A rare mutation in the primer binding region of the amelogenin gene can interfere with gender identification

PCR amplification of part of the X-Y homologous amelogenin gene with a single primer pair has been used as a sex identification test because it generates different length products from the X and Y chromosomes. Using a commercially available kit that contains amelogenin primers, we report a single phenotypically normal Caucasian male out of 327 males tested to date that failed to show an X chromosome-specific PCR product. Using alternative amelogenin primers external to but encompassing the initial amplicon, an X chromosome-specific product was seen. Sequence analysis of this X-specific PCR product revealed a C to G mutation at the most 3' base of the initial reverse amelogenin PCR primer. An alternative reverse PCR primer with this most 3' base deleted showed X- and Y-specific products from the case study male. Rare mutations that result in a failure to amplify sex chromosome-specific products can result in incorrect gender identification.