History of forensic utilization of the X chromosome

The fundamental idea for extensive usage of X-chromosomal markers in forensic practice came from the experiences made during the second half of the last century in the field of clinical genetics. There are many well-known diseases and traits such as haemophilia, Duchenne muscular dystrophy, Lesch-Nyhan syndrome, G6PD deficiency, colour blindness, etc. that follow X-chromosomal inheritance. If a male patient is fertile, all his daughters possess the defective paternal X chromosome and transmit it to half of the next generation. Half of all daughters are again gene carriers and also half of their sons inherit the defective allele and exhibit the trait due to the hemizygote state of their ChrX. Furthermore, when a male exhibits two or more ChrX-linked traits it is obvious that alleles of all relevant loci are unified to one haplotype. This explains why ChrX linkage analysis (Adam et al., 1963) is fairly easy. It is obvious that knowledge of such simple contexts is valuable not in clinical genetics only but also in kinship testing. However, cognition of sex-linked genetic markers usable in forensic genetics rose very slowly.

In the course of forensic kinship testing, which started in the middle of the last century, initially only blood group markers played a role. These were later supplemented by serum protein and enzyme variants. However, all of them were of autosomal inheritance. Regarding X-chromosomal markers, the first significant achievement was made when the Xga blood group was detected by the team of Race and Sanger (Mann et al., 1962; Sanger et al., 1971, 1977; Tippett and Ellis, 1998; Went et al., 1969). Chromosome X linkage of Xga could easily be recognized by comparing the frequencies of the Xga /Xg phenotypes in males and females (males: 0.62/0.38; females: 0.86/0.14). The blood group Xga is a fairly weak antigen. This may be the reason why Xga testing could not be established as a significant method in kinship testing practice. However, some questions of scientific interest, such as identification of the origin of X chromosomes in chromosome aberration syndromes such as Klinefelter and Ullrich-Turner syndrome have been solved using serological Xga testing (Mann et al., 1962; Lewis et al., 1964; Tippett and Ellis, 1998). Later Ellis et al. (1994) identified the Xga antigen derived from the N-terminal domain of a candidate gene, referred to earlier as PBDX.

Two further ChrX loci encoding gene products with polymorphic appearance and therefore with a potential for usage in kinship testing are known. Glucose-6-phosphate dehydrogenase (G6PD) (Adam et al., 1967; Yoshida et al., 1973; Askov et al., 1985; Roychoudhury and Nei, 1988) and phosphoglycerate kinase (PGK) (Beutler, 1969; Chen et al., 1971; Roychoudhury and Nei, 1988) show considerable diversity on the protein level in some geographical regions. However, to our knowledge, with rare exceptions (Bucher and Elston, 1975), X-linked enzyme variants do not play any role in forensic contexts.

In the pre- DNA-technique era forensic scientists used sex chromatin tests for gender assessment in human tissues or single cells (Given, 1976; Duma and Boskovski, 1977). Later this technique was complemented by fluorescence microscopic demonstration of male heterochromatin (Tröger et al., 1976; Mudd, 1984). It can be shown as a quinacrine mustard-stained part of ChrY in met-aphases and even in metaphase cell nuclei.

In the early 1980s clinical geneticists started with genomic linkage analysis aimed at gene carrier detection and prenatal diagnosis (Davies et al., 1963; Davies et al., 1985). Some X-linked diseases, such as Duchenne muscular dystrophy and haemophilia, were in the focus of interest. In the first stage typing targets mainly were single nudeotide polymorphisms (SNPs), which could be detected by the Southern technique and were called restriction fragment length polymorphisms (RFLPs) according to the detection technique involving restriction enzymes. Later SNP linkage markers were supplemented by CA repeat polymorphisms (Lalloz et al., 1991, 1994) and the minisatellite Stl4 (DXS52) (Oberle et al., 1985). Investigation of the latter kind of polymorphism was enabled by creating the polymerase chain reaction (PCR) technique. Whilst usage of dinucleotide repeats is shunned by the forensic community, minisatellite marker DXS52 clearly fulfills the forensic requirements for markers. Nevertheless, DXS52 appeared in the forensic literature only very sporadically (Lamb-ropoulos et al., 1995; Yun and Yun, 1996). The first two ChrX microsatellites that played a significant role were HPRTB (Hearne and Todd, 1991; Edwards et al., 1992; Kishida et al., 1997; Xiao et al., 1998; Szibor et al., 2000) and ARA (Edwards et al., 1992; Sleddens et al., 1992; Kishida and Tamaki, 1997; Desmarais et al., 1998).

Kishida et al. (1997) and Desmarais et al. (1998) almost at the same time created formulas for the calculation of useful parameters such as mean exclusion chance (MEC), which considers the unique inheritance of ChrX. Thirty years before Krüger et al. (1968) had created the MEC for AS. These formulas are summarized in Table 7.1.

One of the challenges in kinship testing is to establish techniques that can bridge large pedigree gaps. We know from observation in clinical genetics that persons who share a very rare genetic feature can be unified to a common pedigree. The famous monarchic haemophilia pedigree can be demonstrated as an example. Some members of the European high nobility show the trait of haemophilia, which leads to the term 'royal disease'. Typing of the haemophilia gene would enables us to show that all affected persons with a certain mutation belong to the same pedigree descending from Queen Victoria (1837-1901). Two reasons ban us from doing this. Firstly, typing of harmful mutations in kinship

Table 7.1 Formulas for evaluation of the forensic efficiency of genetic markers

Formulab

References

I X f (1 - f )2 + If (1 - f )3 +1 f (f + f )(1 - f - f )2

II I f 3 (1 - f ) + If (1 - f )2 + X f (f + f )(1 - f - fj ) m 1-I f 2 + X f -(I /2)2

Krüger et al. (1968) Kishida et al. (1997) Desmarais et al. (1998) Desmarais et al. (1998) Desmarais et al. (1998) Desmarais et al. (1998)

a I: MEC (mean exclusion chance) for AS markers in trios; II: MEC for ChrX markers in trios involving daughters; III: MEC for ChrX markers in trios involving daughters; IV: MEC for ChrX markers in father/daughter duos; V: power of discrimination (PD) in females; VI: PD for ChrX markers in males.

b ff): population frequency of the ith (jth) marker allele.

testing would violate our ethical principles. Secondly, very rare traits would contribute to kinship only very seldomly. However, this example can demonstrate the power of rare alleles. Fortunately, the same effect can be achieved in another way: substituting short tandem repeats (STRs) by haplotypes consisting of clustered STRs provides a comparable power and can be used systematically in kinship testing.

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