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As a novel type of genetic marker, the microhaplotype has shown promising potential in forensic research. In the present study, we analyzed maternal plasma cell-free DNA (cfDNA) samples from twin pregnancies to validate microhaplotype-based noninvasive prenatal testing (NIPT) for paternity, zygosity, and fetal fraction (FF). Paternity was determined with the combined use of the relMix package, zygosity was evaluated by examining the presence of informative loci with two fetal genome complements, and FF was assessed through fetal allele ratios. Paternity was determined in 19 twin cases, among which 13 cases were considered dizygotic (DZ) twins based on the presence of 3~10 informative loci and the remaining 6 cases were considered monozygotic (MZ) twins because no informative locus was observed. With the fetal genomic genotypes as a reference, the accuracy of paternity and zygosity determination were confirmed by standard short tandem repeat (STR) analysis. Moreover, the lower FF, higher FF, and combined FF in each DZ plasma sample were closely related to the estimated value. This present preliminary study proposes that microhaplotype-based NIPT is applicable for paternity, zygosity, and FF determination in twin pregnancies, which are expected to be advantageous for both forensic and clinical settings.

Evaluation of a Microhaplotype-Based Noninvasive Prenatal Test in Twin Gestations: Determination of Paternity, Zygosity, and Fetal Fraction