Forensic Science, Medicine, and Pathology Copyright © 2006 Humana Press Inc. All rights of any nature whatsoever are reserved. ISSN 1547-769X/06/2:283–286/$30.00 (Online) 1556-2891 DOI: 10.1385/Forensic Sci. Med. Pathol.:2:4:283
DNA REVIEWS
Sex Determination Eleanor A. M. Graham Forensic Pathology Unit, University of Leicester, Leicester, United Kingdom Address for correspondence and reprints: Eleanor A. M. Graham Forensic Pathology Unit University of Leicester Robert Kilpatrick Building Leicester Royal Infirmary Leicester LE2 7LX United Kingdom E-mail: [email protected] Accepted for publication: August 21, 2006
Abstract Determining the sex of a given DNA sample can provide criminal investigators with useful intelligence and can aid the identification of missing persons and disaster victims. Polymerase chain reaction-based systems that amplify regions of the amelogenin gene have become the method of choice for sex determination of biological samples. This system can, however, result in false female sex designation when mutations affect primer binding sites of the Y homolog of this target sequence, causing drop out of the Y amplification product. Erroneous sex determination could have drastic consequences when applied to forensic situations by misdirecting investigators or hindering the identification of deceased individuals. Current methods of sex determination are described and possible alternative approaches to avoid errors are discussed. Key Words: Forensic identification; DNA; biological sex; amelogenin; Y-chromosome; X-chromosome. (DOI: 10.1385/Forensic Sci. Med. Pathol.:2:4:283)
INTRODUCTION
METHODS OF SEX DETERMINATION
Routine forensic DNA analysis involves the investigation of short-tandem repeat (STR) markers to individualize biological samples to determine their origin. These systems are not designed to provide any information pertaining to the physical characteristics of the individual being tested, with one major exception: biological sex. On some occasions in both forensic and archaeological situations, human remains are found in a state such that sex assignment via direct inspection or classic anthropological examination is impossible. In missing person’s cases, mass disasters, or dismemberments where corpses are unrecognizable or severely disrupted, it can at times be very difficult and sometimes impossible to determine the sex of an individual by physical examination alone. The sex of a skeleton by anthropological means requires that certain key regions are present, most importantly, the pelvis and skull. In blind tests on skeletal material of known sex, a 100% accuracy of sex assignment is rarely, if ever, achieved. The sexing of adult remains from an intact pelvis is around 95% accurate, 85–90% from an intact skull, and only 80–90% accurate from long bone examination. The accuracy is further reduced when dealing with prepubescent juvenile remains, which can be as low as 50% (1). For this reason, a DNA-based technique for sex assignment is critically important to forensic and archaeological investigation.
The genetic difference between males and females is defined by the presence or absence of theY-chromosome, or more specifically, the Y-chromosome complete with the sex-determining regions. The majority of the DNA of the sex chromosomes is specific to either the X or Y form. There are also regions of homology between the two sex chromosomes that are also useful targets for genetic sex typing of samples. A number of early papers concentrated on polymerase chain reaction (PCR) coamplification of X- and Y-specific regions of the sex chromosomes to give single-banding patterns for female samples and double-banding patterns for male DNA (2–6). Although all of these systems were proven to be accurate for sex determination from test samples, the possibility of obtaining false female results by amplification failure of the Y-specific target was quickly recognized (7). Alternative approaches to sex determination of DNA samples involve investigation of regions of the amelogenin gene. This is the gene that encodes tooth enamel and is present on both the X- (Xp22.1–Xp22.3) (8) and Y- (Yp11.2) (9) chromosomes. Closer inspection of this gene on each of the sex chromosomes identified regions of true homology, but also identified a number of sequence differences between the two forms (10). Because of these homologous regions, PCR systems could now be designed to amplify both X- and Y-chromosomes in a
283
