Lineage markers

Genetic lineage markers comprise polymorphisms that are present on the maternally inherited mitochondrial genome and the paternally inherited Y chromosome. The analysis 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.

Mitochondria

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.

During fertilization of an ovum, the sperm penetrates the egg and the sperm midpiece, which contains between 50-75 mitochondria, enters the egg along with the head [3]. The egg has around 1000-times more mitochondria than the sperm [3]. Although some paternal mtDNA enters the ovum it is actively removed [4]. The process is not always completely effective and very rare cases of paternal mtDNA inheritance have been documented [5].

Copy number

The mtDNA genome is present in multiple copies - individual cells can contain hundreds 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 mitochondrial 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 16 569 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, over hundreds of millions of years, following the formation of the symbiotic relationship between eubacteria and eukaryote cells, most of the genes have been transferred from the mitochondrial to the nuclear genome [13]. 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 nuclear genome [14]. The high mutation ratet is due in part to the exposure of the mtDNA to reactive oxygen species that are produced as by-products in oxidative phosphorylation [15]. 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].

Hypervariable regions

In most forensic investigations the aim of DNA profiling is to differentiate between individuals, therefore the most polymorphic regions are analysed. Following the sequencing 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 control 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 hypervariable 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 [21]. Many of the methods used for SNP detection can also be used (see Chapter 12).

^ Note: mutations in the hypervariable regions are normally referred to as 'a base substitution' as they do not have an effect on any of the products encoded by the mtDNA - for simplicity the term 'mutation' will be used throughout this chapter.

HVS-I

HVS-II

HVS-III

Figure 13.1 The mitochondrial genome is circular and 16 569 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

Figure 13.1 The mitochondrial genome is circular and 16 569 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

There are several scenarios where mtDNA is a valuable genetic marker. These are related to two properties of mtDNA - the high copy number and the maternal inheritance. The high copy number is valuable when the amount of cellular material available for analysis is very small. Crime scene material that is commonly profiled using mtDNA includes hair shafts [22, 23] and faecal samples [24]. 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 [27].

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

Grand Duke Ludwig 4th of Hesse

Tsar Nicholas 2nd

Princess Alice (2nd daughter of Queen Victoria)

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Alexei MariaTatiana Olga Anastasia

Alice of Battenburg

Prince Philip I" Duke of Edinburgh L

Louise of Hesse-Cassel

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Georgij Tsar Romanov Nicholas 2nd

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