The ISHI Student Ambassadors give us a glimpse of where forensic science is headed, and each story reveals a different path into the field. This series highlights their research, their journeys, and the problems they’re determined to solve.
Josefina Castagnola, whose passion for forensic epigenetics is opening new doors for human identification, previews her scientific poster for the upcoming ISHI conference. Her research looks at how DNA methylation in teeth can be used as a tool for age estimation—an especially powerful approach in contexts like mass disasters, where skeletal remains may be the only evidence available.
Guided by curiosity and a drive to contribute to human rights investigations, Josefina’s work is both deeply personal and scientifically ambitious. From troubleshooting pyrosequencing to pushing the limits of low DNA input, she’s learning what it takes to adapt, refine, and move the field forward.
Tell us the story of your research. What sparked the question, and what have you discovered so far?
The starting point of our research at ForenBios was the need to improve age estimation, a key part of the biological profile used for the identification of human remains in forensic anthropology contexts, such as mass disasters. In adults, it remains challenging to obtain accurate results in this parameter with classical anthropological methods. Forensic epigenetics has shown great potential for age estimation, but most studies have focused on biological fluids. That led us to the idea of studying skeletal tissues, which are often the only recoverable tissues in many contexts, and teeth are particularly resilient to environmental insults.
We observed that it is possible to recover substantial amounts of DNA from teeth, specifically from dentin and pulp, as these are the most protected tissues within the tooth. With 100 ng of DNA, we were able to obtain high-quality methylation profiles. Throughout the development of this work, we found that ELOVL2 showed good results in teeth, consistent with what has been reported in most previous studies. Moreover, we faced similar challenges to those reported for other fluids and tissues, such as decreased accuracy in older individuals and limitations in DNA input, knowing that bisulfite conversion tends to degrade the sample. There is a specific challenge in this case: bones and teeth were not included in widely used epigenetic clock models. This means that our work has relied on markers and CpG sites discovered in other fluids and tissues, which highlights the need to search for tissue-specific epigenetic markers for bones and teeth.
Our study is a pilot effort to standardize methylation analysis in dentin for age estimation and to assess whether it is possible to reduce the amount of DNA while still obtaining reliable data.
What drew you to this specific topic—and why does it matter to you personally or professionally?
Contributing to human identification in cases such as mass disasters and human rights investigations is personally very meaningful. Also, I am passionate about forensic epigenetics, and since I started studying this field, I have been fascinated by its potential applications. I would love to collaborate and contribute my part in this discipline, helping to improve methods, validate them, and work together with other scientists.
What’s one method or part of your research process that you found unexpectedly challenging or exciting?
A challenging part of the research was obtaining complete, high-quality methylation profiles, as these were also my first steps working with the pyrosequencer, where I was constantly learning under the guidance of my PI, Dr. Sara Zapico. Each stage has helped me improve the approach through re-evaluation, literature review, and re-testing. The process of troubleshooting and discovery has been both frustrating and exciting, pushing me to think critically and develop further.
Was there a moment during your research where something clicked—or didn’t go as expected? How did you adapt?
Two key moments stood out for me during the research. One was determining the lowest DNA concentration at which methylation profiles could still be reproducible. The other was discovering that some markers chosen from the literature on skeletal tissues were not informative in dentin in this study. These moments showed the need to adapt and optimize the protocol, as well as to standardize it before continuing with lower DNA concentrations or, in the future, working with samples under challenging environmental conditions.
Looking ahead, what’s the next question you’re itching to explore?
Some of the next questions I’m looking forward to exploring are how far we can push the limits of DNA input while still maintaining reliable methylation data. I’m also interested in developing models for age estimation in human remains exposed to extreme conditions, such as fire or other environmental insults, and in expanding the gene panel to include markers more specific to teeth and bones.
If someone only remembers one thing from your poster, what do you hope it is?
What I would like people to take away from my poster is that teeth can be employed in epigenetic DNA methylation studies as a potential tool for age estimation, and that interdisciplinary applications of epigenetics to forensic anthropology can help improve human identification.
What real-world problem do you hope your research helps to solve, and who do you hope it impacts most?
Accurate age-at-death estimation in adult skeletal remains is a major challenge in forensic anthropology. Developing protocols and exploring tissue-specific methylation markers can help improve the accuracy of molecular age estimation in forensic science, supporting human identification and providing answers to those seeking answers.