284 __________________________________________________________________________________________Graham single reaction by placing primers in homologous positions on the amelogenin gene that flank areas of difference between the X and Y forms. This duel amplification from a single set of primers provides an internal PCR control, meaning reaction failure will affect both X- and Y-chromosomes, removing the possibility of false-positive results that could be encountered by separate amplification strategies. Early systems designed to amplify regions of the amelogenin gene focused around an 189bp deletion present in the first intron of the X homolog of the gene (10) and required large amounts of template DNA to be entered into the amplification reaction (11). The starting amount of template DNA was quickly reduced by optimization of the reaction leading to reported success rates from quantities equivalent to a single diploid cell (12). The biggest breakthrough in DNA sex typing came with the publication of an alternative target sequence within amelogenin gene (7). This system was again designed to co-amplify X- and Y-chromosomes in a single reaction, but was based around a shorter, 6bp X-chromosome deletion, allowing for amplification of shorter target sequences. Instead of long-length products (977bp and 788bp), amplified fragments were a mere 106bp and 112bp for the X- and Y-chromosomes respectively, and these targets could also be amplified from minute amounts of template DNA. This system was compatible with both low copy number and degraded DNA, and as such quickly became the system of choice within both forensic and archaeological disciplines. The short product lengths of these redesigned amplicons also had the advantage of falling within the size range of forensically important STR loci, and as such were perfectly suited to multiplexing within DNA profiling kits. Today, forensic DNA profiling is primarily carried out using commercially produced kits, designed to amplify up to 16 STR loci simultaneously. All kit primers for amplification of the amelogenin loci, and all are designed around the 6bp deletion of intron 1 of this gene. An additional bonus of this particular system comes with the detection method of choice. Because the amelogenin DNA fragments can be co-amplified along with autosomal STR loci within commercial DNA profiling kits, the fluorescent labeling and visualization method used for this process provides a degree of quantitative information along with fragment-length measurement. The quantitative data can then be used to determine the ratio of X- to Y-chromosomes present in the analyzed sample. Male/female DNA mixtures are often encountered during forensic investigation, especially in sexual assault investigations. The ability to calculate the relative ratio of male to female DNA present in a given mixture can be extremely useful for downstream analysis. This quantification will also detect chromosome duplication events of the sex chromosomes, for example, to detect the presence of an extra X-chromosome in XXY males. This system is not useful for correct sex determination in sex-reversal syndromes such as XY females and XX males (13), where a mutational event or translocation results in the loss or gain of the amelogenin gene.
PROBLEMS IN CURRENT SYSTEMS As in any PCR technique, problems can be encountered if a nonlethal mutational event occurs in the recognition sequence of either primer. Mutational events of this kind are usually recognized, depending on the position and extent of the mutational change, by inefficient amplification of target sequence or failure of the PCR altogether. For STR loci, the presence of primer binding-site mutations can cause allele drop out and discordance between STR profiles generated using different commercial kits (14). Although far from ideal, problems of this type will usually affect single STR loci in a multiplex reaction. The loss of information at a single position does not affect the overall power of the system. This is not true for the sex-discriminating amelogenin locus. The reliability of amelogenin-based sex testing was first questioned in 1998 with the observation of two phenotypically male individuals being classified as female after PCR analysis (15). A mutation in the amelogenin primer binding region was also reported by Roffey et al. in 2000 (16) when the DNA extracted from a buccal swab taken from a phenotypically normal male was typed as female after STR profiling. After further investigation of this anomaly, it was deduced that a single point mutation in the primer binding region of the Y homolog of the amelogenin locus was responsible for the drop out of the Y-chromosome amplification product in the DNA profile. The authors of this article highlighted the problems that could arise if this result occurred, but was not recognized during forensic casework. The failure of a forensic DNA profiling technique to correctly resolve an issue as simple as sex could seriously misdirect the police investigation, as well as cast doubts on the techniques’ reliability for criminal prosecution. Following this report, further observations of sex testing errors began to emerge in the forensic literature. These observations are summarized in Table 1. Further investigation of Y allele-null males has revealed that primer binding site mutations are not the only cause of amplification failure. Two deletions, one between 304 and 731bp and the second between 712 and 1001bp, have been identified in the amelogenin region of the Y-chromosome (17). A third deletion is reported by a different group; however, the size of this deletion has not been deduced (18). A primer binding site mutation in the X homolog of the amelogenin sequence has also been identified. In this case, one of 327 phenotypically normal males investigated failed to produce an X-specific amplification product after DNA profiling with a commercially available kit (19).
