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 a male sample from Eritrea. The STR profile of this subject was obtained using both the AmpFLSTR™ NGM SElect™ and the PowerPlex® Fusion 6C, obtaining the same result at the amelogenin locus. After X-specific PCR amplification of the amelogenin locus using an in-house primer pair and Sanger sequencing of the AMEL-X null sample, we found that the AMEL-X amplification failure was due to the insertion of an Alu element of 370 bp. We recognised all the hallmarks of the Alu elements, i.e. the TSDs at the boundaries of the inserted element, the 3’ A-tail and the LINE1 endonuclease cleavage site, and we used these elements to precisely identify the insertion point of the Alu. Using this approach, we found that the Alu insertion did not disrupt the primer binding sites of the AMEL-X neither in the AmpFLSTR™ NGM SElect™ nor in the PowerPlex® Fusion 6C system. Instead, the amplification failure of the X-specific amelogenin marker could be explained by the competition of the Yspecific amelogenin locus for the same primers and, consequently, by the sub-optimal amplification of the longer AMEL-X. The insertion allele was not observed in additional 145 eastern African males, 88 genotyped with the PowerPlex® Fusion 6C and 57 analysed by AMEL-X specific PCR assay. To our knowledge, this is the first reported case of an Alu insertion causing an AMEL-X dropout, which is usually consequence of point mutations at the primer binding sites.