Forensic Evaluation of the Ion AmpliSeq MH-74 Microhaplotype Panel in the Portuguese Population

Background/Objectives: Microhaplotypes have emerged as promising genetic markers for forensic applications, combining low mutation rates with high polymorphic information. However, population data remain limited for several regions, including Portugal. This study aimed to generate population data for the Ion AmpliSeq™ MH-74 panel in a Portuguese population and evaluate its performance for forensic identification and kinship analysis. Methods: A total of 237 unrelated individuals were genotyped using massively parallel sequencing on the Ion GeneStudio S5 platform. Allele frequencies and forensic parameters were estimated. Population structure was assessed using principal component analysis (PCA) based on reference data from MicroHapDB. Kinship performance was evaluated through simulations of full siblings, half siblings, and first cousins and compared with STR markers using likelihood ratios (LR) and error rates. Results: The MH-74 panel showed high genetic diversity (mean He = 0.615; mean PIC = 0.565) and strong forensic efficiency, with very high combined power of discrimination and exclusion and a random match probability of 3.86 × 10^-55. PCA revealed clustering consistent with global population structure, with the Portuguese population grouping within European populations. Microhaplotypes produced higher log₁₀(LR) values for related pairs compared to STRs, particularly for closer relationships, with lower false negative rates. Conclusions: The MH-74 panel demonstrates high discriminatory power and reliable performance for kinship analysis in the Portuguese population, supporting the use of microhaplotypes as an effective complement to STR markers in forensic genetics.