ALTERNATIVE APPROACHES The error rates caused by mutational interference observed for sex determination by amplification of regions of the amelogenin gene to date are comparative to rates observed for routinely used autosomal STR loci in forensic DNA profiling (14). The rareness of failures in sex determination provides
Forensic Science, Medicine, and Pathology V2–4
Sex Determination_________________________________________________________________________________285 Table 1 Observed Sex-Typing Failure Frequencies in Different Populations
Population Global Caucasian
Indian Indian Caucasian Malaysian Israeli
Number of individuals tested 350 Unknown (Australian database) 427 270 29,432 338 96
confidence in current techniques, but the amelogenin locus alone cannot anymore be considered as infallible. The phenotypic nature of the information provided by investigation of the amelogenin locus could potentially present massive problems if an error occurs that is not identified. The majority of references cited in this article that describe problem cases do not call for the replacement of the amelogenin locus within commercial DNA profiling kits, they all suggest the supplementation of this locus with Y-chromosome-specific markers. There are a number of candidate DNA markers that are well suited to this auxiliary role. One such target is the sex-determining region of the Y-chromosome (SRY). The potential use of this locus for sex determination was first described in 1990 (20), and has been utilized by numerous groups for this role (see refs. 3 and 4). Zinc finger protein genes (ZFY/ZFX) have also been shown to exhibit homologous regions that can be simultaneously targeted in amelogenin-type amplification reactions (6,21,22). DNA markers of this type can effectively perform the same function as the amelogenin locus for sex determination of biological samples; they cannot, however, provide additional statistical support for the individualization of a biological sample. There is another class of DNA marker that is capable of providing both sex typing and individualistic information— sex chromosome STR markers. Y-chromosome analysis is a rapidly expanding speciality within forensic DNA profiling. Alone, as a result of their mode of paternal inheritance YSTRs are not as informative as autosomal STR loci and are predominantly used for paternity testing and evolutionary genetics. The commercialization and widespread usage of these core loci is, however, resulting in the accumulation of large amounts of population data for these YSTR markers. A standardized panel of 12 YSTR markers has been validated for forensic use (23). The inclusion of a single YSTR locus within the currently available autosomal STR profiling kits would therefore provide a duel function of confirming the biological sex of the sample being analyzed and provide additional
Number of observations of incorrect sex typing
Reference
2 1
15 16
10 6 6 6 1
18 24 25 26 27
statistical support for the individualization of that sample. This type of marker could easily be incorporated into current DNA profiling kits to provide a back up for sex determination and could, as a result, prevent errors occurring during criminal investigation and human identification.
Educational Message 1. The sex of a DNA sample can easily be determined by analysis of the X- and Y-chromosomes. 2. A 6bp deletion present within intron 1 of the X homolog of the amelogenin gene is primarily used as a sex-typing target in forensic and archaeological DNA analysis. 3. Primer binding site mutations and deletions present in the Y homolog of the amelogenin gene can result in errors in sex determination by DNA analysis. 4. Inclusion of Y-chromosome-specific amplification targets within commercial STR profiling kits would provide a back up to the amelogenin locus for accurate sex determination for forensic and archaeological investigation.
The author has stated that she does not have a significant financial interest or other relationship with any product manufacturer or provider of services discussed in this article.
