APPLICATIONS OF mtDNA PROFILING
Figure 13.1 The mitochondrial genome is circular and 16569 bp long. It encodes for 13 proteins, 22 transfer RNAs and two ribosomal RNAs. The polymorphic hypervariable sequence regions I, II and III (HVS-I, HVS-II and HVS-III) are located within the control region. Other regions of the genome that are utilized in forensic casework for species identification are the coding regions for the 12S and 16S ribosomal RNAs and cytochrome b gene
Applications of mtDNA profiling
ThereareseveralscenarioswheremtDNAisavaluablegeneticmarker.Thesearerelated to two properties of mtDNA – the high copy number and the maternal inheritance. The highcopynumberisvaluablewhentheamountofcellularmaterialavailableforanalysis is very small. Crime scene material that is commonly profiled using mtDNA includes hair shafts [22, 23] and faecal samples . mtDNA is also useful for the analysis of human remains that are highly degraded and not amenable to standard STR typing [25, 26]. The maternal inheritance is a useful trait for human identification when there are no direct relatives to use as a reference sample – the identification of some of the Romanov family using Prince Philip as a reference sample provides a powerful illustration (Figure 13. 2) of the use of maternal inheritance . A series of historical cases has followed that demonstrates the application of mtDNA when linking relatives to human remains [28-30].
Homoplasmy and heteroplasmy
Normally an individual contains only one type of mtDNA – this is termed homoplasmy. Mutations will inevitably occur within some of the thousands of copies of mtDNA within a cell and if these mutated copies of the genome were passed onto future generations, a mixture of different mtDNA genomes would occur. The process that maintains homoplasmy as the norm is not precisely understood but at some point a
Figure 13.2 The family tree of the Romanov royal family. (a) The maternal lineage of the Tsarina and her children, which provides a direct link to Prince Philip. (b) The maternal lineage of Tsar Nicholas, linking him to two living maternal relatives, the Duke of Fife and Xenia Cheremeteff-Sfiri. The squares represent males and the circles represent females, the transmission of the relevant mtDNA type is indicated by shading
genetic bottleneck occurs before the formation of a mature oocyte . The bottleneck allows only a few mtDNA molecules to pass into the oocyte during its formation [32, 33], thereby reducing the possibility of passing on a mixture of wild type and mutant genomes. It is, however, possible to find more than one type of mtDNA within a cell – this is known as heteroplasmy and it arises when a mother passes on a normal version of her mtDNA genome (wild type) and also a version of the genome that contains a mutation. An individual will therefore posses two versions on their mtDNA, maybe only differing by one base. Two factors, the severity of the bottleneck and subsequent genetic drift, determine the relative levels of wild to mutated mtDNA [16, 17]. Heteroplasmy can be
Figure 13.3 The above sequence shows the presence of heteroplasmy at position 16189. Two bases, a C and an A, are present in approximately equal amounts (see plate section for full-colour version of this figure)
Table 13.1 An example where an mtDNA profile has been generated from the HVS-I of five bones that were found in close proximity. The mtDNA profiles of three women who were maternal relatives of three missing individuals are also shown. Maternal reference 1 clearly matches the bone profiles, while maternal references 2 and 3 can be excluded as potential maternal relatives as they have different mtDNA types. In this particular case the mtDNA profiling helped to establish the identification of the human remains, and that the bones all came form the same person 
stable through several generations before one of the mtDNA versions becomes fixed [16, 17, 29, 33, 34]. When heteroplasmy is detected, the two types of mtDNA genomes normally only differ at one position [22, 23].
Interpretation and evaluation of mtDNA profiles
mtDNA is used for both associating crime scene samples with individuals and also in the identification of human remains. In both cases the profile that has been generated from the unknown sample has to be compared to a reference profile. In the case of a crime scene investigation, the reference sample will be from a suspect. In the case of human identification, a sample taken from a maternal relative or a personal artefact such as a toothbrush can be used . The first step is to turn the information into a more manageable form.
The data after sequencing consists of upwards of 350 DNA bases from both HVS-I and HVS-II and around 150 bases from HVS-III. Once the sequencing data have been checked to ensure that there is confidence in the sequence data and no errors, it is compared to the Cambridge Reference Sequence (CRS) [11, 12]. The CRS was the first complete sequence of the mtDNA genome to be published in 1981. Differences between the questioned sequence and the CRS are noted and only these differences are recorded. Table 13.1 shows an example of where a HVS-I profile generated from a set of human remains is compared to profiles generated from three reference samples. The mtDNA profile is called a haplotype.
Declaring a match is straight forward but exclusions can be more problematic. When a questioned sample and a reference sample differ at only one position the likelihood of that one base difference occurring though a mutation has to be assessed. In such an instance the results are usually classified as inconclusive – when there are two or more differences between a questioned and known sample it is normally classified as an exclusion . If a match is declared, the statistical significance of the match has to be assessed. The mtDNA genome is inherited as a single locus and this limits the evidential value of the marker in forensic cases. Haplotype frequencies have to be measured directly by counting the occurrence of a particular haplotype in a database and reporting the size of the database.
When databases are relatively small, for example 100, many of the less common haplotypes that are within a population will not be represented. There are mechanisms that compensate for the limitations of reference databases, such as minimum haplotype frequencies, and employing standard error calculations and correction factors to allow for subpopulations [37, 38]. When reporting the results of mtDNA analysis, the caveats associated with mtDNA have to be clearly explained so that there is no confusion with ‘standard’ (autosomal STR typing) analysis. In particular that ‘it is inherited only from one’s mother, and therefore all individuals who are related by a maternal link will have the same mtDNA profile’, and that ‘it varies less between individuals, and therefore more individuals chosen at random from the population will have the same mtDNA profile’ should be made very clear.