Identification of male lineages

Most of the human Y chromosome (i.e. the non-recombining region of the Y chromosome, NRY) is male specific and is inherited unchanged from fathers to sons, unless a rare mutational event occurs. DNA recombination, a genetic process that reshuffles genetic material between homologue chromosomes to create additional variation, is not acting on the NRY because of the absence of a homologue chromosome. Due to the lack of recombination, a Y-chromosomal mutation creating a new allele is always inherited by male offspring in subsequent generations. On the other hand, the lack of recombination also means that all male relatives carry the same Y chromosome, independent of the degree of paternal relationship. This makes Y chromosome polymorphisms very useful for male identification but also means that male lineages (i.e. groups of paternally related males), but not individual males, can be identified, at least with the currently available Y markers.

The use of Y-chromosomal polymorphisms for male identification in forensics started 40 years ago with the analysis of whole Y-chromosome length polymorphisms to detect exclusion constellations in paternity cases (Nuzzo et al., 1966), even though the molecular basis of the underlying polymorphisms was unknown at the time. Later, and with increased knowledge about the molecular biology of the human Y chromosome, the use of whole Y-chromosome length differences was abolished from forensic applications due to the discovery of Y-chromosome length differences between cells from the same individual (Daiger and Chakraborty, 1985). The real breakthrough came in the early 1990s with the identification of the first Y-chromosomal microsatellite or short tandem repeat (Y-STR) polymorphism, DYS19 (Roewer et al., 1992), and its immediate application to a rape case, revealing an exclusion constellation (Roewer and Epplen, 1992). However, as known from the application of autosomal STRs to forensics, the value of a single marker for human individualization is limited. Instead, many STRs are needed to achieve high resolution and confidence. In 1997 a first attempt towards the forensic application of Y-STR haplotypes was made by the Forensic Y-Chromosome Research Group coordinated by Lutz Roewer from the Humboldt University in Berlin with the characterization of 13 Y-chromosomal STRs in 3825 unrelated males from 48 population samples (Kayser et al., 1997). This study was recently rated as the second most highly cited publication in the five leading forensic science and legal medicine journals, according to the ISI Web of Science database (Jones, 2007), but in fact it is the most cited paper ever published in a leading forensic journal (306 citations) based on a database query in July 2006, reflecting the success of Y-STR markers, especially in forensic science.

By 1997, 13 Y-chromosomal STRs were available for forensic applications. Of these, nine describe the so-called 'minimal haplotype' recommended by the International Forensic Y User Group as the minimal set of Y-STRs to be used for human male individualization in forensics (Kayser et al., 1997). The advantage of genetic markers from the non-recombining part of the Y chromosome compared with those from any other chromosome is that single marker information can be combined as haplotype information since the male-specific part of the Y chromosome is inherited completely linked from fathers to sons. For forensic applications this means that the multiplication of single locus allele frequencies for obtaining combined DNA profile matching probabilities cannot be applied to Y-chromosomal markers, but instead compound haplotype frequencies must be used for establishing matching probabilities in cases of non-exclusions. The combination of single loci in compound Y-chromosomal haplotypes, such as using the nine Y-STRs from the minimal haplotype, leads to an enormous increase in informativity. Consequently, the number of individuals that need to be investigated for obtaining representative haplotype frequencies is expected to be enormous and much larger than needed for autosomal markers. This has led to the establishment of Y-STR haplotype databases for obtaining more accurate and reliable haplotype frequencies. The largest database is the 'Y-Chromosome Haplotype Reference Database, YHRD'. This database started out as a European initiative (Roewer et al., 2001), was later expanded by mirror databases for U.S. populations (Kayser et al., 2002), and Asian populations (Lessig et al., 2003) and today exists as a combined and further expanded database with 40 108 haplotypes in a set of 320 populations worldwide, including population samples from all continental regions (Release '19' from August 2006).

