Background: Since the early days of PCR techniques, sex identification, “sex-typing,” of genomic DNA samples
has been a fundamental part of human forensic analysis but also in animal genetics aiming at strategic livestock
breeding. Most analyses are employing the AMELX/AMELY gene loci on the X and Y chromosomes present in most
mammals. We hypothesize that sex-typing in humans is also possible based on the genes NLGN4X and NLGN4Y,
which represent X and Y chromosome-specific copies of a common ancestral neuroligin-4 orthologue.
Methods: Genomic DNA was isolated from human blood and buccal cell samples (total n = 111) and submitted
to two different strategies: (a) a traditional two-primer PCR approach detecting an insertion/deletion (indel)
polymorphism immediately upstream of the translational start on exon 1 and (b) detection of a single nucleotide
polymorphism, SNP, on the translational stop carrying exon 7. The SNP detection was based on a quantitative PCR
approach (rhAMP genotyping) employing DNA/RNA hybrid oligonucleotides that were blocked and which could
only be activated upon perfect annealing to the target DNA sequence.
Results: All indel PCR-tested human DNA samples showed two bands for males representing X- and Y-specific
copies of NLGN4 and a single band for female samples, i.e., homozygosity of NLGN4X and absence of NLGN4Y, in
accordance with the self-reported sex of the donors. These results were in perfect agreement with the results of
the rhAMP-based SNP-detection method: all males were consequently positive for both alleles, representing either
SNP variant, and females were interpreted as homozygous regarding the SNP variant found in NLGN4X. Both
methods have shown reliable and consistent results that enabled us to infer the sex of donor DNA samples across
different ethnicities.
Conclusions: These results indicate that the detection of human NLGN4X/Y is a suitable alternative to previously
reported methods employing gene loci such as AMELX/Y. Furthermore, this is the first report applying successfully
the rhAMP-genotyping strategy as a means for SNP-based sex-typing, which consequently will be applicable to
other gene loci or different species as well