REFERENCES 1. 1 Brown KA. Mini Review: Gender and Sex - What can ancient DNA tell us? Ancient Biomolecules 1998;2:3–15. 2. 2 Neeser D, Liechti-Gallati S. Sex determination of forensic samples by simultaneous PCR amplification of alpha-satellite DNA from both the X and Y chromosomes. J Forensic Sci 1995; 40:239–241. 3. 3 Naito E, Dewa K, Yamanouchi H, Kominami R. Sex typing of forensic DNA samples using male- and female-specific probes. J Forensic Sci 1994;39:1009–1017.
Forensic Science, Medicine, and Pathology V2–4
286 __________________________________________________________________________________________Graham 4. 4 Cui KH, Warnes GM, Jeffrey R, Matthews CD. Sex determination of preimplantation embryos by human testis-determininggene amplification. Lancet 1994;343:79–82. 5 Pfitzinger H, Ludes B, Mangin P. Sex determination of forensic 5. samples: co-amplification and simultaneous detection of a Y-specific and an X-specific DNA sequence. Int J Legal Med 1993;105:213–216. 6 Stacks B, Witte MM. Sex determination of dried blood stains 6. using the polymerase chain reaction (PCR) with homologous X–Y primers of the zinc finger protein gene. J Forensic Sci 1996;41:287–290. 7 Sullivan KM, Mannucci A, Kimpton CP, Gill P. A rapid and 7. quantitative DNA sex test: fluorescence-based PCR analysis of X–Y homologous gene amelogenin. Biotechniques 1993; 15:636–638, 640–641. 8 Lau EC, Mohandas TK, Shapiro LJ, Slavkin HC, Snead ML. 8. Human and mouse amelogenin gene loci are on the sex chromosomes. Genomics 1989;4:162–168. 9 Salido EC, Yen PH, Koprivnikar K, Yu LC, Shapiro LJ. The 9. human enamel protein gene amelogenin is expressed from both the X and the Y chromosomes. Am J Hum Genet 1992;50: 303–316. 10 Nakahori Y, Takenaka O, Nakagome Y. A human X–Y homolo10. gous region encodes “amelogenin.” Genomics 1991;9:264–269. 11 Akane A, Shiono H, Matsubara K, et al. Sex identification of 11. forensic specimens by polymerase chain reaction (PCR): two alternative methods. Forensic Sci Int 1991;49:81–88. 12 Akane A, Seki S, Shiono H, et al. Sex determination of foren12. sic samples by dual PCR amplification of an X–Y homologous gene. Forensic Sci Int 1992;52:143–148. 13 Mohammed F, Tayel SM. Sex identification of normal persons 13. and sex reverse cases from bloodstains using FISH and PCR. J Clin Forensic Med 2005;12:122–127. 14 Clayton TM, Hill SM, Denton LA, Watson SK, Urquhart AJ. 14. Primer binding site mutations affecting the typing of STR loci contained within the AMPFlSTR SGM Plus kit. Forensic Sci Int 2004;139:255–259.
