Genome-wide DNA methylome profiling to differentiate monozygotic twins

In the current forensic environment, short tandem repeat (STR) profiling originating from genomic differences is highly accurate and widely used to identify individuals. However, if the person of interest is a monozygotic (MZ) twin, his or her DNA profile is identical to that of his or her twin; thus, STR profiling cannot discriminate between them. Therefore, DNA methylation, which is known to have different patterns even in MZ twins, has attracted attention as a promising marker to differentiate MZ twins. These epigenetic patterns are affected by environmental factors and age, and distinct DNA methylation patterns have been observed among the three types of MZ twins, i.e., dichorionic-diamniotic, monochorionic-diamniotic, and monochorionic-monoamniotic. To compare DNA methylation profiles among these three types of MZ twins and identify common markers to differentiate MZ twins, we collected cord blood samples from 54 pairs of MZ twins and analyzed their DNA methylation profiles using the Human MethylationEPIC v2.0 platform. The differences in DNA methylation observed among the three types of MZ twins occurred in immune-related regions. Differentially methylated genes identified in both monochorionic-diamniotic and monochorionic-monoamniotic twins were enriched in cytokine signaling and interleukin signaling-related regions. However, differentially methylated genes in dichorionic-diamniotic twins were enriched in PPI at synapse and the neuronal system. To facilitate twin differentiation, we selected a combination of CpG sites that differed between MZ twins and validated this CpG combination in two independent cohorts comprising 118 British MZ twin pairs and 47 Korean MZ twin pairs. Additionally, these selected DNA methylation markers were evaluated in 60 independent samples of MZ twins using pyrosequencing. Our results suggest that the methylation differences observed between MZ twins at birth persist throughout life. Consequently, these CpG site combinations could serve as valuable methylation markers in forensic cases where a suspect is a MZ twin.