Tn Tn

Xenia Cheremeteff-Sfiri

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

Duke of Fife genetic bottleneck occurs before the formation of a mature oocyte [31]. 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 16 189. 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 [36]

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 [36]

Sample

HVS-I sequence

Right femur

16189C

16 223T

16 271C

16 278T

Left femur

16189C

16 223T

16 271C

16 278T

Right pelvis

16189C

16 223T

16 271C

16 278T

Left ulna

16189C

16 223T

16 271C

16 278T

Left tibia

16189C

16 223T

16 271C

16 278T

Maternal Reference 1

16189C

16 223T

16 271C

16 278T

Maternal Reference 2

Same as Cambridge Reference Sequence

Maternal Reference 3

16,278T

16,293G

16,311C

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 [35].

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 [21].

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.

The Y chromosome

In humans the Y chromosome is approximately 60 Mb long (million base pairs) long and contains just 78 genes [39]. The SRY gene (sex-determining region Y) located on the Y chromosome encodes a protein that triggers the development of the testes and through an extended hormonal pathway causes a developing foetus to become male [40].

With the exception of two regions, PAR 1 and 2 (PAR = pseudoautosomal region), located at the tips of the chromosome, no recombination occurs during meiosis. The remaining 95% of the Y chromosome is non-recombining, male specific, and is passed from father to son unchanged, except when mutations occur. The lack of recombination may be the reason why there are relatively few genes on the Y chromosome. If there is no chromosome crossing over, mutations within genes have little chance to be repaired or rectified and hence will be passed onto the next generation.

Y chromosome polymorphisms

The Y chromosome contains a large number of polymorphisms including variable number [41] and short tandem repeats (VNTRs and STRs), insertions, deletions and single nucleotide polymorphisms (SNPs).

The first STR locus to be identified on the Y chromosome was DYS19 [42]. Since then hundreds of Y chromosome STR have been described. The development of Y STR typing has mirrored the development of the autosomal STRs, and multiplexes have been developed with increasing numbers of robust and highly discriminating Y STR multiplexes [43-46]. The growth in interest in Y STR loci has led to numerous

FORENSIC APPLICATIONS OF Y CHROMOSOME POLYMORPHISMS

Table 13.2 The Y chromosome STR Loci that are commonly used in forensic analysis

Minimal haplotype

Extended haplotype

PowerPlex® Y

AmpF/STR®

DYS19

DYS19

DYS19

DYS19

DYS385 a/b

DYS385 a/b

DYS385 a/b

DYS385

DYS389 I

DYS389 I

DYS389 I

DYS389 I

DYS389 II

DYS389 II

DYS389 II

DYS389 II

DYS390

DYS390

DYS390

DYS390

DYS391

DYS391

DYS391

DYS391

DYS392

DYS392

DYS392

DYS392

DYS393

DYS393

DYS393

DYS393

DYS438

DYS437

DYS437

DYS439

DYS438

DYS438

DYS439

DYS439

DYS448

DYS456

DYS458

DYS635

GATA H4

population studies to establish allele frequency databases. The 'Y Chromosome Haplo-type Reference Database' was established to collate STR haplotypes (www.yhrd.org).

To ensure comparability between datasets, minimal and extended haplotypes were

defined. Two commercial kits, the PowerPlex Y (Promega Corporation) and the

AmpF/STR Yfiler (Applied Biosystems) incorporate all of the extended haplotype loci (Table 13.2).

Forensic applications of Y chromosome polymorphisms

That the Y chromosome is only found in males makes it a valuable tool, in particular for the analysis of male and female mixtures after sexual assaults when differential DNA extraction is not possible; Y STR analysis has been successful with female:male ratios of up to 2000:1 [47]. The presence of male DNA has also been detected when vaginal swabs are analysed, even when no spermatozoa have been detected - either through the assailant being azoospermic (1-2% of rape cases) or through the deterioration of the spermatozoa [44,48]. The Y STRs can also be used to detect the presence of two male profiles - the interpretation of the mixtures depends on the presence of major and minor contributors [47].

In addition to using Y chromosome testing for the identification of evidential samples, it has also been used for paternity testing and is particularly valuable in deficient cases, where the alleged father is not available for testing. In these cases, any male relative who is paternally related to the alleged father can be used as a reference. An extreme example of where this has been used is the paternity analysis that linked the third US president, Thomas Jefferson to the child of one of his slaves, Sally Hemings [49]. Cases involving human identification have also used the Y chromosome as a tool to link remains to paternal family members, and as with deficient paternity cases the use of the Y chromosome is particularly advantageous when there are no parents or children to use as reference material; it also simplifies the sorting of the material following mass disasters [50]. The mutation rate in Y STR loci is similar to autosomal STRs, at approximately 2.8 x 10-3 [51, 52]. The Y chromosome will accumulate mutations as it is passed through the patrilineal line and direct comparison between males on the same lineage may result in a false exclusion if mutations are not considered.

The non-random distribution of the Y chromosome among global populations, due largely to the widespread practice of patrilocality [53, 54] (where the female moves to the male's birth place/residence after marriage), makes it a useful tool for inferring the geographical origins of biological material recovered from a crime scene and human remains [55]. In some cultures, where the male name is passed onto male children, there is also the potential of attributing surnames to Y profiles [56, 57].

Interpretation and evaluation of Y STR profiles

When the Y chromosome profiles from a reference and an unknown sample match, the significance of the match has to be assessed. The first step is to assess the frequencies of the Y STR haplotypes in the population of interest. The simplest method is to report the frequency of the Y STR haplotype in the population, known as the counting method. The figure quoted is entirely dependent upon the size of the database and is normally based on frequency databases that are constructed for the major ethnic groups represented within individual countries, although comparisons can also be made to the combined data in the yhrd databases with over 40 000 haplotypes (representing at least the minimal haplotype). So, for example, a match can be reported as 'the haplotype has been seen twice in 400 UK Caucasian individuals'.

Difficulties arise in the interpretation of the Y chromosome. This is primarily caused by the patrilineal inheritance and clustering of male family members in relatively small geographic areas. This geographical clustering of male relatives coupled with the limited size of the haplotype frequency databases (many haplotypes are seen only once) makes the estimation of profile frequencies hazardous. An alternative method for assessing the significance of a match is to use a likelihood ratio and to incorporate population subdivisions with the increased potential for common co-ancestry [38]. Regardless of the method used to calculate the matching frequency, when presenting the results of Y chromosome analysis, as with mtDNA, there is a need clearly to state how the use of Y STR typing varies from that of autosomal markers and that there will be other males in the population with the same Y STR haplotype.

Further reading

Jobling, M., Hurles, M., and Tyler-Smith, C. (2004) Human Evolutionary Genetics: Origins, Peoples and Disease. Garland Science. Cavalli-Sforza, L.L., Menozzi, P., and Piazza, A. (1996) The History and Geography of Human Genes. Princeton University Press.

REFERENCES

WWW resources

The Y Chromosome Haplotype Reference Database: http://www.ystr.org/index.html EMPOP - Mitochondrial DNA Control Region Database: http://www.empop.org/

MITOMAP: http://www.mitomap.org/

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