15 Santos FR, Pandya A, Tyler-Smith C. Reliability of DNA-based 15. sex tests. Nat Genet 1998;18:103. 16 Roffey PE, Eckhoff CI, Kuhl JL. A rare mutation in the amelo16. genin gene and its potential investigative ramifications. J Forensic Sci 2000;45:1016–1019. 17 Mitchell RJ, Kreskas M, Baxter E, Buffalino L,Van Oorschot, RA. 17. An investigation of sequence deletions of amelogenin (AMELY), a Y-chromosome locus commonly used for gender determination. Ann Hum Biol 2006;33:227–240. 18 Kashyap VK, Sahoo S, Sitalaximi T, Trivedi R. Deletions in the 18. Y-derived amelogenin gene fragment in the Indian population. BMC Med Genet 2006;7:37. 19 Shadrach B, Commane M, Hren C, Warshawsky I. A rare muta19. tion in the primer binding region of the amelogenin gene can interfere with gender identification. J Mol Diagn 2004;6:401–405. 20 Sinclair AH, Berta P, Palmer MS, et al. A gene from the human 20. sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 1990;346:240–244. 21 Reynolds R, Varlaro J. Gender determination of forensic sam21. ples using PCR amplification of ZFX/ZFY gene sequences. J Forensic Sci 1996;41:279–286. 22 Roy R, Steffens D.L. Infrared fluorescent detection of PCR 22. amplified gender identifying alleles. J Forensic Sci 1997; 42:452–460. 23 Krenke BE, Viculis L, Richard ML, et al. Validation of male23. specific, 12-locus fluorescent short tandem repeat (STR) multiplex. Forensic Sci Int 2005;151:111–124. 24 Thangaraj K, Reddy AG, Singh L. Is the amelogenin gene reli24. able for gender identification in forensic casework and prenatal diagnosis? Int J Legal Med 2002;116:121–123. 25 Steinlechner M, Berger B, Niederstatter H, Parson W. Rare fail25. ures in the amelogenin sex test. Int J Legal Med 2002;116:117–120. 26 Chang YM, Burgoyne LA, Both K. Higher failures of amelo26. genin sex test in an Indian population group. J Forensic Sci 2003;48:1309–1313. 27 Michael A, Brauner P. Erroneous gender identification by the 27. amelogenin sex test. J Forensic Sci 2004;49:258–259.
Forensic Science, Medicine, and Pathology V2–4
DNA REVIEWS
Sex Determination Eleanor A. M. Graham Forensic Pathology Unit, University of Leicester, Leicester, United Kingdom Address for correspondence and reprints: Eleanor A. M. Graham Forensic Pathology Unit University of Leicester Robert Kilpatrick Building Leicester Royal Infirmary Leicester LE2 7LX United Kingdom E-mail: [email protected] Accepted for publication: August 21, 2006
Abstract Determining the sex of a given DNA sample can provide criminal investigators with useful intelligence and can aid the identification of missing persons and disaster victims. Polymerase chain reaction-based systems that amplify regions of the amelogenin gene have become the method of choice for sex determination of biological samples. This system can, however, result in false female sex designation when mutations affect primer binding sites of the Y homolog of this target sequence, causing drop out of the Y amplification product. Erroneous sex determination could have drastic consequences when applied to forensic situations by misdirecting investigators or hindering the identification of deceased individuals. Current methods of sex determination are described and possible alternative approaches to avoid errors are discussed. Key Words: Forensic identification; DNA; biological sex; amelogenin; Y-chromosome; X-chromosome. (DOI: 10.1385/Forensic Sci. Med. Pathol.:2:4:283)
INTRODUCTION
METHODS OF SEX DETERMINATION
Routine forensic DNA analysis involves the investigation of short-tandem repeat (STR) markers to individualize biological samples to determine their origin. These systems are not designed to provide any information pertaining to the physical characteristics of the individual being tested, with one major exception: biological sex. On some occasions in both forensic and archaeological situations, human remains are found in a state such that sex assignment via direct inspection or classic anthropological examination is impossible. In missing person’s cases, mass disasters, or dismemberments where corpses are unrecognizable or severely disrupted, it can at times be very difficult and sometimes impossible to determine the sex of an individual by physical examination alone. The sex of a skeleton by anthropological means requires that certain key regions are present, most importantly, the pelvis and skull. In blind tests on skeletal material of known sex, a 100% accuracy of sex assignment is rarely, if ever, achieved. The sexing of adult remains from an intact pelvis is around 95% accurate, 85–90% from an intact skull, and only 80–90% accurate from long bone examination. The accuracy is further reduced when dealing with prepubescent juvenile remains, which can be as low as 50% (1). For this reason, a DNA-based technique for sex assignment is critically important to forensic and archaeological investigation.
