Identification and molecular characterisation of an AMEL-X null allele due to an Alu insertion

The amelogenin locus is used in most forensic STR multiplex kits as sex-typing marker. In the present work, we performed the molecular characterisation of an AMEL-X null allele, observed in an Eritrean male profile when using either the PowerPlex® Fusion 6C or the AmpFLSTR™ NGM SElect™ multiplexes. After X-specific PCR amplification of the amelogenin locus using an in-house primer pair and Sanger sequencing, we found that the AMEL-X amplification failure was due to an insertion of a 370 bp Alu element. We recognised all the hallmarks of the Alu elements, i.e. the target specific duplications (TSDs) at the boundaries of the insertion, the 3’ A-tail and the LINE1 endonuclease cleavage site, and we used these elements to precisely identify the insertion point of the retro-transposon. We found that the Alu insertion did not disrupt the primer binding sites of the AMEL-X neither in the PowerPlex® Fusion 6C nor in the AmpFLSTR™ NGM SElect™ system. Instead, the amplification failure of the X-specific amelogenin marker could be explained by the competition of the Y-specific amelogenin locus for the same primer pair, which lead to the sub-optimal amplification of the longer AMEL-X. The analysis of 145 additional eastern African males did not reveal other AMEL-X null alleles. To our knowledge, this is the first reported case of an Alu insertion causing an AMEL-X dropout, which is usually due to point mutations at the primer binding sites.