Triple sex marker failure in male DNA: AMELY, Y-InDel, and terminal Yq deletion resolved by integrated STR/NGS analysis.

Sex-inference in forensic DNA analysis is a critical investigative parameter, commonly relying on the Amelogenin locus, along with the Y-Indel marker (rs2032678) and a limited set of Y-chromosomal markers included in autosomal STR multiplexes and quantitative PCR assays. However, structural rearrangements of the Y chromosome can result in discordant profiles that misidentify phenotypically male individuals as "false females" in standard autosomal kits. We report an exceptionally rare case from central India exhibiting three convergent genetic anomalies in a phenotypic male blood sample: the loss of primary forensic sex markers (AMELY/Y-Indel), X-chromosomal heterozygosity and an extensive Yq deletion. This "triple-failure" haplotype (AMELY-/Y-InDel-/Yq-) represents unprecedented complexity combining South Asian-pattern Yp11.2 microdeletion with massive Yq loss and X-chromosomal diploidy. Moreover, Quantification discordance occurs between Quantifiler Trio and PowerQuant confirmed selective Yq loss, while SRY positivity and NGS validation established male genetic sex. YHRD Release R69 analysis of the partial Yfiler Plus profile revealed no matches worldwide (n = 106,444; RMP <1/106,444, 95% CI up to 1/28,856) or in Eurasian-Indian (n = 1240; RMP <1/1240, 95% CI up to 1/337) and Indian national (n = 1238; RMP <1/1238, 95% CI up to 1/336) databases. The case exposes critical diagnostic limitations of single-target sex typing and Yq-biased quantification in structural variants, underscoring the need for redundant markers (SRY, Yp STRs, multi-Y qPCR targets). Integrated STR/NGS analysis with database frequency assessment remains essential for resolving such "ghost" Y-profiles in forensics.