP, of the whole database is recommended to avoid alignment differences and reporting bias.
The database used to assess the significance of a match should be considered, relevant to the case circumstances. Whatever is chosen should be able to be justified considering any uncertainties, taking into account the fact that mtDNA frequencies may vary significantly at a local population level [ 42 ].
Techniques developed to analyse ancient DNA can provide useful approaches to sequence significantly degraded material associated with a crime scene. Bones must be carefully and thoroughly cleaned to remove external contaminants and the analysis undertaken in laboratories dedicated to the analysis of extremely low-level DNA. Over time DNA degrades and can produce false sequences. Hydrolytic damage can lead to sequence miscoding through deamination. Deamination of cytosine produces uracil, which pairs with adenine producing thymine through PCR; miscoding produces the majority of sequencing errors.
Treatment with N-glycosylase to remove uracils can assist here [ 43 ]. This is particularly problematic if the amplification is initiated only from a few copies and suspicion should be raised if there are unexpectedly successful products. When sample quality is an issue a capture-hybridisation method [ 44 ] can be used to enrich the mtDNA.
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