Monozygotic twins are difficult to differentiate in a forensic laboratory because they come from the same zygote. This causes the standard STR typing method used within forensic laboratories to be unable to distinguish monozygotic twins. Cassandra’s research assessed the usefulness of pyrosequencing CpG sites to determine if DNA methylation can be used to discriminate monozygotic twins. She will be presenting this research during her poster presentation at ISHI 34 this September. We chatted with her to learn a little more, and if you’ll be at the conference, be sure to stop by poster #56 to learn more!
Briefly describe your work/area of interest.
Monozygotic twins are difficult to discriminate in forensic DNA laboratories with the standard STR typing method because they have identical STR profiles. As a result, multiple professionals and researchers in the forensic field have focused on finding a way to differentiate monozygotic twins. One method of interest is epigenetics. Even though monozygotic twins share identical genomes, multiple epigenetic mechanisms can change the genome depending on the environmental conditions the individual faces. An epigenetic mechanism is DNA methylation. DNA methylation involves a methyl group being added to the C5 position in cytosine to regulate gene expression. It commonly occurs in CpG islands and promoter regions.
To investigate if DNA methylation can discriminate monozygotic twins, Towson University started this research project with multiple previous students under the direction of Dr. Cynthia Zeller and Dr. Kelly Elkins. One student analyzed the methylation level of cg00211609 (body of FAM132A) in 13 monozygotic twin pair samples with bisulfite pyrosequencing, and found it discriminated three out of 13 pairs of monozygotic twins. Since the first loci showed promising results, both Dr. Cynthia Zeller and Dr. Kelly Elkins decided to continue the project by adding a locus to determine if sequencing multiple loci will allow more or all the twin pairs to be discriminated.
The differentially methylated site chosen was cg26287080 (body of EXOC7). To evaluate the methylation level at this specific locus, each sample was first extracted via phenol-chloroform or provided as extracted DNA from the Coriell Institute for Medical Research. Then the samples were treated with bisulfite with the Qiagen Epitect Plus Bisulfite Kit. After bisulfite treatment, the samples were amplified with the PyroMark PCR Kit at the specific site and sequenced on the PyroMark Q48 AutoPrep with the PyroMark Q48 Advanced CpG Reagents. For Cg26287080 (body of EXOC7), the methylation level of the four CpG sites was determined by the PyroMark Q48 Autoprep software. The methylation levels determined by the software for the monozygotic twin pair were then inputted into a paired t-test to determine if the methylation level was significantly different enough to discriminate the twins. After statistical analysis, it was found that another pair of twins were differentiated. Overall, with both sites, it was found that four out of the thirteen pairs of monozygotic twins were differentiated. In the future, this research project will be continued by adding more monozygotic twin pairs and by sequencing more loci.
How did you get interested in this work? Why did this particular project appeal?
I first got involved in this research project when the previous Towson student graduated, and my research advisor, Dr. Kelly Elkins, wanted someone to continue the project because of the promising results. In addition, both the first and the second site used in this project were shown to be differentially methylated in a study by Park et al., 2017 (https://doi.org/10.1016/j.fsigss.2017.09.177). After being introduced to the project, I was immediately interested and honored to continue the research because I had always found DNA methylation fascinating.
Previously, I had learned about how DNA methylation can be useful for body fluid identification and age prediction, but I was not aware of how it can be used for twin discrimination. Dr. Kelly Elkins. The project also made me aware of how monozygotic twins cannot be differentiated with the standard DNA workflow and how it can cause issues in some instances where the case relies on DNA evidence. Although cases involving monozygotic twins that rely heavily on DNA evidence are rare, it is still important that forensic laboratories find a method capable of discriminating monozygotic twins. After being introduced to this project and its significance, I knew I wanted to continue this project so that I could contribute to helping find the best way to analyze monozygotic twin cases.
Can you summarize the impact of your work for the audience (ISHI attendees and some general forensic enthusiasts)? How might this advance the field?
This research project is intended to provide an additional method for discriminating monozygotic twins. As mentioned previously, STR typing is not capable of discriminating monozygotic twins. Therefore, this research aims to evaluate the effectiveness of bisulfite pyrosequencing two CpG sites for twin discrimination. The two sites sequenced have proven to discriminate some monozygotic twin pairs. In the future, more sites will be evaluated to determine the best sites so that most or all twin pairs can be differentiated. In the larger scheme of things, this research project along with other research on differentially methylated sites can help find a standard method for twin discrimination so that cases where it is necessary to discriminate monozygotic twin can be solved and analyzed fully.
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