Genetic lineage markers comprise polymorphisms that are present on the maternally inherited mitochondrial genome and the paternally inherited Y chromosome. The anal- ysis of lineage markers is limited in most forensic casework because they do not possess the power of discrimination of autosomal markers. Even so, there are some features of both mtDNA and the Y chromosome that make them valuable forensic markers.
The mitochondria are organelles that exist in the cytoplasm of eukaryotic cells. They carry out the vital job of producing approximately 90% of the energy required by the cell through the process of oxidative phosphorylation.
Inheritance of the mitochondrial genome
Mitochondria contain their own genome (mtDNA) which is maternally inherited [1, 2]. This was discovered in the 1950s after unusual patterns of inheritance of certain phe- notypes were explained by the existence of extra nuclear genomes that did not obey Mendel’s laws of inheritance. Duringfertilizationofanovum,thespermpenetratestheeggandthespermmidpiece, which contains between 50-75 mitochondria, enters the egg along with the head . The egg has around 1000-times more mitochondria than the sperm . Although some paternal mtDNA enters the ovum it is actively removed . The process is not always completely effective and very rare cases of paternal mtDNA inheritance have been documented .
The mtDNA genome is present in multiple copies – individual cells can contain hun-dreds of mitochondria and a single human mitochondrion can contain several copies of the genome [6-8]. Somatic cells, therefore, have thousands of copies of the mitochon- drial genome and approximately 1% of total cellular DNA comprises mtDNA [9, 10]. This compares with only two copies per cell of the nuclear genome.
The mtDNA genome
The human mitochondrial genome is a 16569 bp circular molecule. It encodes for 22 transfer RNAs (tRNAs), 13 proteins and two ribosomal RNAs (the 12S and 16S rRNA) [11, 12]. The majority of mitochondrial proteins is encoded by the nuclear genome as, overhundredsofmillionsofyears,followingtheformationofthesymbioticrelationship between eubacteria and eukaryote cells, most of the genes have been transferred from the mitochondrial to the nuclear genome . Analysis of the human mtDNA genome revealed a very economic use of the DNA and there are very few non-coding bases within the genome except in a region called the D-loop. The D-loop is the region of the genome where the initial separation, or displacement, of the two strands of DNA during replication occurs. The regulatory role of the D-loop has led to the other name by which it is known – the control region. It is approximately 1100 bp long.
Polymorphisms in mtDNA
The mtDNA genome accumulates mutations relatively rapidly as compared with the nucleargenome.Thehighmutationrate† isdueinparttotheexposureofthemtDNA toreactiveoxygenspeciesthatareproducedasby-productsinoxidativephosphorylation . Direct analysis of mother-to-children transmissions has estimated that a mutation in the hypervariable regions is passed from mother to child approximately once in every 30 to 40 events. In the vast majority of cases where a mutation is detected, there is only one base change between the mother and child [16, 17].
In most forensic investigations the aim of DNA profiling is to differentiate between individuals, therefore the most polymorphic regions are analysed. Following the se- quencing of the human mtDNA genome it was apparent that the D-loop was not under the same functional constraints as the rest of the genome. Some blocks within the con- trol region are highly conserved but large parts are not. Two main regions are the focus of most forensic studies, these are known as hypervariable sequence regions I and II (HVS-I and HVS-II) and they contain the highest levels of variation within the mtDNA genome. Both the hypervariable blocks are approximately 350 bp long. A third hyper- variable region, HVS-III has also been used in some cases. Within the hypervariable regions the rate of mutation is not constant and some sites are hotspots for mutation, while others show much lower rates of change [18-20]. Because the polymorphic sites are concentrated within relatively small regions of the mtDNA genome, they can be analysed using PCR amplification followed by Sanger sequencing . Many of the methods used for SNP detection can also be used (see Chapter 12).