The genetic difference between males and females is defined by the presence or absence of theY-chromosome, or more specifically, the Y-chromosome complete with the sex-determining regions. The majority of the DNA of the sex chromosomes is specific to either the X or Y form. There are also regions of homology between the two sex chromosomes that are also useful targets for genetic sex typing of samples. A number of early papers concentrated on polymerase chain reaction (PCR) coamplification of X- and Y-specific regions of the sex chromosomes to give single-banding patterns for female samples and double-banding patterns for male DNA (2–6). Although all of these systems were proven to be accurate for sex determination from test samples, the possibility of obtaining false female results by amplification failure of the Y-specific target was quickly recognized (7). Alternative approaches to sex determination of DNA samples involve investigation of regions of the amelogenin gene. This is the gene that encodes tooth enamel and is present on both the X- (Xp22.1–Xp22.3) (8) and Y- (Yp11.2) (9) chromosomes. Closer inspection of this gene on each of the sex chromosomes identified regions of true homology, but also identified a number of sequence differences between the two forms (10). Because of these homologous regions, PCR systems could now be designed to amplify both X- and Y-chromosomes in a
283
284 __________________________________________________________________________________________Graham single reaction by placing primers in homologous positions on the amelogenin gene that flank areas of difference between the X and Y forms. This duel amplification from a single set of primers provides an internal PCR control, meaning reaction failure will affect both X- and Y-chromosomes, removing the possibility of false-positive results that could be encountered by separate amplification strategies. Early systems designed to amplify regions of the amelogenin gene focused around an 189bp deletion present in the first intron of the X homolog of the gene (10) and required large amounts of template DNA to be entered into the amplification reaction (11). The starting amount of template DNA was quickly reduced by optimization of the reaction leading to reported success rates from quantities equivalent to a single diploid cell (12). The biggest breakthrough in DNA sex typing came with the publication of an alternative target sequence within amelogenin gene (7). This system was again designed to co-amplify X- and Y-chromosomes in a single reaction, but was based around a shorter, 6bp X-chromosome deletion, allowing for amplification of shorter target sequences. Instead of long-length products (977bp and 788bp), amplified fragments were a mere 106bp and 112bp for the X- and Y-chromosomes respectively, and these targets could also be amplified from minute amounts of template DNA. This system was compatible with both low copy number and degraded DNA, and as such quickly became the system of choice within both forensic and archaeological disciplines. The short product lengths of these redesigned amplicons also had the advantage of falling within the size range of forensically important STR loci, and as such were perfectly suited to multiplexing within DNA profiling kits. Today, forensic DNA profiling is primarily carried out using commercially produced kits, designed to amplify up to 16 STR loci simultaneously. All kit primers for amplification of the amelogenin loci, and all are designed around the 6bp deletion of intron 1 of this gene. An additional bonus of this particular system comes with the detection method of choice. Because the amelogenin DNA fragments can be co-amplified along with autosomal STR loci within commercial DNA profiling kits, the fluorescent labeling and visualization method used for this process provides a degree of quantitative information along with fragment-length measurement. The quantitative data can then be used to determine the ratio of X- to Y-chromosomes present in the analyzed sample. Male/female DNA mixtures are often encountered during forensic investigation, especially in sexual assault investigations. The ability to calculate the relative ratio of male to female DNA present in a given mixture can be extremely useful for downstream analysis. This quantification will also detect chromosome duplication events of the sex chromosomes, for example, to detect the presence of an extra X-chromosome in XXY males. This system is not useful for correct sex determination in sex-reversal syndromes such as XY females and XX males (13), where a mutational event or translocation results in the loss or gain of the amelogenin gene.