Numerous laboratories, mostly from the international forensic genetics community, have contributed Y-STR haplotype data under controlled quality criteria to the YHRD and the number of contributed haplotypes is constantly increasing. The YHRD allows complete and partial Y-STR haplotype profiles to be searched for population-based and region-based frequencies, and provides useful graphical representations of the geographical distribution of the respective haplotypes, as well as lists with the number of matches per population sample. In addition to the search function, information about typing methods, molecular characteristics, including mutation rate estimates, and several statistical tools, including a haplotype frequency surveying method, are available through the public-domain website of the database (http://www.yhrd.org). This makes the YHRD unique not only in size but also in data authenticity, compared with databases established from published Y-STR data or other databases without quality control requirements. Due to the introduction of various commercial Y-STR kits (Yfiler from Applied Biosystems, Power-Plex Y from Promega, genRES DYSplex from Serac, Y-PLEX from Reliagene, Menplex Argus Y from Biotype), company-based Y-STR haplotype databases have become available recently, mostly collecting data from individuals living in the USA:

http://www.promega.com/techserv/tools/pplexy/

http://www.appliedbiosystems.com/yfilerdatabase/

http://www.reliagene.com/index.asp?menu_id=rd&content_id=y_frq

In the future it would be desirable if all data could be included in a single database allowing user-friendly single access for a comprehensive Y-STR haplotype frequency search.

The number of scientifically known Y-STRs has dramatically increased over recent years. In 2004, results from a comprehensive survey of Y-STRs were published using the nearly complete Y chromosome sequence for a systematic search for all useful Y-STR markers (Kayser et al., 2004). In this study, 166 previously unknown Y-STR markers were found, increasing the total number of verified Y-STRs to 215 (Figure 9.2). Although the 9-11 commonly used Y-STRs provide high haplotype diversity, and thus high probability of male lineage identification, additional Y-STRs will increase the haplotype discrimination, depending on the marker added and the population analysed (Beleza et al., 2003; Park et al., 2005; Turrina et al., 2006). Furthermore, studies have reported population samples with a high number of identical 9-16 loci Y-STR haplotypes as a result of severe bottlenecks in the history of those populations, e.g. 14% of males in a Pakistani population sample (Mohyuddin et al., 2001) and 13% of males in a Finish population sample (Hedman et al., 2004) share the same 16-loci Y-STR haplotype. Therefore, in cases of matching haplotypes, typing additional Y-STRs can be useful for forensic applications, and currently many of the newly described Y-STRs are investigated with respect to their haplotype discrimination potential, population genetic diversity, mutation rates, as well as their suitability for multiplex analyses.

A number of Y-STR markers are located in multicopy regions of the Y chromosome and thus consistently show more than one male-specific allele (Kayser et al., 2004). These multicopy Y-STRs, although often very variable due to the simultaneous detection of multiple polymorphic loci (Redd et al., 2002), are less

Figure 9.2 Cumulative number of Y-STRs, as published in scientific journals. Markers are counted separately only when amplifiable separately; consequently, multicopy Y-STRs that cannot be typed separately are counted only once. Those Y-STR markers that were submitted to public databases but not verified through scientific publications were not included

Figure 9.2 Cumulative number of Y-STRs, as published in scientific journals. Markers are counted separately only when amplifiable separately; consequently, multicopy Y-STRs that cannot be typed separately are counted only once. Those Y-STR markers that were submitted to public databases but not verified through scientific publications were not included useful for forensic stain analyses since they might cause interpretation difficulties in mixed stains with more than one male involved (Butler et al., 2005). Some of these multicopy markers are located in Y-chromosomal regions where the number of copies is assumed to be associated with male fertility problems (such as AZF). Consequently, a Y-STR profile including these loci can potentially be informative for the fertility status of a man (Bosch and Jobling, 2003). Such loci should be omitted from forensic tests because of the additional information they potentially reveal.

