Amplification failure of the amelogenin gene (AMELX) caused by a primer binding site mutation.

In Quantitative Fluorescent Polymerase Chain Reaction (QF-PCR), genetic identification of sex and detection of chromosome X and Y major numerical disorders is routinely performed analyzing PCR products generated from the amplification of the amelogenin gene, in conjunction with other sex-typing markers and short tandem repeat (STR) loci (Cirigliano et al., 1999). The amelogenin gene is located in the pseudoautosomal region of the sex chromosomes, on Xp22.3 and on Yp11.2, respectively; sequence differences between the two homologs have been commonly used to differentiate males from females. In particular, PCR products generated from the X- (AMELX) and Y- (AMELY) chromosome can be discriminated from one another using primers flanking a 6 bp deletion in the first intron of the X chromosome (Sullivan et al., 1993). Almost all QF-PCR assays have implemented primer sets of variable amplicon size targeting this deletion. However, the reliability of the amelogenin test in prenatal diagnosis may sometimes be challenged, mainly because of occasional amplification failure of AMELY in normal males, possibly leading to false gender identification, because of different Yp deletions encompassing the amelogenin locus. Several types of rare nonrecurrent deletions have been observed in Caucasians, whereas a single recurrent deletion is particularly frequent in the Indian subcontinent, involving about 2–8% of males (Jobling et al., 2007). In contrast, PCR drop-out of the X-homologue of the amelogenin gene, remaining undetected in females carrying a heterozygous genotype and thus not affecting DNA-based sex tests, have rarely been reported in the literature, and always as a consequence of point mutations in the PCR primers binding sites (Shadrach et al., 2004; Alves et al., 2006).