PROBLEMS IN CURRENT SYSTEMS As in any PCR technique, problems can be encountered if a nonlethal mutational event occurs in the recognition sequence of either primer. Mutational events of this kind are usually recognized, depending on the position and extent of the mutational change, by inefficient amplification of target sequence or failure of the PCR altogether. For STR loci, the presence of primer binding-site mutations can cause allele drop out and discordance between STR profiles generated using different commercial kits (14). Although far from ideal, problems of this type will usually affect single STR loci in a multiplex reaction. The loss of information at a single position does not affect the overall power of the system. This is not true for the sex-discriminating amelogenin locus. The reliability of amelogenin-based sex testing was first questioned in 1998 with the observation of two phenotypically male individuals being classified as female after PCR analysis (15). A mutation in the amelogenin primer binding region was also reported by Roffey et al. in 2000 (16) when the DNA extracted from a buccal swab taken from a phenotypically normal male was typed as female after STR profiling. After further investigation of this anomaly, it was deduced that a single point mutation in the primer binding region of the Y homolog of the amelogenin locus was responsible for the drop out of the Y-chromosome amplification product in the DNA profile. The authors of this article highlighted the problems that could arise if this result occurred, but was not recognized during forensic casework. The failure of a forensic DNA profiling technique to correctly resolve an issue as simple as sex could seriously misdirect the police investigation, as well as cast doubts on the techniques’ reliability for criminal prosecution. Following this report, further observations of sex testing errors began to emerge in the forensic literature. These observations are summarized in Table 1. Further investigation of Y allele-null males has revealed that primer binding site mutations are not the only cause of amplification failure. Two deletions, one between 304 and 731bp and the second between 712 and 1001bp, have been identified in the amelogenin region of the Y-chromosome (17). A third deletion is reported by a different group; however, the size of this deletion has not been deduced (18). A primer binding site mutation in the X homolog of the amelogenin sequence has also been identified. In this case, one of 327 phenotypically normal males investigated failed to produce an X-specific amplification product after DNA profiling with a commercially available kit (19).
ALTERNATIVE APPROACHES The error rates caused by mutational interference observed for sex determination by amplification of regions of the amelogenin gene to date are comparative to rates observed for routinely used autosomal STR loci in forensic DNA profiling (14). The rareness of failures in sex determination provides
Forensic Science, Medicine, and Pathology V2–4
Sex Determination_________________________________________________________________________________285 Table 1 Observed Sex-Typing Failure Frequencies in Different Populations
Population Global Caucasian
Indian Indian Caucasian Malaysian Israeli
Number of individuals tested 350 Unknown (Australian database) 427 270 29,432 338 96
confidence in current techniques, but the amelogenin locus alone cannot anymore be considered as infallible. The phenotypic nature of the information provided by investigation of the amelogenin locus could potentially present massive problems if an error occurs that is not identified. The majority of references cited in this article that describe problem cases do not call for the replacement of the amelogenin locus within commercial DNA profiling kits, they all suggest the supplementation of this locus with Y-chromosome-specific markers. There are a number of candidate DNA markers that are well suited to this auxiliary role. One such target is the sex-determining region of the Y-chromosome (SRY). The potential use of this locus for sex determination was first described in 1990 (20), and has been utilized by numerous groups for this role (see refs. 3 and 4). Zinc finger protein genes (ZFY/ZFX) have also been shown to exhibit homologous regions that can be simultaneously targeted in amelogenin-type amplification reactions (6,21,22). DNA markers of this type can effectively perform the same function as the amelogenin locus for sex determination of biological samples; they cannot, however, provide additional statistical support for the individualization of a biological sample. There is another class of DNA marker that is capable of providing both sex typing and individualistic information— sex chromosome STR markers. Y-chromosome analysis is a rapidly expanding speciality within forensic DNA profiling. Alone, as a result of their mode of paternal inheritance YSTRs are not as informative as autosomal STR loci and are predominantly used for paternity testing and evolutionary genetics. The commercialization and widespread usage of these core loci is, however, resulting in the accumulation of large amounts of population data for these YSTR markers. A standardized panel of 12 YSTR markers has been validated for forensic use (23). The inclusion of a single YSTR locus within the currently available autosomal STR profiling kits would therefore provide a duel function of confirming the biological sex of the sample being analyzed and provide additional
Number of observations of incorrect sex typing
Reference
2 1
15 16
10 6 6 6 1
18 24 25 26 27
statistical support for the individualization of that sample. This type of marker could easily be incorporated into current DNA profiling kits to provide a back up for sex determination and could, as a result, prevent errors occurring during criminal investigation and human identification.
