Exploring the efficiency of different sets of microhaplotypes for first to third degree kinships

Microhaplotypes (MHs) are short, multi-allelic genetic markers that combine high genomic abundance with low mutation rates and the absence of stutter artefacts in PCR. These properties make them promising genetic markers for resolving complex kinships in forensic genetics. In this study, 202 MHs were genotyped in 181 family samples. By integrating empirical genotyping with extensive in-silico simulations, we evaluated the ability of these 202 MHs to identify first-, second-, and third-degree relationships from unrelated pairs. Additionally, four previously selected sets of MHs (190, 301, 337 and 703 MHs) were re-examined to determine their respective effectiveness across these kinship classes. The 202 MHs reliably distinguished first-degree relatives from unrelated individuals and other types of kinships. In addition, these 202 MHs could also discriminate second-degree relatives from unrelated individuals with the accuracy > 0.99. In contrast, the panel's performance declined markedly for third-degree relationships, and obtained accuracy was less than 0.5 when analytical threshold of cumulative likelihood ratio was set as 10⁴ and 10^-4. The four alternative panels (190, 301, 337 and 703 MHs) exhibited similarly high effectiveness for first- and second-degree relationships with the accuracy > 0.99. For third-degree relationships, accuracy increased monotonically with the number of microhaplotypes. Specifically, the 337-MH and 703-MH sets correctly assigned the majority of third-degree pairs regardless of the threshold adopted, indicating their suitability for third-degree kinship testing. In summary, the 202-MH panel could serve as a highly efficient tool for first- and second-degree kinship testing in forensic practice. In addition, panels containing 337 or 703 MHs are recommended for third-degree analyses. Future work should further improve performance by integrating MHs with other types of genetic markers to address intricated kinship analysis.