Mobile element-based assay for human gender determination.

Determination of gender from human DNA samples is a common problem in forensic laboratories. While several PCR-based assays are currently available for human sex typing, each of the current approaches has limitations. Methods based on male-specific amplification, such as the amplification of the SRY locus [1], lack an internal positive control to discriminate between female DNA and male DNA which has failed to amplify for technical reasons. Restriction fragment length polymorphism assays based on sex-specific mutations at the ZFX/ZFY locus [2] require a second enzyme digestion or hybridization step following the initial PCR amplification. A recent method proposed by Cali et al. [3] based on a single adenine insertion within a tandem repeat array at the DXYS156 locus requires access to allele detection equipment potentially unavailable to forensic labs with limited resources. The most widely used approach is based on the Amelogenin locus, which yields different-sized PCR amplicons for the X and Y chromosome versions of the Amelogenin gene [4]. However, this method misidentifies males as females in some cases due to a deletion in the AMEL Y region [5– 7]. This deletion has previously been reported to be present at a frequency of 0.018% in Caucasian males, 1.85% among Indians, and as high as 8% in Sri Lankans [5–7]. While the frequency of the deletion is relatively low, the crucial nature of forensic test results in circumstances such as rape and prenatal gender determination where there is risk for male-specific inherited disorders makes any source of error a legitimate cause for concern. This has led several researchers to recommend that Amelogenin should not be relied upon as the sole determinant of gender [5–8]. Here, we present an alternative PCR method of human gender identification based on the presence/absence of Alu sequences. Alu elements are transposable elements which have amplified throughout primate evolution and comprise roughly 10% of the human genome [9]. Alu insertions are generally considered to be homoplasy free with respect to human population genetics, as the probability of two Alu elements independently inserting in the same genomic location is extremely small [9]. The insertion of an Alu element into a nonrecombining X–Y homologous region creates a way of differentiating between inserted and noninserted chromosomes based on PCR amplicon size. While some recently integrated Alu insertions remain polymorphic in the human population, many ultimately reach fixation for the presence of the Alu insertion [9]. Fixed insertions on either the X or the Y chromosome provide a way of identifying the respective chromosome, as the inserted chromosome yields a larger fragment when the homologous region is amplified with PCR (Fig. 1). By screening X–Y homologous Alu insertions for levels of insertion polymorphism, we identified twomonomorphic Alu insertions thatmeet the necessary criteria for a gender determination assay, one fixed on the X chromosome, AluSTXa, and one fixed on theY chromosome,AluSTYa. Both of theAlu elements presumably inserted and reached fixation in the human lineage prior to the radiation of modern humans from Africa. Amplification of DNA samples from 778 diverse (African-American, EuropeanAmerican, and Hispanic-American) individuals of defined sex from paternity/identity cases for both the AluSTYa and theAluSTXa loci showed 100% accuracy in gender identification. TheDNA samples used in the study consisted of 389 females (278 African-American, 102 European-American, and 9 Hispanic-American) and 389 males (288 African-American, 90 European-American, and 11 Hispanic-American). Corresponding author. Fax: 1-225-578-7113. E-mail address: [email protected] (M.A. Batzer). Analytical Biochemistry 312 (2003) 77–79