Educational Message 1. The sex of a DNA sample can easily be determined by analysis of the X- and Y-chromosomes. 2. A 6bp deletion present within intron 1 of the X homolog of the amelogenin gene is primarily used as a sex-typing target in forensic and archaeological DNA analysis. 3. Primer binding site mutations and deletions present in the Y homolog of the amelogenin gene can result in errors in sex determination by DNA analysis. 4. Inclusion of Y-chromosome-specific amplification targets within commercial STR profiling kits would provide a back up to the amelogenin locus for accurate sex determination for forensic and archaeological investigation.
The author has stated that she does not have a significant financial interest or other relationship with any product manufacturer or provider of services discussed in this article.
REFERENCES 1. 1 Brown KA. Mini Review: Gender and Sex - What can ancient DNA tell us? Ancient Biomolecules 1998;2:3–15. 2. 2 Neeser D, Liechti-Gallati S. Sex determination of forensic samples by simultaneous PCR amplification of alpha-satellite DNA from both the X and Y chromosomes. J Forensic Sci 1995; 40:239–241. 3. 3 Naito E, Dewa K, Yamanouchi H, Kominami R. Sex typing of forensic DNA samples using male- and female-specific probes. J Forensic Sci 1994;39:1009–1017.
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286 __________________________________________________________________________________________Graham 4. 4 Cui KH, Warnes GM, Jeffrey R, Matthews CD. Sex determination of preimplantation embryos by human testis-determininggene amplification. Lancet 1994;343:79–82. 5 Pfitzinger H, Ludes B, Mangin P. Sex determination of forensic 5. samples: co-amplification and simultaneous detection of a Y-specific and an X-specific DNA sequence. Int J Legal Med 1993;105:213–216. 6 Stacks B, Witte MM. Sex determination of dried blood stains 6. using the polymerase chain reaction (PCR) with homologous X–Y primers of the zinc finger protein gene. J Forensic Sci 1996;41:287–290. 7 Sullivan KM, Mannucci A, Kimpton CP, Gill P. A rapid and 7. quantitative DNA sex test: fluorescence-based PCR analysis of X–Y homologous gene amelogenin. Biotechniques 1993; 15:636–638, 640–641. 8 Lau EC, Mohandas TK, Shapiro LJ, Slavkin HC, Snead ML. 8. Human and mouse amelogenin gene loci are on the sex chromosomes. Genomics 1989;4:162–168. 9 Salido EC, Yen PH, Koprivnikar K, Yu LC, Shapiro LJ. The 9. human enamel protein gene amelogenin is expressed from both the X and the Y chromosomes. Am J Hum Genet 1992;50: 303–316. 10 Nakahori Y, Takenaka O, Nakagome Y. A human X–Y homolo10. gous region encodes “amelogenin.” Genomics 1991;9:264–269. 11 Akane A, Shiono H, Matsubara K, et al. Sex identification of 11. forensic specimens by polymerase chain reaction (PCR): two alternative methods. Forensic Sci Int 1991;49:81–88. 12 Akane A, Seki S, Shiono H, et al. Sex determination of foren12. sic samples by dual PCR amplification of an X–Y homologous gene. Forensic Sci Int 1992;52:143–148. 13 Mohammed F, Tayel SM. Sex identification of normal persons 13. and sex reverse cases from bloodstains using FISH and PCR. J Clin Forensic Med 2005;12:122–127. 14 Clayton TM, Hill SM, Denton LA, Watson SK, Urquhart AJ. 14. Primer binding site mutations affecting the typing of STR loci contained within the AMPFlSTR SGM Plus kit. Forensic Sci Int 2004;139:255–259.
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