The most important application of Y-chromosome markers for male lineage identification in forensics is in cases of sexual assault. The nature of the material available from sexual assault cases, usually mixed stains from the female victim's epithelial cells and the male perpetrator's sperm cells, makes autosomal STR profiling challenging. Normally, to separate the male and female genetic components, differential lysis is applied to extraction DNA from mixed stains. Often, and especially when the number of sperm cells is low, this approach fails, resulting in potential overlap of the victim's and perpetrator's autosomal STR profiles, making male perpetrator identification impossible. Recent technological advances, e.g. using laser dissection microscopy to specifically collect sperm cells, are promising but the success of autosomal STR analysis from such material depends on the number of sperm cells collectable in each particular case. If the number of sperm cells is low, technical problems of low copy number (LCN) analysis are expected, and the approach will not be successful when no sperm cells can be collected. However, the specific detection of male DNA by analysing Y-chromosomal STRs in principle avoids the problem of profile overlap (since females do not carry a Y chromosome) and additionally is much more sensitive. Mixing experiments have shown that Y-STRs can still be amplified successfully and reliably up to malefemale DNA mixtures of 1 : 2000 (Prinz et al., 1997). In addition, even in the absence of sperm (i.e. in cases of oligospermic or azoospermic males involved) but the presence of mostly or only male epithelial cells in a mixed stain, Y-STR analysis has proven to be highly successful (Betz et al., 2001). It has also been shown that Y-STR haplotype profiling in rape cases can be successful from cervicovaginal samples recovered up to 4 days post-coitus (Hall and Ballantyne, 2003).

The use of Y-STR markers in forensics is regulated by two recommendations of the DNA Commission of the International Society of Forensic Genetics in collaboration with expert Y chromosome scientists (Gill et al., 2001; Gusmao et al., 2006). Because of the above-mentioned advantages and the enormous research effort that has been undertaken, Y chromosome markers (especially Y-STRs) are routinely used for male lineage identification in many forensic laboratories all over the world, and have been for many years already. One case shall be mentioned as an example to demonstrate the power of Y-STRs in forensic male lineage identification. To identify a serial rapist who had raped 14 young women and murdered one of them in north-western Poland, Y-STR haplotype profiles were obtained from >400 suspects as part of an elimination process (Dettlaff-Kakol and Pawlowski, 2002). A man was identified with a

Y-STR haplotype profile identical to that obtained from the crime scene, but with an autosomal STR profile matching that of the crime scene in only 9 of 10 markers. This finding suggested that the perpetrator must be one of this man's close relatives and DNA analysis of his brother revealed a complete match of both the autosomal STR and Y-STR profiles, identifying the brother as the perpetrator. It should be pointed out that Y chromosome (or mtDNA)-based mass screenings (which are on a voluntary basis) are also seen critically since legislation in many countries provides the right to refuse testimony in cases where close relatives are involved. However, Y chromosome (and mtDNA)-based male (and female) lineage identification is able to identify close and distant male (and female) relatives.

Although there is general agreement on the use of Y-STR markers to exclude suspects, there is still an ongoing discussion about how to use Y-STR haplotype information in the courtroom when a match between the crime scene sample and a suspect is established. Usually it is common practise to place some significance on the probability of such a match. This can be achieved by a method that extrapolates frequency estimates based on observed data stored in a database such as the YHRD (Krawczak, 2001; Roewer et al., 2000). This 'haplotype surveying method', available via the YHRD website, generates estimates of the prior and posterior frequency distributions of Y-STR haplotypes (Roewer et al., 2000). A simplified use of Y-STR haplotype databases for obtaining confidence in the statistical meaning of a haplotype match can be obtained from mismatch distribution analysis (Pereira et al., 2002), whereas a more conservative approach is to simply count the number of times the haplotype exists in the database and establish a confidence interval by taking into account the size of the database (Budowle et al., 2003). However, some scientists argue that existing Y-STR databases are not representative of real populations because of their limited size and because the databases are normally based on unrelated individuals, whereas real populations are not only bigger in size but also do contain related individuals. The latter is especially important for Y chromosome markers since close and distantly related men can share the same Y-STR haplotype. Such a perspective leads to the most conservative use of Y-STR haplotype information in cases of established matches, stating that the suspect cannot be excluded from being the donor of the crime scene sample (de Knijff, 2003). It is noteworthy that official recommendations by the DNA Commission of the International Society of Forensic Genetics on the estimation of the weight of the evidence of Y-STR typing have not yet been provided but are expected in the near future, as announced elsewhere (Gusmao et al., 2006).

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